The role of YAP1 in neuroblastoma tumor progression
vorgelegt von
Dipl.-Ing.
Kerstin Ahrens
an der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktorin der Naturwissenschaften
- Dr. rer. nat. -
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Jens Kurreck
Gutachter: Prof. Dr. Johannes H. Schulte
Gutachter: Prof. Dr. Roland Lauster
Gutachterin: Prof. Dr. Angelika Eggert
Gutachter: Prof. Dr. Hyun-Dong Chang
Tag der wissenschaftlichen Aussprache: 01. November 2021
Berlin 2022
Summary
Summary
Neuroblastoma is a pediatric solid tumor, that originates from the developing sympathetic
nervous system and typically occurs in the adrenal medulla and paraspinal ganglia with
secondary locations in the bone marrow and lymph nodes among others. After significant
therapeutic advances over the past decades, neuroblastoma patients with refractory and
relapse tumors still possess a poor survival prognosis and their therapy remains a major
therapeutic challenge. The transcriptional co-activator Yes1 associated transcriptional
regulator 1 (YAP1) has been implicated in neuroblastoma relapse development and thus, may
be a notable regulator of drug resistance and tumor cell migration. This study outlines the
functional role of YAP1 in neuroblastoma tumor progression.
YAP1 shows a highly heterogeneous expression profile in neuroblastoma cell lines and tumor
tissues. Transient siRNA-mediated YAP1 knockdown reduced the proliferative capacity of
neuroblastoma cells expressing high level of YAP1. Migration assays and RNA sequencing of
tetracycline-inducible YAP1S127A-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
revealed that cell motility depends on YAP1. The application of standard chemotherapeutic
drugs causes less-reduced cell viability in YAP1S127A-activated cells, indicating a YAP1-mediated
cell protection against chemotherapeutic toxicity. Furthermore, YAP1 provoked a
mesenchymal phenotype switch in one cell line, whereas genes affecting the amino acid
metabolism and mitochondrial folate cycle were upregulated in the other. The enforced
expression of YAP1S127A enhanced the glucose consumption and extracellular acidification
rate. Accompanied by an upregulation of key glycolytic genes, these results delineate a YAP1-
triggered metabolic switch towards aerobic glycolysis (Warburg effect) in neuroblastoma cells.
Since the Hypoxia inducible factor 2
𝛼
(HIF-2
𝛼
) encoding gene EPAS1 was upregulated in
YAP1S127A-activated cells, a YAP1-mediated stabilization of HIF-2
𝛼
with the subsequent
regulation of glucose metabolism was hypothesized. Contrary to our assumption, an increased
HIF-2
𝛼
abundance could only be detected in hypoxic YAP1S127A-activated cells. These
metabolic changes induced by YAP1 accompanied by increased cell motility, enhanced
chemotherapy tolerance and hypoxic adaption outline an aggressive and resistant tumor-cell
phenotype, that likely favors neuroblastoma progression and relapse development.
Overall, the results of this thesis highlight the unfavorable role of YAP1 in neuroblastoma
progression. Thus, our work provides new evidence of YAP1 as a promising therapeutic target
in neuroblastoma treatment. Further efforts are needed to verify our findings in a complex
biological system with focus on neuroblastoma relapse development on the one hand and the
mechanism and timing of YAP1 activation on the other.
Zusammenfassung
Zusammenfassung
Das Neuroblastom ist eine maligne Krebserkrankung des jungen Kindesalters. Die Tumore ent-
stehen im sich entwickelnden sympathischen Nervengewebe und zeigen sich typischerweise
im Mark der Nebennieren und im Grenzstrang. Metastasen werden unter anderem in
Knochenmark und Lymphknoten gefunden. Trotz erheblicher Therapieoptimierungen in den
letzten Jahrzehnten haben Patienten mit rezidivierenden und therapieresistenten
Neuroblastomen noch immer schlechte Prognosen und ihre Therapie bleibt eine der größten
therapeutischen Herausforderungen. Der transkriptionelle Co-Aktivator Yes1 associated
transcriptional regulator 1 (YAP1) wurde mit der Entwicklung rezidivierender Neuroblastome
in Zusammenhang gebracht und könnte somit ein entscheidender Regulator von
Medikamentenresistenzen und Tumorzellmigration sein. Die vorliegende Arbeit untersucht
den Einfluss von YAP1 auf die Entstehung von Neuroblastomen.
Die YAP1-Expression variiert stark in Neuroblastom-Zelllinien und Tumorgeweben. Eine
transiente siRNA-vermittelte Minderung der YAP1-Expression reduzierte die Zellproliferation
von YAP1 hoch exprimierenden Zellen. Migrationsassays und eine RNA-Sequenzierung von
SH-EP-TR-YAP1S127A und SK-N-AS-TR-YAP1S127A Zellen, die nach Zugabe von Tetrazyklin
konstitutiv aktives YAP1S127A synthetisieren, zeigten eine Abhängigkeit der Zellmotilität von
YAP1. Die Exposition gegenüber häufig eingesetzten Chemotherapeutika reduzierte die
Zellviabilität YAP1S127A-aktivierter Zellen in geringerem Maße als die der Kontrollzellen, was
auf einen YAP1-vermittelten Schutz der Zelle gegen die Toxizität der Chemotherapeutika
hindeutet. Weiterhin bewirkte die Induktion von YAP1S127A in einer der beiden Zelllinien einen
Wechsel der Zellidentität hin zu einem mesenchymalen Phänotyp, wohingegen in der Anderen
eine Hochregulation von Genen beobachtet wurde, die den Aminosäure-Stoffwechsel und den
mitochondrialen Teil des Folatzyklus regulieren. Die Überexpression von YAP1S127A erhöhte
außerdem den Glukoseverbrauch und die extrazelluläre Versauerungsrate, was zusammen
mit der erhöhten Transkription von Schlüsselgenen der Glykolyse eine YAP1-vermittelte
Aktivierung der aeroben Glykolyse (Warburg Effekt) beschreibt. EPAS1, welches den Hypoxie-
induzierbaren Faktor 2
𝛼
(HIF-2
𝛼
) kodiert, war in beiden YAP1S127A-aktivierten Zelllinien
hochreguliert. Die Vermutung, YAP1 stabilisiere das Protein HIF-2
𝛼
und reguliere damit den
Glukosemetabolismus, konnte nicht bestätigt werden. Erhöhte HIF-2
𝛼
Abundanz wurde nur
in hypoxischen YAP1S127A-aktivierten Zellen detektiert. Die beschriebenen
Stoffwechselveränderungen, die erhöhte Zellmotilität, die induzierte Toleranz von
Chemotherapeutika und die Adaption an hypoxische Bedingungen durch die Aktivität von
Zusammenfassung
YAP1 resultiert in einem aggressiven und resistenten Tumorzelltyp, der das Fortschreiten des
Neuroblastoms und die Entwicklung von Rezidiven fördert.
Zusammenfassend zeigen die Ergebnisse dieser Arbeit einen nachteiligen Effekt von aktivem
YAP1 in der Progredienz des Neuroblastoms. Damit liefern wir neue Hinweise, YAP1 als ein
vielversprechendes Zielmolekül in der Neuroblastom-Therapie zu betrachten. Für die
Validierung der hier dargelegten Ergebnisse sind weitere Untersuchungen nötig, die den
Beitrag von YAP1 zur Entwicklung von Neuroblastom-Rezidiven und den Mechanismus sowie
das Timing der YAP1-Aktivierung in einem komplexen biologischen System betrachten.
Table of Contents
i
Table of Contents
List of Figures v
List of Tables viii
List of Abbreviations ix
1.Introduction 1
1.1Neuroblastoma ....................................................................................................... 1
1.1.1Clinical presentation and assessment ........................................................ 1
1.1.2Staging systems and risk stratification ....................................................... 2
1.1.3Tumor biology ............................................................................................ 2
1.1.4Therapy ...................................................................................................... 4
1.2Yes1 associated transcriptional regulator 1 ............................................................ 6
1.2.1YAP1 structure and transcriptional activity ................................................ 6
1.2.2Regulation of YAP1 activity ........................................................................ 8
1.2.3Cell growth under control of YAP1 ........................................................... 11
1.2.4YAP1 regulates cancer cell migration and metastasis .............................. 13
1.2.5YAP1-mediated antitumor-therapy resistance ........................................ 15
1.2.6YAP1-induced epithelial-mesenchymal transition ................................... 18
1.2.7YAP1 and cancer cell metabolism ............................................................ 19
2.Scope and objectives 26
3.Materials 27
3.1Cell lines ................................................................................................................ 27
3.2Bacterial strains ..................................................................................................... 28
3.3Cell culture media ................................................................................................. 28
3.4Chemicals and enzymes ........................................................................................ 29
3.5Antibodies ............................................................................................................. 32
3.6Kits ......................................................................................................................... 33
3.7Disposables ........................................................................................................... 34
3.8Laboratory equipment .......................................................................................... 35
Table of Contents
ii
3.9Plasmids ............................................................................................................... 37
3.10Oligonucleotides .................................................................................................. 38
4.Methods 40
4.1Cell culture ........................................................................................................... 40
4.1.1Cell lines .................................................................................................. 40
4.1.2Cultivation of neuroblastoma cell lines ................................................... 40
4.1.3Cultivation of non-neuroblastoma cell lines ........................................... 41
4.1.4Cell passaging .......................................................................................... 41
4.1.5Cell stock preparation ............................................................................. 42
4.1.6Cell thawing ............................................................................................. 42
4.1.7Cell counting ............................................................................................ 42
4.1.8Silencing RNA-mediated gene knockdown .............................................. 43
4.1.9Lentiviral cell transduction with short hairpin RNA ................................ 43
4.1.10Generation and YAP1S127A activation of neuroblastoma cell lines
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A .................................................... 44
4.2Functional in vitro assays ..................................................................................... 46
4.2.1Cell viability assay .................................................................................... 46
4.2.2Colorimetric cell proliferation immunoassay (BrdU-ELISA) ..................... 46
4.2.3Photometric apoptosis immunoassay (DNA-histone ELISA) ................... 47
4.2.4Flow cytometry analysis of apoptotic cells ............................................. 48
4.2.5Cell cycle analysis .................................................................................... 49
4.2.6Wound healing assay .............................................................................. 50
4.2.7Transwell migration assay ....................................................................... 51
4.2.8Colony formation assay ........................................................................... 52
4.2.9Serum starvation of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A ...... 52
4.2.10Detection of senescence via β-galactosidase staining ........................... 53
4.2.11Measurement of pH value, glucose and lactate concentration in
supernatants of in vitro cell cultures .................................................................... 54
4.3Molecular biology methods ................................................................................. 54
4.3.1Gateway® Cloning of pDEST30-YAP1(S127A)-2xFlag/-3xFlag for
overexpression of constitutively active YAP1 ...................................................... 54
Table of Contents
iii
4.3.2Cloning of shRNA vectors for lentiviral transduction ............................... 55
4.3.3Preparation of chemically competent bacteria cells ................................ 56
4.3.4Transformation of E. coli cells .................................................................. 57
4.3.5Agarose gel electrophoresis ..................................................................... 58
4.3.6Small scale plasmid DNA extraction ......................................................... 58
4.3.7Large scale plasmid DNA extraction ......................................................... 59
4.3.8Production and titration of lentiviral particles ......................................... 59
4.3.9RNA extraction ......................................................................................... 61
4.3.10Nucleic acid quantification ...................................................................... 63
4.3.11RNA quantification and RIN determination ............................................ 63
4.3.12Reverse transcription .............................................................................. 64
4.3.13Quantitative real-time PCR ..................................................................... 64
4.3.14RNA sequencing and data analysis .......................................................... 65
4.4Biochemical methods ............................................................................................ 66
4.4.1Protein isolation of whole-cell lysates ..................................................... 66
4.4.2Measurement of protein concentration with bicinchonic acid assay ...... 67
4.4.3Protein separation by SDS-PAGE .............................................................. 68
4.4.4Western Blot ............................................................................................ 69
4.4.5Immunofluorescent staining of YAP1 and Flag-Tag ................................. 70
4.5Statistics ................................................................................................................ 71
4.6Software ................................................................................................................ 71
5.Results 73
5.1Endogenous YAP1 expression in neuroblastoma cell lines and tumor tissues ..... 73
5.2Neuroblastoma cell density has no effect on YAP1 in vitro .................................. 76
5.3Inhibition of YAP1 protein synthesis via RNA interference ................................... 77
5.3.1Small interfering RNA-mediated YAP1 knockdown .................................. 78
5.3.2Short hairpin RNA-mediated YAP1 knockdown ....................................... 80
5.4Impaired cell proliferation and viability upon YAP1 knockdown .......................... 81
5.5Reduced YAP1 levels hampered migratory potential of neuroblastoma cells ...... 84
5.6Influence of YAP1 knockdown on extracellular glucose and lactate concentrations
86
Table of Contents
iv
5.7Establishment of a tetracycline-inducible overexpression system for constitutively
active YAP1S127A in neuroblastoma cell lines SH-EP and SK-N-AS .................................. 87
5.8Transcriptome analysis of YAP1-activated neuroblastoma cells .......................... 90
5.9Gene ontology enrichment analysis of YAP1-activated neuroblastoma cells ...... 93
5.10Effect of YAP1 induction on tumor cell growth .................................................... 94
5.11Enhanced cell migration in YAP1-activated neuroblastoma cells ........................ 99
5.12YAP1 activation induces a mesenchymal/neural crest cell-like phenotype in
SK-N-AS-TR-YAP1S127A .................................................................................................. 103
5.13Induction of genes regulating serine de novo synthesis, folate cycle and cysteine
synthesis in YAP1-activated SH-EP-TR-YAP1S127A ......................................................... 105
5.14YAP1 enhances amino acid metabolism in neuroblastoma cells ....................... 106
5.15Active YAP1 reduces the sensitivity to chemotherapy-induced cytotoxicity in
neuroblastoma cells .................................................................................................... 108
5.16YAP1 enables neuroblastoma cells to survive growth factor limitations ........... 111
5.17YAP1 initiates a glycolytic switch in neuroblastoma cells .................................. 113
5.18Does YAP1 synergize with HIF-2
α
? ..................................................................... 117
6.Discussion 121
6.1YAP1 co-determines cell proliferation and viability of neuroblastoma cells ..... 121
6.2YAP1 mediates a reduced sensitivity to cytotoxicity of chemotherapeutic drugs to
neuroblastoma cells .................................................................................................... 125
6.3YAP1 promotes motility of neuroblastoma cells ................................................ 128
6.4Does YAP1 induce EMT in neuroblastoma? ....................................................... 132
6.5YAP1 intervenes in the metabolism of neuroblastoma cells .............................. 136
6.6Conclusions and Outlook .................................................................................... 146
7.Bibliography 149
8.Appendix 177
8.1Supplemental Figures ......................................................................................... 177
8.2Supplemental data of gene analysis ................................................................... 180
8.3Acknowledgement / Danksagung ...................................................................... 183
List of Figures
v
List of Figures
Figure 1 Mammalian Hippo-YAP1 pathway ............................................................................ 8
Figure 2 Gating strategy for FACS analysis of apoptosis ....................................................... 48
Figure 3 Gateway cloning strategy ........................................................................................ 54
Figure 4 Transduction efficiency of lentivirus carrying shRNA targeting YAP1 in neuroblastoma
cells ....................................................................................................................................... 61
Figure 5 Various YAP1 protein synthesis in neuroblastoma cell lines ................................... 73
Figure 6 YAP1 expression levels are variable in 19 neuroblastoma cell lines ....................... 74
Figure 7 Neuroblastoma tumors exhibit various YAP1 expression levels ............................. 74
Figure 8 Event-free survival probability of neuroblastoma patients negatively correlates with
YAP1 mRNA expression levels in INSS 3 and 4s tumors ........................................................ 75
Figure 9 Cell-cell contact has no effect on YAP1 levels and YAP1 protein phosphorylation . 77
Figure 10 Establishment of a siRNA-mediated YAP1 knockdown in neuroblastoma cells .... 79
Figure 11 Insufficient YAP1 knockdown was achieved by shRNA delivery ........................... 80
Figure 12 The cell viability of neuroblastoma cells is reduced upon YAP1 knockdown ........ 81
Figure 13 Cell proliferation is impaired upon YAP1 knockdown ........................................... 82
Figure 14 Cell cycle, but not apoptosis is marginally affected by YAP1 knockdown ............. 83
Figure 15 Cell viability of neuroblastoma cells with shRNA-mediated YAP1 knockdown ..... 83
Figure 16 YAP1 knockdown hampers cell motility of neuroblastoma cells .......................... 85
Figure 17 Unaltered pH values, lactate and glucose levels in the supernatant of neuroblastoma
cells after YAP1 knockdown .................................................................................................. 86
Figure 18 Experimental validation of a successful tetracycline-induced YAP1 activation in
SH-EP-TR-YAP1S127A cells ....................................................................................................... 87
Figure 19 Immunofluorescent staining of YAP1 and Flag-Tag demonstrates activated YAP1S127A
predominantly in and around the cell nuclei ....................................................................... 88
Figure 20 The expression of YAP1 and its target genes could be induced by tetracycline-
treatment of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells ....................................... 89
Figure 21 The reduced cell viability upon YAP1 knockdown could be reversed by additional
YAP1S127A activation in SH-EP-TR-YAP1S127A cells ................................................................... 90
List of Figures
vi
Figure 22 YAP1 activation in RNA sequencing samples ........................................................ 91
Figure 23 Genes are differentially expressed upon YAP1S127A activation in SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A cells ............................................................................................. 92
Figure 24 Cell viability and cell numbers are not affected in YAP1-activated cells ............... 95
Figure 25 Cell proliferation is not altered in YAP1-activated cells ........................................ 95
Figure 26 Apoptosis and cell cycle analysis revealed no effect of YAP1 activation in
neuroblastoma cells .............................................................................................................. 96
Figure 27 Clonogenicity of neuroblastoma cells is not affected upon YAP1 activation ........ 97
Figure 28 YAP1 overexpression does not induce senescence ............................................... 98
Figure 29 Cell motility is elevated in YAP1-activated SH-EP-TR-YAP1S127A cells .................... 99
Figure 30 Neuroblastoma cell migration is increased upon YAP1 activation ..................... 100
Figure 31 Migration-associated genes are upregulated in YAP1-activated SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A cells ........................................................................................... 101
Figure 32 Assignment of YAP1-induced differentially expressed genes to MES and ADRN
signatures ............................................................................................................................ 102
Figure 33 RNA expression of transcription factors defines neuroblastoma subpopulations in
YAP1-activated cells ............................................................................................................ 103
Figure 34 Gene regulating serine/cysteine synthesis and the folate cycle are upregulated in
YAP1-activated SH-EP-TR-YAP1S127A ................................................................................... 104
Figure 35 YAP1 induces the upregulation of amino acid transporter genes in neuroblastoma
cells .................................................................................................................................... 105
Figure 36 Cell viability of YAP1-activated cells under treatment with etoposide, doxorubicin
and vincristine ..................................................................................................................... 108
Figure 37 Failed activation of YAP1 in SH-EP-TR-YAP1S127A clone #14 ................................ 109
Figure 38 YAP1 activation increased the cell viability of serum-starved neuroblastoma cells
............................................................................................................................................ 110
Figure 39 YAP1 activation has no effect in cell proliferation of serum-starving cells ......... 111
Figure 40 YAP1 activation in SH-EP-TR-YAP1S127A cells alters the extracellular glucose and
lactate concentration
Figure 41 YAP1S127A overexpression affects the glycolytic rate in neuroblastoma cell lines113
List of Figures
vii
Figure 42 Glycolysis-associated genes are upregulated in YAP1-activated SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A cells ........................................................................................... 115
Figure 43 EPAS1 is upregulated in YAP1-activated SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells .................................................................................................. 116
Figure 44 No HIF-2
𝛼
stabilization in YAP1-activated neuroblastoma cells under normoxic
conditions ........................................................................................................................... 117
Figure 45 HIF-2
𝛼
protein levels in normoxic and hypoxic YAP1-activated neuroblastoma cells
............................................................................................................................................ 118
Figure 46 HIF and TEAD binding site enrichment in YAP1-induced SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells .................................................................................................. 119
List of Tables
viii
List of Tables
Table 1 Cell lines ..................................................................................................................... 27
Table 2 Bacterial Strains .......................................................................................................... 28
Table 3 Cell culture media and reagents ................................................................................ 28
Table 4 Chemicals ................................................................................................................... 29
Table 5 Enzymes ...................................................................................................................... 32
Table 6 Antibodies used for Western Blot .............................................................................. 32
Table 7 Antibodies used for Immunostaining ......................................................................... 32
Table 8 Kits .............................................................................................................................. 33
Table 9 Disposables ................................................................................................................. 34
Table 10 Laboratory equipment ............................................................................................. 35
Table 11 Plasmids ................................................................................................................... 37
Table 12 qPCR Primer ............................................................................................................. 38
Table 13 DNA Sequencing Primer ........................................................................................... 38
Table 14 siRNAs ....................................................................................................................... 38
Table 15 shRNA ....................................................................................................................... 39
Table 16 Components of lentiviral transduction medium ...................................................... 44
Table 17 Components of transfection medium for HEK293T cells ......................................... 60
Table 18 Components of HEK293T post transfection growth medium .................................. 60
Table 19 Components of qPCR master mix ............................................................................. 64
Table 20 Temperature profile SYBRgreen-based qPCR ........................................................... 65
Table 21 SDS-PAGE separation and stacking gel composition for a single gel ........................ 68
Table 22 Characteristics of ten neuroblastoma cell lines used in this project1. ..................... 76
Table 23 Gene Ontology analysis of up- and downregulated genes in YAP1-induced SH-EP-TR-
YAP1S127A 1. .............................................................................................................................. 93
Table 24 Gene Ontology analysis of up- and downregulated genes in YAP1-induced SK-N-AS-
TR-YAP1S127A 1. ......................................................................................................................... 94
List of Abbreviations
ix
List of Abbreviations
Abbreviation
Designation
-Tet
Ethanol-treated control
#
Clone of a plasmid-transfected cell line
+Tet
Tetracycline
2-DG
2-Deoxyglucose
7-AAD
7-Amino-actinomycin D
ABC
ATP-binding cassette
Abdom.
Abdominal
ABTS
2,2-Azinodi-3-ethylbenzthiazoline sulfonate
Adren.
Adrenal
ADRN
Adrenergic
AKT
Protein kinase B
ALDH1L2
Aldehyde dehydrogenase 1 family member L2
ALK
Anaplastic lymphoma kinase
AMOT
Angiomotin
Amp.
Amplified
AP-1
Activator protein 1
APC
Allophycocyanin
APS
Ammonium persulfate
ARNT
Aryl hydrocarbon receptor nuclear translocator
ATP
Adenosine 5-triphosphate
BCA
Bicinchonic acid
BM
Bone marrow
bp
Base pairs
BrdU
5-bromo-2-deoxyuridine
BSA
Bovine serum albumin
CBS
Cystathionine
𝛽
-synthetase
cDNA
Complementary DANN
List of Abbreviations
x
Abbreviation
Designation
CRC
Core regulatory circuitries
Ct
Threshold Cycle
CTH
Cystathionine 𝛾-lyase
ddH2O
Double-distilled water
Del
Deletion
DEPC
Diethyl carbonate
DMEM
Dulbecco´s modified eagle medium
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
DTT
Dithiothreitol
ECAR
Extracellular acidification rate
ECM
Extracellular matrix
EDTA
Ethylenediaminetetraacetic acid
EFS
Event-free survival
eGFP
Enhanced GFP
EGTA
Ethylene glycol-bis(𝛽-aminoethyl ether)-N,N,Nʹ,Nʹ-tetra
acetic acid
ELISA
Enzyme-linked immunosorbent assay
EMT
Epithelial mesenchymal transition
ERK
Extracellular-signal regulated kinase
f
Female
FACS
Fluorescence Associated Cell Sorting
FAK
Focal adhesion kinase
FCS
Fetal calf serum
FCS
Fetal calf serum
FDR
False discovery rate
FPKM
Fragments per kilobase million
FSC
Forward scatter
G-phase
Gap phase
GFP
Green fluorescent protein
List of Abbreviations
xi
Abbreviation
Designation
GLS
Glutaminase
GLUD
Glutamate dehydrogenase
GLUL
Glutamine synthetase
GLUT
Glucose transporter
GlycoPER
Glycolysis-derived PER
GO
Gene ontology
GOT
Glutamic-oxaloacetic transaminase
GPCR
G-protein coupled receptor
GPI
Glucose-6-phosphate isomerase
GPI
Glucose-6-phosphate isomerase
GPT
Glutamic—pyruvic transaminase
GSH
Glutathione
HEPES
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
HIF
Hypoxia inducible factor
HK
Hexokinase
HRP
Horseradish peroxidase
IC50
Half maximal inhibitory concentration
IgG
Immunoglobulin G
INRG
International Neuroblastoma Risk Group
INSS
International Neuroblastoma Staging System
JAK
Janus kinase
kb
Kilobase
kDa
Kilodalton
LATS
Large tumor suppressor
LB
Lysogeny broth
LDHA
Lactate dehydrogenase A
LOH
Loss of heterozygosity
LV
Empty vector
m
Male
M-phase
Mitosis phase
List of Abbreviations
xii
Abbreviation
Designation
MAPK
Ras/mitogen-activated protein kinase
MCT
Monocarboxylate transporter
MES
Mesenchymal
Metast.
Metastasis
mIBG
Metaiodobenzylguanidine
miRNA
MicroRNA
mmHg
Millimeter of mercury
MOB
MOB kinase activator
mRNA
Messenger RNA
MST
STE20-like protein kinase
MTFMT
Mitochondrial methionyl-tRNA formyl transferase
MTHFD
Methylene THF dehydrogenase
MTHFR
Methylene THF reductase,
mTOR
Mammalian target of rapamycin
mTORC
mTOR complex
N/A
Not available
NA
Non-Amplified
NADH
Nicotinamide adenine dinucleotide + hydrogen
NADPH
Nicotinamide adenine dinucleotide phosphate
NC
Negative control
NCC
Neural crest cells
NEAA
Non-essential amino acids
NF2
Merlin
NSCLC
Non-small cell lung cancer
OCR
Oxygen consumption rate
OD
Optical density
PAGE
Polyacrylamide Gel electrophoresis
PBS
Phosphate buffered saline
PCR
Polymerase chain reaction
PEG
Polyethylene glycol
List of Abbreviations
xiii
Abbreviation
Designation
pEMT
Partial EMT
PER
Proton efflux rate
PET
Polyethylene terephthalate
PFK
Phosphofructokinase
PFKFB
PFK/Fructose-2,6-bisphosphatase
PGK
Phosphoglycerate kinase
PHGDH
Phosphoglycerate dehydrogenase
PI
Propidium iodide
PI3K
Phosphatidylinositol-3-kinase
pO2
Oxygen partial pressure
Prim.
Primary
PSAT
Phosphoserine aminotransferase
PSPH
Phosphoserine phosphatase
PTPN14
Protein tyrosine phosphatase non-receptor type 14
PVDF
Polyvinylidene difluoride
pYAP1S127
YAP1 phosphorylated at serine at position 127
qPCR
Quantitative real-time PCR
RIN
RNA integrity number
RLU
Relative luminescence units
ROI
Region of interest
ROS
Reactive oxygen species
Rot/AA
Rotenone and Antimycin A
RPMI
Roswell Park Memorial Institute
RT
Room temperature
RTK
Receptor tyrosine kinase
S-phase
DNA synthesis phase
SA-β-Gal
Senescence-associated β-galactosidase
SAV1
Salvador homolog 1
SD
Standard deviation
SDHA
Succinate-dehydrogenase complex, subunit A
List of Abbreviations
xiv
Abbreviation
Designation
SDS
Sodium dodecyl sulfate
SHMT
Serine hydroxylmethyltransferase
shRNA
Short hairpin RNA
siRNA
Small interfering RNA
siYAP1
siRNA targeting YAP1
SLC
Solute carrier family
SNV
Single-nucleotide variants
SOC
Super optimal broth
SSC
Sideward scatter
SSP
Serine synthesis pathway
STAT
Signal transducer and activator of transcription
TAE-buffer
Tris/acetic acid/EDTA-buffer
TBS
Tris-buffered saline
TBS-T
TBS with 10% Tween-20 (v/v) (TBS-T)
TE-buffer
Tris/EDTA-buffer
TEAD
TEA domain transcription factor
TERT
Telomerase reverse transcriptase
Tet-ON
Tetracycline-inducible gene expression
TF
Transcription factor
THF
Tetrahydrofolate
TKI
Tyrosine kinase inhibitor
TMB
3,3,5,5-Tetramethylbenzidin
TR
Tet-repressor
TYMS
Thymidylate synthetase
U
Unit
v/v
Volume per volume
w/v
Weight per volume
wt
Wildtype
X-Gal
5-Bromo-4-chlor-3-indolyl β-D-galactopyranoside
YAP1
Yes1 associated transcriptional regulator 1
List of Abbreviations
xv
Abbreviation
Designation
ZEB
Zinc-finger E-box binding homeobox
𝛼KG
𝛼
-Ketoglutarate
Introduction
1
1. Introduction
1.1 Neuroblastoma
1.1.1 Clinical presentation and assessment
Neuroblastoma is an embryonal solid tumor originating from either neural crest cells (NCC) or
more evolved sympathoadrenal progenitors (Tsubota and Kadomatsu, 2018), which is not yet
fully clarified. Primary tumors typically emerge along the sympathetic nervous system in
adrenal glands and paraspinal ganglia with lesions in the abdomen, pelvis, chest or neck
(Maris, 2010). One per 100,000 children under 15 years of age is diagnosed with
neuroblastoma in Germany with a median age of 18 month (London et al., 2005;
Kinderkrebsinfo.de, 2021). Beside a minimal proportion <2% of cases with familiar
predispositions, most neuroblastomas occur sporadically (Maris et al., 2002). Clinical outcome
ranges from spontaneous regression to highly metastatic and refractory malignancy, whereas
children younger than 18 months possess a higher overall survival probability than the older
(London et al., 2005). Patients can display a poor overall condition presenting with pain, fever
and swellings at tumor sites, albeit about 40% of the children show no symptoms at all (Simon
et al., 2017). In case a neuroblastoma is suspected, laboratory testing for increased levels of
urinary catecholamines and its metabolites are accompanied by imaging-based and
histological evaluation of the tumor (Matthay et al., 2016). Tumor masses are preferably
assessed by computer tomography and magnetic resonance tomography. Utilizing the
enrichment of radiolabeled metaiodobenzylguanidine (mIBG) in 90% of all neuroblastoma,
especially bone metastasis and hardly detectable soft tissue lesions can be visualized by mIBG
scintigraphy (Maris et al., 2007). The remaining mIBG non-avid neuroblastoma can be spotted
by positron-emission-tomography scans with predominant administration of radioactive
tracer such as 18F-L-dihydroxyphenylalanine (18F-DOPA) or 68Ga-DOTATATE (Matthay et al.,
2016). Tumor pathology according to the International Neuroblastoma Pathology
Classification finally confirms the type of tumor and is a crucial tool for risk stratification
(Simon et al., 2017).
Introduction
2
1.1.2 Staging systems and risk stratification
The neuroblastoma treatment stratification presupposes the staging and risk group
classification of neuroblastoma patients. To capture the expanse of the tumor, two different
systems coexist: the surgical and pathological International Neuroblastoma Staging System
(INSS) and the pre-therapeutic clinical International Neuroblastoma Risk Group (INRG) Staging
System (Matthay et al., 2016; Simon et al., 2017). Since 1986, INSS classifies patients into six
tumor stages (stage 1, 2a, 2b, 3, 4 and 4S) after tumor resection depending on the extend of
surgical resection and metastasis. Patients with localized tumors are categorized into stage 1,
2 and 4S with respect to lymph nodes that are positive for tumor cells, tumor dissemination
and patient age. Multiple distant metastatic disease and MYCN amplification is a criterion for
stage 4 categorization (Brodeur et al., 1988, 1993). For a better comparison of clinical trial
data and also due to emerging omission of initial complete tumor resection, a pre-treatment
risk stratification approach was developed and resulted in the INRG staging system, that only
identifies image-based risk factors to classify patients into the four categories L1, L2, M and
MS (Cohn et al., 2009). Tumor staging together with clinical and biological parameters
including age, ploidy and MYCN copy number variations allows a neuroblastoma risk group
classification with a subsequent treatment stratification (Liang et al., 2020).
1.1.3 Tumor biology
Neuroblastoma is a clinically very heterogeneous disease with a broad spectrum of disease
outcome between spontaneous regression without any therapeutic intervention and life-
threatening high-risk disease. Despite a rather low mutational burden in high-risk
neuroblastoma, some recurrent genomic alterations are significantly enriched in those (Pugh
et al., 2013).
1.1.3.1 Genetic mutations and amplifications
With an incidence of 20-25% of all neuroblastoma cases, the amplification of oncogenic MYCN
is one of the most prominent genetic aberrations and one of the major predictors for patient’s
outcome (Brodeur et al., 1984). Murine animal models of neural-crest specific MYCN
Introduction
3
overexpression revealed that MYCN alone can drive neuroblastoma development (Weiss et
al., 1997; Althoff et al., 2015). MYCN is a transcription factor (TF) forming heterodimers with
MAX to activate gene transcription affecting cell proliferation, apoptosis, cell cycle and
survival, angiogenesis and metabolism among others (Huang and Weiss, 2013; Xia et al.,
2019). Second most frequent genetic aberration activates the tyrosine kinase receptor
anaplastic lymphoma kinase (ALK), which is mutated in 6-7% of all neuroblastomas (Molenaar
et al., 2012). ALK regulates the balance between proliferation and differentiation via
downstream signaling including Ras/mitogen-activated protein kinase (MAPK),
phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (AKT) and Janus kinase (JAK)/signal
transducer and activator of transcription (STAT) pathways and cooperates with MYCN to drive
neuroblastoma progression (Trigg and Turner, 2018). In familiar neuroblastoma cases ALK is
the major driver, whereas the most frequent and aggressive point-mutation ALKF1174L was only
found in sporadic tumors (Schulte and Eggert, 2015). In a conditional mouse model, ALKF1174L
transgenic mice developed neuroblastoma from neural crest-derived cells, presenting the
oncogenic potential of it (Heukamp et al., 2012). In addition to MYCN amplification and ALK
mutation, further genomic rearrangements upregulating TERT (telomerase reverse
transcriptase) define another high-risk subgroup of neuroblastoma patient. Subsequent
activation of telomerase activity leads to unfavorable telomere maintenance associated with
poor outcome (Peifer et al., 2015). Inactivating mutations of the X-chromosomal ATRX
represent another mechanism of alternative telomere lengthening in high-risk neuroblastoma
(Cheung et al., 2012). Moreover, further genes are described to be mutated in neuroblastoma
such as PHOX2B, LIN28B, ARID1A, ARID1B, NRAS and PTPN11, all being detrimental for
patients survival (Schulte and Eggert, 2015).
1.1.3.2 Chromosomal instability
Partial or complete chromosomal rearrangements are common in neuroblastoma (Koneru et
al., 2020). Loss of chromosome 1p and 11q can be found in nearly one fourth of all cases and
are associated with poor outcome (Vandesompele et al., 1998; Pugh et al., 2013). Gain of 17q
is also associated with advanced disease stages (Bown et al., 1999). In contrast, whole
chromosome gains or losses, that are mainly detected in stage 1, 2 and 4S tumors, promise a
Introduction
4
more favorable outcome (Cheung and Dyer, 2013). One factor that is incorporated into risk
stratification is ploidy of the tumor cells: hyper diploid or triploid tumors show better
outcomes than diploid ones (Cohn et al., 2009). Shredding of a chromosomal region followed
by random reassembly, so called chromotripsis, occurs in high stage neuroblastoma and is
associated with poor prognostics (Molenaar et al., 2012).
1.1.3.3 Mutational landscape of relapse neuroblastoma
The survival rate of post-relapse patients still remains under 20%, highlighting the
aggressiveness and lethality of recurrences (London et al., 2011). In a comparative study of
primary and relapse neuroblastoma, Schramm et al. found a higher load of mutations in the
latter with single-nucleotide variants (SNV) of DOCK8, CHD5, ANK3, SPTA1, PLXNA3, PLXND1
and Yes1 associated transcriptional regulator (YAP1)-regulator PTPN14, as well as segmental
variants such as chromosome 9p deletion and de novo MYCN amplification (Schramm et al.,
2015). Mutations of NF1, PTPN11, NRAS, HRAS, KRAS and BRAF, all activating the Ras/MAPK
pathway, as well as ALK aberrations and MYCN amplification were found in relapsed
neuroblastoma as well (Eleveld et al., 2015; Padovan-Merhar et al., 2016). A recent study
extended the list of relapse-associated mutations by FAT1, TERT, CCND1, LSAMP among
others, indicating a huge diversity of potentially pathogenic mutations in recurrent tumor
masses (Fransson et al., 2020). Although YAP1 gene was not directly modified, a YAP1-
activated transcriptional signature was found in relapse tumors in one study (Schramm et al.,
2015).
1.1.4 Therapy
Neuroblastoma treatment stratification is based on risk group classification and follows the
Guidelines for diagnosis and treatment of patients with neuroblastic tumors published by
Society of Pediatric Oncology and Hematology in Germany (Simon et al., 2017).
Low-risk patients, which includes INSS stage 1 as well as stage 4S
≤
18 months of age without
MYCN amplification, INSS stage 2 with additional lack of chromosome 1p aberration and INSS
stage 3 diagnosed under two years of age with lack of MYCN amplification and 1p aberration,
Introduction
5
reach well survival prognosis with either observation or tumor resection only. If symptoms are
menacing, additional multiagent low or moderate intensity chemotherapy is suggested to
induce tumor regression. A complete resection requiring extensive surgery is not necessary
(Simon et al., 2017). Radiotherapy is avoided completely in low-risk cases (Tonini, Nakagawara
and Berthold, 2012).
Intermediate risk is estimated for patients suffering from MYCN non-amplified INSS stage 2
tumors and patients younger than two years of age with INSS stage 3 tumors exhibiting 1p
aberrations. In addition, MYCN non-amplified INSS stage 3 patients
≥
2 years of age and INSS
stage 4
≤
18 months are also considered to be intermediate risk (Simon et al., 2017). These
children all receive chemotherapy, that is reduced in intensity (Tonini, Nakagawara and
Berthold, 2012). If not necessary through absence of image-based risk factor analysis, surgery
is delayed to until after cytotoxic therapy aiming for complete tumor resection. If the danger
of substantial organ damage is too high, radiotherapy can substitute the surgery (Simon et al.,
2017).
High-risk neuroblastoma comprises all MYCN-amplified tumors and INSS stage 4 patients older
than 18 months and share poor survival prognosis. The three-phasic treatment strategy starts
with induction therapy to shrink initial tumor masses by high-dose administration of cytotoxic
drug combinations of cisplatin, carboplatin, cyclophosphamide, ifosfamide, doxorubicin,
etoposide, teniposide, vincristine and vinblastine, often followed by surgical resection of
primary tumor. The second treatment phase aims for consolidation by myeloablative
chemotherapy and autologous stem cell transplantation, previously expanded by
131Iodin metaiodobenzylguanidine (131I-mIBG) therapy in cases of residual lesions positive for
123I-mIBG-uptake. Consolidation phase is recommended to be completed with radiotherapy
(Simon et al., 2017). Maintenance therapy reaches for elimination of minimal residual disease
and encompasses antibody immunotherapy (anti-GD2, also known as CH14.18), additional
cytokine immunotherapy (IL-2, GM-CSF) and differentiation therapy (13-cis retinoic acid)
(Matthay et al., 2016).
Although first line high-risk neuroblastoma treatment elicits partial or complete remission, up
to 60% of those patients relapse (Cohn et al., 2009; Simon et al., 2011). Most effective
treatments include combinations of cytotoxic drugs such as topotecan with
Introduction
6
cyclophosphamide (London et al., 2010) or temozolomide (Di Giannatale et al., 2014) and
irinotecan with temozolomide (Bagatell et al., 2010) as proven in several clinical phase II trials.
In addition, 131I-mIBG is also administered to relapsed neuroblastoma patients with positive
(partial and complete) response rates (Zhou et al., 2015). Despite extensive affords to improve
neuroblastoma therapy, the five-year overall survival rate of relapsed patients receiving a
second line treatment still remains about 20% (Basta et al., 2016). Since “quick and easy” DNA
sequencing approaches are exploitable, molecular targeted therapy gained importance. ALK
inhibitors such as crizotinib (effective only against ALKR1275Q) and ceritinib are already under
investigation in clinical trials against relapsed and refractory neuroblastoma (Trigg and Turner,
2018; Foster et al., 2021). Inhibitors against Aurora Kinase A, BET, MDM2, PI3K/mTOR, BRD4,
Ras/MAPK, MEK1/2, TrkA, TrkB, HDAC and Survivin among others are tested preclinically for
efficacy in neuroblastoma disease (Esposito et al., 2017). Still under investigation in phase I/II
clinical trials is the function and efficacy of chimeric antigen receptor T cell therapy with T cells
engineered to target tumor-derived L1CAM or GD2 (Richards, Sotillo and Majzner, 2018).
1.2 Yes1 associated transcriptional regulator 1
1.2.1 YAP1 structure and transcriptional activity
In 1994, YAP1 was described for the first time by M. Sudol as a protein that binds the Src
homology domain of protein-tyrosine kinase Yes1 (Sudol, 1994). Lacking a DNA binding
domain, the protein acts as a transcriptional co-activator for many transcription factors (TF).
The TEA domain transcription factor (TEAD) interaction domain in the N-terminal region of
YAP1 enables highly conserved interaction with TEADs to stimulate cell growth, epithelial
mesenchymal transition (EMT) and gene expression (Vassilev et al., 2001; Zhao et al., 2008;
Hilman and Gat, 2011). Interaction with TFs SMAD (Ferrigno et al., 2002; Grannas et al., 2015),
RUNX (Yagi et al., 1999; Zaidi et al., 2004), ERBB4 (Komuro et al., 2003), PAX3 (Manderfield et
al., 2014), TBX5 (Rosenbluh et al., 2012) and p73 (Strano et al., 2001) are realized via one or
two WW domains, that each contain two highly conserved tryptophane residues and
recognize PPxY motifs. Further TFs such as activator protein 1 (AP-1) (Zanconato et al., 2015;
Koo et al., 2020) and BRD4 (Zanconato et al., 2018) associate with YAP1-TEAD complex on
Introduction
7
gene promoter or enhancer regions. Cooperation of YAP1 with these and other TFs induces
expression of a plethora of target genes regulating proliferation, cell growth, organ size
control, cell migration, drug and stress resistance mechanisms, metabolic changes, EMT and
cell immunity (Zanconato, Cordenonsi and Piccolo, 2016; Szulzewsky, Holland and Vasioukhin,
2021). Commonly described YAP1 target genes include CTGF, CYR61, COL8A1, SKP2, SNAPC
and AXL (Zhang, Smolen and Haber, 2008; Xu et al., 2011; Stein et al., 2015). For nuclear
translocation of YAP1, the C-terminal PDZ motif is crucial (Oka and Sudol, 2009).
Posttranscriptional modifications such as serine and tyrosine phosphorylation additionally
affect YAP1 activity and intracellular localization. The most important modification is the
phosphorylation of serine 127 (pYAP1S127) by different kinases such as LATS1, LATS2 and AKT,
which creates a binding consensus for protein 14-3-3 and sequesters YAP1 in the cytoplasm
(Piccolo, Dupont and Cordenonsi, 2014). Activating phosphorylation of tyrosine Y407 by
Yes/Src and c-Abl kinases is reported to trigger YAP1 activity (Levy et al., 2008; Rosenbluh et
al., 2012), whereas complex-building of tyrosine phosphatase PTPN14 with YAP1 inhibits its
transcriptional activity (Liu et al., 2013). As YAP1 is categorized as pathogenic protein and
investigated extensively in the past decade, meanwhile many more proteins are described to
interact with YAP1 including TFs (e.g., ZEB-1, AP-1,
𝛽
-catenin), kinases (e.g., MEK, PI3K),
phosphatases (e.g., PP1A, PP2A), adaptor proteins (FAT4, FRMD4/6, NF2), ubiquitin ligases
(e.g., NEDD4), Rho GTPases (e.g., RAC1, RHOA), G-protein coupled receptors (GPCR),
chromatin modulators and miRNAs (e.g., miR-141, miR-375) (Varelas, 2014; Wang et al.,
2017). In neuroblastoma, the transcriptional activity of YAP1 is not yet elucidated. YAP1 is
located at the long arm of chromosome 11 at position 11q22, and reports of activating
mutations or gene amplifications are not known in this tumor entity. Paradoxically, deletion
of 11q frequently occurs in neuroblastoma patients (Pugh et al., 2013), indicating further
regulatory mechanisms explaining increased YAP1 activity in relapsed tumors (Schramm et al.,
2015).
Introduction
8
1.2.2 Regulation of YAP1 activity
Together with homolog TAZ, YAP1 is key effector of the highly conserved canonical Hippo
signaling pathway. The serine kinase signaling cascade was initially discovered in Drosophila.
It was identified as tumor suppressor pathway, that controls organ growth via regulation of
cell cycle and cell proliferation (Justice et al., 1995; Xu et al., 1995). The core of the mammalian
Hippo-YAP1 pathway (Figure 1) consists of STE20-like protein kinase 1 (STK4/MST1, Hippo in
Drosophila) and STE20-like protein kinase 2 (STK3/MST2, Hippo in Drosophila), that interact
with and phosphorylate adaptor protein salvador homolog 1 (SAV1). This complex adds a
phosphate group to large tumor suppressor 1 (LATS1) and large tumor suppressor 2 (LATS2),
which interact with MOB kinase activator 1 (MOB1) (Chan et al., 2011). Finally, YAP1 is
phosphorylated by LATS1/2 at the five serine residues S61, S109, S127, S164, and S381. These
posttranslational modifications lead to an inactivation of YAP1 by different mechanisms,
although only S127 and S381 play a role in inhibiting oncogenic potential of YAP1 (Zhao et al.,
2010). The binding of 14-3-3 protein at phosphorylated S127 sequesters YAP1 in the
cytoplasm and prevents transcriptional activity (Basu et al., 2003; Dong et al., 2007).
Controversially, Ege et al. recently demonstrated a dominant nuclear export of pYAP1S127 via
LATS1/2
MST1/2
YAP1
YAP1
TF Target genes
P
P
P
LATS1/2
MST1/2
YAP1
Proliferation
Survival
Migration
Invasion
Resistance
EMT
nucleus
cytosol
Hippo pathway
ON
Hippo pathway
OFF
SAV1
MOB1 MOB1
SAV1
Figure 1 Mammalian Hippo-YAP1 pathway. Active STE20-like protein kinases 1/2 (MST1/2) phosphorylate protein salvador
homolog 1 (SAV1). This complex adds a phosphate to large tumor suppressor 1/2 (LATS1/2), which interacts with MOB
transcriptional activator (MOB1). LATS1/2 phosphorylate serine residues of YAP1 to prevent its translocation into the
nucleus, where it forms a complex with various transcription factors (TF). Induced target gene expression regulates cellular
processes including cell growth, cell motility and resistance mechanisms.
Introduction
9
exportin 1 over its constant active import, culminating in increased cytoplasmic pYAP1S127
levels (Ege et al., 2018). Mechanical forces acting on a cell result in flattening of the nucleus
and formation of nuclear pores, that trigger the import and thus, Hippo-independent
activation of YAP1 (Elosegui-Artola et al., 2017). Phosphorylation at S381 induces a
subsequent phosphorylation by casein kinase 1, which entails the initiation of a
phosphodegron and the ubiquitination of YAP1 catalyzed by
SCF!"#$%&
E3 ubiquitin ligase.
Ultimately, this cascade results in YAP1 protein degradation (Zhao et al., 2010). When the
Hippo pathway is inactive, YAP1 remains active by absence of its phosphorylation and is
translocated into the nucleus, if no other mechanisms inactivate the protein. After nuclear
entry, YAP1 cooperates with various TFs to induce target gene expression (Hansen, Moroishi
and Guan, 2015).
Crucial regulators of Hippo-pathway activity are i) cell polarity and tight junctions, ii) adherens
junctions with cadherin-catenin complexes, iii) mechanotransduction and iv) receptor
signaling (Gumbiner and Kim, 2014). Cell-cell contact leads to inhibition of proliferation via
activation of the kinase LATS (Zhao et al., 2007). In epithelial cells, the adherens-junction
component
𝛼
-catenin linking the cadherin/catenin complex to actin cytoskeleton prevents
dephosphorylation and subsequent activation of YAP1 by complex building with pYAP1 and
14-3-3 (Schlegelmilch et al., 2011). Angiomotin (AMOT), a tight junction associated protein,
interacts with merlin (NF2) and kibra to activate Hippo-core proteins LATS1/2 and MST1/2 and
thereby inhibits transcriptional activity of YAP1 (Zhao et al., 2011). Merlin alone is a strong
negative regulator of YAP1 (Plouffe et al., 2016). Mechanical forces emerging from stiff
extracellular matrix (ECM) and high cell density reduce stress fiber formation and thereby
activate MST and LATS kinases (Wada et al., 2011). The adhesion of cellular integrins with ECM
protein fibronectin 1 (FN1) leads to the stimulation of a LATS1/2-inhibiting FAK-SRC-PI3K
cascade (Kim and Gumbiner, 2015). Signaling initiation of G-protein coupled receptors (GPCR)
can regulate Hippo-YAP activity bidirectionally, dependent on the G-protein that is coupled to
the receptor. Receptors coupled to G12/13, Gq/11 or Gi/o inhibit LATS1/2, whereas Gs-bound
receptors lead to a protein kinase A and Rho GTPase-dependent YAP1 phosphorylation (Yu et
al., 2012, 2013). Receptor tyrosine kinases (RTK) such as fibroblast growth factor receptor,
vascular endothelial growth factor (VEGFR) or AXL diminish LATS1/2 activity and thereby
promote YAP1 activation (Azad et al., 2020). Because cell-cell contact is closely linked to tight
Introduction
10
junctions, adherens junctions and mechanotransduction, these regulators are all interrelated
and depend on each other. In addition to MST1/2-mediated phosphorylation of LATS1/2,
mitogen-activated protein kinase kinase kinase kinase (MAP4K) proteins are a second group
of physiological LATS1/2 activators, which are induced by shared as well as distinct upstream
signals (Meng et al., 2015). As some YAP1 upstream regulatory mechanisms are shown to work
independent of MST kinases, the MAP4Ks and other kinases gain impact on the complexity of
Hippo-YAP1 regulation (Yu et al., 2012, 2013; Meng et al., 2015).
Although Hippo pathway is established as the main regulator, the fate of YAP1 does not only
depend on that signaling cascade. Even conditions regulating MST1/2 and LATS1/2 can directly
influence the YAP1 activity. The nuclear translocation of YAP1 can be inhibited by Amot by
direct recruitment and detention to tight junctions and actin cytoskeleton (Zhao et al., 2011).
Protein tyrosine phosphatase non-receptor type 14 (PTPN14) and
𝛼
-catenin can also
sequester YAP1 at cell junctions (Yu, Zhao and Guan, 2015). Cell contact inhibition can be
overcome by ECM stiffness and cell spreading, which activates YAP1 dependent on Rho
activity and stress fiber formation (Dupont 2011). Tensional forces acting on cells can activate
YAP1 directly by loss of F-actin capping and severing proteins cofilin, CapZ (Aragona et al.,
2013) or via protein shuttling through emerging nuclear pores (Elosegui-Artola et al., 2017;
Ege et al., 2018).
Many signaling cascades other than Hippo are known to affect YAP1 transcriptional activity.
Alternative Wnt signaling increases nuclear transport of YAP1 via Rho-mediated inhibition of
LATS kinases (Park et al., 2015), whereas cytoplasmic YAP1 inhibits Wnt pathway by binding
𝛽
-catenin (Imajo et al., 2012). The mevalonate pathway generates geranylgeranyl
pyrophosphate, which is required for protein synthesis of Rho GTPases, that in turn activate
YAP1 (Sorrentino et al., 2014). The activation of GPCRs and RTKs induces PI3K/PDK1 signaling,
which activates YAP1 directly (Roy et al., 2019) or via LATS inhibition (Fan, Kim and Gumbiner,
2013). Furthermore, YAP1 cross-talks with Notch (Slemmons et al., 2017), sonic hedgehog
(Fernandez-L et al., 2009), bone morphogenetic proteins (Kumar, Nitzan and Kalcheim, 2019)
and mammalian target of rapamycin (mTOR) signaling (Liang et al., 2014).
MicroRNAs, which are sequence-specific small RNAs repressing post-transcriptional gene
expression, are described to regulate YAP1 activity both, positively and negatively (Lin et al.,
Introduction
11
2013; Shen et al., 2015; Ruan et al., 2016; Mohamed et al., 2018; Torrini et al., 2019). Either
YAP1 translation is impaired directly or upstream regulators such as
SCF!"#$%&
E3 ubiquitin
ligase, LATS or MST kinases are targeted and suppressed.
Other YAP1 protein modifications such as methylation at lysine 494 by Set-7 (Oudhoff et al.,
2013), ubiquitination by Amot130 and Nedd4 family ubiquitin ligase AIP4 (Adler, Heller et al.,
2013) and acetylation by CBP/p300 acetyltransferase (Hata et al., 2012) entail YAP1 inhibition.
The protein inactivating phosphorylation of YAP1 is the core regulatory mechanism of Hippo
pathway. This posttranslational modification is also utilized by many other proteins such as
c-Abl (Levy et al., 2008), AKT (Basu et al., 2003), JNK1/2 (Tomlinson et al., 2010) and AMPK
(Wang et al., 2015) to directly impair YAP1 activity. Enabling flexibility in cellular protein
response, this phosphorylation can actively be reversed by phosphatases PP1A (Wang et al.,
2011) and PP2A (Schlegelmilch et al., 2011).
In some cancers, YAP1 signaling is further regulated by mutations, amplifications or gene
fusions of YAP1, its upstream regulators (e.g., NF2, LATS1/2) and interacting TFs (e.g., TEAD1-
4) (Asthagiri et al., 2009; Van Raamsdonk et al., 2009; Murakami et al., 2011; Y. Wang et al.,
2018). However, activating mutations or gene fusions of YAP1 are not present in
neuroblastoma.
1.2.3 Cell growth under control of YAP1
The Hippo pathway and YAP1 control organ growth via regulation of cell proliferation and
apoptosis. In Drosophila, inactivating mutations of hpo, sav and wts lead to an upregulation
of cell cycle regulators (e.g., CCNE1) and apoptosis inhibitors (e.g., DIAPH1), thus promoting
cell growth (Tapon et al., 2002; Harvey, Pfleger and Hariharan, 2003). The overexpression of
YAP1 ortholog yki in Drosophila resulted in the same phenotype (Huang et al., 2005). In
mammalian cells, doxycycline-controlled overexpression of human YAP1 under control of a
liver-specific promotor resulted in liver overgrowth, which could be reversed by inhibition of
YAP1 expression (Camargo et al., 2007; Dong et al., 2007). In other normal tissues such as
epithelial cells and cardiomyocytes, ectopic YAP1 expression increases the cell proliferation
and partially induces tumorigenic growth via TEAD-dependent induction of cell cycle genes
(Schlegelmilch et al., 2011; von Gise et al., 2012; Nishimoto et al., 2019). Furthermore,
Introduction
12
aberrant proliferation and tumor growth of various cancers such as sonic hedgehog-mediated
medulloblastoma (Fernandez-L et al., 2009), glioblastoma (Castellan et al., 2021), pediatric
hepatoblastoma (LaQuaglia et al., 2016), non-small cell lung cancer (NSCLC) (Yu et al., 2018),
ovarian cancer (Xia et al., 2014) and breast cancer (Lamar et al., 2012) rely on active YAP1
levels. Mechanistically, YAP1 cooperates with several TFs and other proteins (see section
1.2.1) to promote proliferation by upregulation of cell cycle genes (CDC25A, CDC25C, CCNA2,
CCND1, CCND2) and genes regulating DNA synthesis and repair (CDC6, GINS1, MCM3/4/5/6/7,
POLA2, POLE3, TOP2A, RAD18, RAD51) (Fernandez-L et al., 2009; Kapoor et al., 2014;
Zanconato et al., 2015; Zhao et al., 2019). Additional protection of the tumor cells is managed
by a YAP1-driven induction of anti-apoptotic genes (BIRC5, BCL2L1) (Rosenbluh et al., 2012;
Lin et al., 2015; Zhao et al., 2016). Although YAP1 is predominantly discussed as a candidate
oncogene, there are some reports of YAP1 acting as a tumor suppressor by initiating
apoptosis. Recruitment of p73 and p300 protein to apoptotic target genes (P53AIP1, BAX) in
response to chemotherapeutic DNA damage is managed by and dependent on YAP1 (Strano
et al., 2005), if YAP1 is released from AKT/14-3-3 protein complex (Basu et al., 2003). In
hematological cancer cells, that were shown to exhibit pervasive DNA damage, the YAP1-
induced apoptosis is mediated by ABL1 and p73 (Cottini et al., 2014).
In neuroblastoma cells, knockdown of YAP1 resulted in decreased cell proliferation in vitro.
Pharmacological YAP1 inhibition together with cisplatin administration delayed tumor growth
in vivo (Yang et al., 2017). The overexpression of YAP1 leads to an AKT-mediated cytoplasmic
translocation of cyclin-dependent kinase inhibitor p27Kip1. The extranuclear trapping of p27Kip1
leads to an enrichment of KI67 positive neuroblastoma cells, indicating higher proliferation
rates (Shen et al., 2020). The cytotoxic alkaloid tetrandrine was discovered to have an
apoptotic effect on neuroblastoma cell lines SH-SY5Y and SK-N-AS, which was partially
mediated by activation of the Hippo pathway and subsequent inhibition of YAP1. In turn, the
overexpression of YAP1 in both cell lines resulted in BCL2 induction and thereby promoted
higher cell viability under tetrandrine treatment (Zhao et al., 2019). Further biological studies
of YAP1-driven proliferation in neuroblastoma are still missing.
Introduction
13
1.2.4 YAP1 regulates cancer cell migration and metastasis
Cell migration is a physiological process that ensures the development and maintenance of
multicellular organisms. Tissue homeostasis, wound healing, stem cell homing and immune
cell trafficking rely on the proper movement of individual cells. Migration and tissue invasion
of cancer cells, in turn, is prerequisite for development of metastasis and distant relapses.
Migration is a multi-step process including reorganization of the cytoskeleton, assembly and
disassembly of cell-substrate adhesions, remodeling of the ECM and generation as well as
translation of mechanic forces. These characteristics are common in all types of migration, be
it mesenchymal, amoeboid, blebbing or collective cell migration (Friedl and Alexander, 2011).
Physiological migration is triggered by gradients of soluble (chemotaxis) or immobilized
(haplotaxis) attractants, substrate rigidity (durotaxis), shear forces (mechanotaxis) or electric
fields (galvanotaxis) (Devreotes and Horwitz, 2015). Tumor cells harness all modes of
migration to invade surrounding tissues and to metastasize. The formation of invadopodia,
that are actin-rich areas surrounded by adhesion and scaffolding proteins, is an additional
specialty of cancer cells to cross anatomical barriers by degradation of ECM (Murphy and
Courtneidge, 2011).
YAP1 is one regulator of cell migration and tumor metastasis formation. In early embryonal
development, YAP1 determines the migration of neural crest cells together with Wnt and BMP
signaling (Kumar, Nitzan and Kalcheim, 2019). Confirming the decisive role in neural crest
development, dominant-negative YAP1 expression in avian embryos caused impaired EMT of
neural crest cells and inhibited their subsequent delamination and migration (Bhattacharya,
Azambuja and Simoes-Costa, 2020). Canine non-transformed epithelial cells can disseminate
upon mechanical stimuli through a YAP1 initiated program, which transcriptionally
upregulates WT1 and subsequently inhibits E-cadherin, core member of adherens junctions.
In addition to the EMT initiation, the activation of Ras-related C3 botulinum toxin substrate 1
(Rac1) leads to cell migration and dissemination (Park et al., 2019). In various carcinomas such
as melanoma (Lamar et al., 2012), mammary carcinoma (Lamar et al., 2012), NSCLC (Yu et al.,
2018), ovarian carcinoma (Xia et al., 2014), breast cancer (Shen et al., 2018), gastric cancer
(Zhou et al., 2016) and also non-epithelial tumor types such as glioma (Y. Zhang et al., 2018),
Ewing sarcoma (Bierbaumer et al., 2021) and neuroblastoma (Yang et al., 2017), YAP1 is
modulator of cell motility and metastasis formation. In gastric cancer cells, YAP1
Introduction
14
transcriptionally induces the synthesis of Rho-activating GTPase protein ARHGAP29, which
leads to inhibition of the RhoA-ROCK-LIMK axis. The subsequent disintegration of the actin
cytoskeleton enables cell migration and thus, increases the risk for metastasis (Qiao et al.,
2017). In adipose tissue-derived mesenchymal stroma as well as breast cancer cells, YAP1
transcriptionally activates genes of focal adhesion-related integrins, vinculin, zyxin and talin,
which are obligatory for the interaction of a cell with surrounding ECM. Breast cancer cells
lacking YAP1 failed to migrate and invade surrounding matrices (Nardone et al., 2017; T. Wang
et al., 2018).
Thrombosponin-1 (THSB1) is a further transcriptional YAP1 target, that stimulates the focal
adhesion kinase (FAK) and promotes the formation of focal adhesions (Shen et al., 2018). Focal
adhesions, which link the ECM to the cellular actin cytoskeleton, sense mechanic tensions
from substrate rigidity or cell geometry. Induced signaling cascades via Rho, FAK and other
focal adhesion-associated proteins as well as the formation of actin stress fibers activate YAP1
in a LATS-dependent and independent manner (Low et al., 2014; Panciera et al., 2017;
Dobrokhotov et al., 2018). The complex interplay of YAP1 and focal adhesion remodeling
allows tumor cells to detach from primary tumor bulk and migrate through surrounding
tissues. The formation of invadopodia and subsequent cell migration is also supported by
active YAP1. In nasopharyngeal cancer cells, the leukemia inhibitory factor receptor inhibits
YAP1 phosphorylation and thus promotes invadopodia formation, focal adhesion assembly
and cancer dissemination in vitro and in vivo (Liu et al., 2018). In glioblastoma and renal
carcinoma cell lines, YAP1 is additionally activated by cell detachment and prevents anoikis,
the cell-detachment induced apoptosis (Zhao et al., 2012). In vivo, orthotopically injected
ovarian cancer cells, that were genetically modified for stable YAP1S127A expression, revealed
increased anoikis resistance and metastasis formation via a platelet-mediated RhoA-myosin
phosphatase target subunit 1-PP1 axis (Haemmerle et al., 2017).
Migrating tumor cells use the vascular or lymphatic systems to shuttle through the organism.
The shear stress induced by those fluids can activate YAP1 and matrix metalloproteinases
through activation of a signaling cascade downstream of ROCK and LIMK, which further
promotes cell movement and matrix degradation (H. J. Lee et al., 2017). The YAP1 target genes
AXL, CTGF and CYR61 encode for pericellular matrix proteins, that function predominantly as
modulators of surface receptors. Their YAP1-mediated upregulation contributes to a
Introduction
15
migratory phenotype of cancer cells (Zhao et al., 2008; Xu et al., 2011; Hsu et al., 2015; Malik,
Liszewska and Jaworski, 2015). Furthermore, the activation of GPCRs coupled to G12/13,
Gq/11 or Gi/o, either by lisophosphatic acid, sphingosine-1-phosphophate and thrombin
among other ligands or by receptor-activating mutations such as GNAQ or GNA11, are
reported to induce YAP1-dependent cell migration (Mo et al., 2012; Yu et al., 2012, 2014).
The first indication of YAP1 as a potential modulator of neuroblastoma cell migration was
provided by Schramm et al., who demonstrated a YAP1-activated mRNA signature in relapse
tumor tissues (Schramm et al., 2015). Silencing of YAP1 in two neuroblastoma cell lines
reduced cell invasion potential in vitro (Yang et al., 2017). YAP1 overexpression in a human
neuroblastoma cell line promotes an early neural crest cell phenotype and increases its
migration potential (Hindley et al., 2016). In a mouse model mimicking neuroblastoma
metastatic relapse formation and progression, Hippo signaling was found to be repressed in
the metastatic subpopulation. Thus, YAP1/TAZ levels were increased in these metastatic cells
and YAP1/TAZ double knockdown reduced cell growth dramatically. In addition, YAP1/TAZ
silencing suppressed the cell migration in metastatic cells, yet not in the parental cell lines.
This finding indicates a crucial role of YAP1 and TAZ in mediating the migratory phenotype of
metastatic neuroblastoma cells (Seong et al., 2017). The activation of YAP1 in lymph node
metastasis developed from xenografted cells of human melanoma, breast cancer and lung
cancer, together with the failure of lymph node metastasis development upon YAP1
knockdown, highlighted the role of YAP1 generally in metastasis development (Lee et al.,
2019).
1.2.5 YAP1-mediated antitumor-therapy resistance
The deregulation of Hippo-YAP1 signaling is one mechanism of intrinsic and acquired therapy
resistance in cancer cells. Different mechanisms are described how active YAP1 mitigates the
efficacy of antitumor therapies including chemotherapeutic agents, immune checkpoint
inhibitors and targeted-therapy molecules. Additionally, dependent on the cell type and the
applied molecule, YAP1 is also reported to convey drug sensitivity to the cell.
Introduction
16
YAP1 mediates drug resistance to the cytostatic DNA damaging agents cisplatin, etoposide or
doxorubicin in leukemia (Kawahara et al., 2008), meningiomas (Baia et al., 2012), oral
squamous cell carcinoma (Yoshikawa et al., 2015) and urothelial cell carcinoma (Ciamporcero
et al., 2016). The YAP1-mediated upregulation of genes in the membrane transporter
superfamily of ATP-binding cassette (ABC)-transporters (e.g., ABCG2), which are described as
drug efflux pumps, provokes doxorubicin resistance in lung cancer cells (Dai et al., 2017). The
same chemotherapeutic agent resulted in elevated antiapoptotic Bcl-xL protein levels as well
as increased phosphorylation of extracellular-signal regulated kinases (ERK) and AKT in YAP1-
overexpressing hepatocellular cancer cells (Huo et al., 2013). Cisplatin treatment of the same
cell type leads to a SIRT1-mediated deacetylation of YAP1, which increases its nuclear
accumulation and mediates resistance (Mao et al., 2014). Further, YAP1 is reported to induce
cytoprotective autophagy to cisplatin-treated ovarian cancer cells (Xiao et al., 2016). In
neuroblastoma cells, YAP1 provokes resistance to therapy-induced apoptosis to cisplatin
(Yang et al., 2017), cyclophosphamide (Shim et al., 2020) and topoisomerase 1 inhibitor
irinotecan (Seong et al., 2017).
Resistance to antimicrotubular drugs such as paclitaxel and docetaxel also depends on YAP1
activity, although that effect remains controversial. While YAP1-dependent drug resistance
can be induced by miRNA-363-mediated suppression of LATS2 in ovarian cancer cells
(Mohamed et al., 2018), CDK1 can phosphorylate YAP1 independent of LATS and sensitizes
the same cells to taxane treatment (Zhao et al., 2014). Another group of chemotherapeutic
drugs interfering with the metabolism of cells include antifolates, fluorouracil and
gemcitabine and are also associated to YAP1-induced resistance. Fluorouracil resistance of
breast cancer cells is mediated by YAP1 nuclear translocation and transcriptional upregulation
of HDAC2 via the TF RUNX1 (Z. Zhang et al., 2020). The application of YAP1 inhibitor
verteporfin on pancreatic ductal adenocarcinoma cells enhances the sensitivity of
gemcitabine (Donohue et al., 2013). In pancreatic carcinoma cells, the YAP1 activation upon
gemcitabine treatment sensitizes the cells via the downregulation of multidrug transporters
and cytidine deaminase, a gemcitabine metabolizing enzyme (Gujral and Kirschner, 2017).
Additionally, well-characterized YAP1 target genes such as CTGF and CYR61 are also described
to induce therapy resistance in different cancer entities via induction of antiapoptotic proteins
such as XIAP, Bcl-xL or survivin (Lin et al., 2004; Yin et al., 2010; Song et al., 2021).
Introduction
17
Targeted therapies aim for an inhibition of one tumor-driving molecule or pathway. The
dysregulation of YAP1 often leads to inhibitor resistance via activation of an alternative
pathway and/or a reshaping of cells via EMT induction. The acquired resistance to KRAS
inhibition in a KRASG12D-driven mouse model depends on transcriptionally active YAP1, which
promotes upregulation of the AP-1 subunit FOS and, together with TF AP-1, induces an EMT-
like program (Shao et al., 2014). The direct targeting of MAPK signaling with vemurafenib (RAF
inhibitor) and trametinib (MEK inhibitor) in BRAF-mutant and RAS-mutant tumor cells is
bypassed by antiapoptotic YAP1 signaling (Lin et al., 2015). Further, the survival of tyrosine
kinase-driven cancer cells treated with epidermal growth factor receptor inhibitors afatinib,
gefitinib or erlotinib is mediated by YAP1 activation and subsequent upregulation of AXL
(Ghiso et al., 2017; Lee et al., 2018; Saab et al., 2019). In neuroblastoma cells, YAP1
accumulates in the cell nucleus upon MEK inhibition and promotes cell cycle gene expression
to overcome trametinib-induced cytotoxicity (Coggins et al., 2019). Neuroblastoma xenografts
were sensitized to cyclophosphamide or trametinib treatment after YAP1 silencing, partially
mediated through upregulation of pro-apoptotic protein Harakiri (Shim et al., 2020). As the
tyrosine kinase gene ALK is frequently mutated in neuroblastoma (Pugh et al., 2013), and ALK
inhibitor resistance is mediated by YAP1-dependent upregulation of anti-apoptotic genes
(Tsuji et al., 2020), a YAP1-mediated resistance development in neuroblastoma cells is likely,
but not elucidated yet. Only the upregulation of the YAP1 target gene AXL and the induction
of mesenchymal features in ALK inhibitor-resistant cell lines is described yet (Debruyne et al.,
2016).
Furthermore, programmed cell death ligand 1 (CD274) and multiple cytokines (IL6, CXCL5,
CSF1/2/3) are direct transcriptional YAP1/TAZ targets and known to convey drug resistance
and immune evasion of various cancers by intrinsic as well as ligand-dependent signaling
(Murakami et al., 2016; Wang et al., 2016; B. S. Lee et al., 2017; Miao et al., 2017; Kim et al.,
2018; Li et al., 2020).
Introduction
18
1.2.6 YAP1-induced epithelial-mesenchymal transition
The transition of cells from epithelial to mesenchymal phenotype and the inverse (MET) are
crucial processes for embryonal development and pathologically reactivated in development
of fibrosis and cancer. EMT comprises the assembly and disassembly of cell junctions and an
intricated reorganization of the cellular cytoskeleton with subsequent changes in motility
capability, all orchestrated by EMT-related transcription factors such as Snail1, Slug, Twist1/2
and Zinc-finger E-box binding homeobox 1/2 (ZEB1/2) (Lamouille, Xu and Derynck, 2014). YAP1
is one regulator of EMT. Together with various TFs such as TEADs, ZEB1 or AP-1, YAP1
promotes transcription of some EMT-related genes (SNAI2, RUNX, FOS) in normal and tumor
tissues and thereby promotes EMT-like phenotype of cells, leading to enhanced cell motility,
drug resistance and tumor progression (Zhao et al., 2008; Lamar et al., 2012; Diepenbruck et
al., 2014; Shao et al., 2014; Yu et al., 2018; Feldker et al., 2020).
In Ras-activated cancer cells, YAP1 and AP-1 family transcription factor FOS interact to induce
the expression of mesenchymal genes such as SNAI2, FN1, VIM and ZEB1 and downregulate
epithelial gene CDH1 (E-cadherin) and OCLN (Occludin). Thus, YAP1 mediates apoptosis
resistance to Ras inhibitors in Ras-driven cancer cells (Shao et al., 2014). In NSCLCs, YAP1
cooperates with TEADs to induce a Slug-driven EMT program (Yu et al., 2018). Mammary
carcinoma cells overexpressing active YAP1S127A revealed the TEAD-dependent transcriptional
activation of EMT-related genes and additionally displayed enhanced colonization of
disseminated tumor cells in xenograft experiments (Lamar et al., 2012).
Neuroblastoma tumors originate from sympathoadrenal precursors and have already
experienced the EMT process to migrate from neural tube (Matthay et al., 2016). Thus,
neuroblastoma cells are unlikely able to undergo this type of transition anymore. However,
neuroblastoma and well-established cell lines seem to comprise two different cell identities,
one being the sympathetic noradrenergic phenotype and the other a NCC-like or
mesenchymal phenotype. The mesenchymal cell type is characterized by protein synthesis of
Slug, fibronectin 1, vimentin and YAP1 (Van Groningen et al., 2017) and exhibits an mRNA
cluster including FOSL1, FOSL2, RUNX1, RUNX2 and PRRX1 (Boeva et al., 2017). In line with the
transcriptome analysis, this phenotype is shown to be more resistant to chemotherapeutic
agents doxorubicin, etoposide and cisplatin than the noradrenergic subpopulation (Boeva et
al., 2017; Van Groningen et al., 2017). YAP1 together with TEAD2 and Slug are strongly
Introduction
19
increased upon PRRX1-overexpression in neuroblastic SK-N-BE(2)-C cells (Szemes,
Greenhough and Malik, 2019), which suggests that YAP1 might induce the mesenchymal
phenotype switch described for the PRRX1-active cells (Van Groningen et al., 2017).
1.2.7 YAP1 and cancer cell metabolism
Metabolic reprogramming of rapidly proliferating cancer cells warrants the supply of energy
demands and biomolecules such as amino acids, proteins and nucleotides. YAP1 is a
transcriptional co-activator that regulates a plethora of cellular processes including glucose
metabolism, hypoxic adaption, lipid metabolism, amino acid synthesis, one-carbon
metabolism as well as nucleotide synthesis.
1.2.7.1 Glycolysis
The energy demand of normal cells is predominantly supplied by oxidative phosphorylation,
that yields 36 mol of energy equivalents adenosine 5-triphosphate (ATP) per glucose molecule.
Aerobic glycolysis, which is preferred by various cancer cells, only yields 2 ATP per glucose
molecule. Cells therefore increase their glucose uptake and convert the hexose into lactate
via the glycolysis pathway (Nelson et al., 2009). This metabolic phenomenon of tumor cells
has been described around 100 years ago by Otto Warburg and is nowadays referred to as
Warburg effect (Warburg, Wind and Negelein, 1927). It remains partially vague, why cancer
cells switch to the “inefficient” aerobic glycolysis compared to ATP-generating normal cells,
but evidences consolidate that highly proliferating tumor cells are not only in need of energy,
but they also require biomolecules to generate biomass (Vander Heiden, Cantley and
Thompson, 2009). In addition, tumor-derived lactate inhibits the function of tumor-attacking
immune cells by reduced microenvironmental pH values (Lardner, 2001; Fischer et al., 2007).
YAP1 has been shown to bidirectionally interact with cellular glucose metabolism. It
transcriptionally activates GLUT3 (glucose transporter 3) in a Glut3-addicted subset of
glioblastoma tumors (Cosset et al., 2017). Also in HEK293A cells, the overexpression of YAP1
promotes aerobic glycolysis via upregulation of GLUT3 and glycolysis gene HK2 (hexokinase 2)
(Wang et al., 2015). In breast cancer cells, YAP1 and TEAD1 regulate GLUT1 (glucose
Introduction
20
transporter 1) and the extracellular acidification rate of the cells (Lin and Xu, 2017). In NF2-
mutant cancer cells, knockdown of YAP1 reduced the gene expression of GLUT3 as well as
glycolysis genes and suppressed some glycolytic metabolites including lactate (White et al.,
2019). Taken together, YAP1 transcriptionally activates glucose transporters and glycolysis
enzymes to provoke the Warburg effect. In turn, YAP1 is also strongly affected by the glucose
metabolism of a cell. In early embryonal development, a glycolytic switch that is predominant
for premigratory neural crest cells to undergo EMT and start migration, induces YAP1 and
TEAD to initiate the EMT process (Bhattacharya, Azambuja and Simoes-Costa, 2020). YAP1
activity is regulated by PFK1 binding TEADs and regulating the interaction with YAP1 (Enzo et
al., 2015). In addition, the LATS1/2 dependent posttranslational inhibition of YAP1 by AMPK
phosphorylation is another mechanism of energy-metabolism regulated YAP1 activity in
cancer cells (Mo et al., 2015; Wang et al., 2015). In response to high glucose levels, YAP1 is
activated by transfer of N-acetyl-glucosamine to serine residues (O-GlcNAcetylation) and thus,
promotes tumorigenesis (Peng et al., 2017).
1.2.7.2 Amino acid metabolism
Amino acids serve as building blocks for synthesis of proteins and other macromolecules, they
control redox status and antioxidant systems, and they serve as substrates for post-
translational and epigenetic modifications. Cancer cell reprogramming thus includes
reorganization of amino acid acquisition and utilization to meet altered metabolic
requirements. Cellular uptake of non-essential and essential amino acids is achieved through
upregulation of numerous amino acid transporters. An import of amino acids against the
concentration gradient is realized by co-transport of ions (H+, K+, Na+, Cl-) or different amino
acids (Kandasamy et al., 2018; L. Zhang et al., 2020). In addition to amino acid transport, the
intracellular volume of non-essential amino acids is balanced through its de novo synthesis
(see section 1.2.7.3) and catabolism such as glutaminolysis.
Enabling a sufficient metabolite supply for YAP1-induced cancer cell proliferation, YAP1 does
not only activate glucose transporters, but also amino acid transporters as described for
hepatocellular carcinoma (Park et al., 2016), hepatoblastoma (Liu et al., 2017) and breast
cancer (Edwards et al., 2017). YAP1 activates amino acid transporters solute carrier family
Introduction
21
(SLC) 38 member 1 (SLC38A1, NAT1), SLC7A5 (LAT1), SLC1A5 (ASCT2) and SLC3A2 (CD98 heavy
chain) in different cellular backgrounds (Hansen et al., 2015; Park et al., 2016; Edwards et al.,
2017; Liu et al., 2017), thereby facilitating the transport of large and neutral amino acids
including glutamine (Pochini et al., 2014). The provoked increase of amino acid import
activates mTOR complex 1 (mTORC1) and thus promotes cell growth and tumor development
(Hansen et al., 2015; Park et al., 2016; Liu et al., 2017). Closing a positive feedback loop,
serine/threonine kinase mTOR in turn activates YAP1 as demonstrated in perivascular
epithelioid cell tumors (Liang et al., 2014). Mechanistically, an mTOR-mediated
phosphorylation of Hippo-pathway member MST1 at threonine120 limits its inhibitory
function on the YAP1 protein, as demonstrated in prostate cancer cells (Collak et al., 2012). In
non-transformed epithelial cells MCF10A, YAP1 has been shown to induce mTOR signaling
independent of Hippo pathway. The miRNA hsa-miR-29 is transcriptionally induced by YAP1
and inhibits the translation of the phosphatase and tensin homolog (PTEN), which in turn
activates mTOR1/2 (Tumaneng et al., 2012). In bladder cancer, YAP1 activates the mTOR
signaling and inhibits its ubiquitination dependent on the upregulation of E3 ubiquitin ligase
and direct YAP1 target, SKP2. Additionally, the overexpression of mTOR resulted in an
upregulation of YAP1 and TEAD and nuclear accumulation of YAP1 (Xu et al., 2020).
Glutamine is a conditional essential amino acid for many cancer cells, serving as precursor
molecule for amino acids, proteins, amino sugars, nucleotides and antioxidative glutathione.
Many cancer cells meet their metabolic requirements by increased glutamine uptake and
metabolization (Yuneva et al., 2007; Scalise et al., 2017). Glutamine is mainly imported via
amino acid transporters (e.g., SLC1A5, SLC38A2) and metabolized via the glutaminolysis
pathway. Here, it is converted into glutamate and ammonium ion by mitochondrial
transaminases kidney-type glutaminase (GLS1) or liver-type glutaminase (GLS2). Glutamate is
further converted into
𝛼
-ketoglutarate by aminotransferases glutamic-oxaloacetic
transaminase 1/2 (GOT1/2), glutamic—pyruvic transaminase 2 (GPT2), phosphoserine
aminotransferase 1 (PSAT1) or by glutamate dehydrogenase 1 (GLUD1), which then fuels the
citric acid cycle. Additionally, glutamate supplies glutathione synthesis and amino acid
synthesis.
Introduction
22
YAP1 transcriptionally activates GLS1 to maintain glutaminolysis-dependent cell proliferation
in pulmonary arterial endothelial cells (Bertero et al., 2016). In HER2-positive breast cancer
cells, overexpression of receptor tyrosine kinase EphA2 induces YAP1 and TEAD, which in turn
results in an upregulation of GLS and glutamine transporter SLC1A5 and subsequently
promotes glutamine metabolism (Edwards et al., 2017). The reverse reaction is catalyzed by
the glutamine synthetase (GLUL). In zebrafish livers, GLUL is a direct target gene of YAP1
together with TEAD4 and forces nucleotide synthesis, ammonia or glutamate detoxification
and acid-base homoeostasis (Cox et al., 2016). Yang et al. demonstrated the YAP1-induced
upregulation of GOT1/2, GPT2 and PSAT1, that further convert glutamate into
𝛼
-ketoglutarate
and amino acids serine, aspartate and alanine. An increased synthesis of these proteins
supports glutamate metabolism, amino acid synthesis and cell growth of breast cancer cells
(Yang et al., 2018). The inhibition of transaminases in YAP1-active cells resulted in diminished
cell proliferation, indicating a potential vulnerability of YAP1-activated cancer cells (Du et al.,
2018; Yang et al., 2018).
1.2.7.3 De novo serine synthesis pathway and folate cycle
Serine is an important precursor molecule for the non-essential amino acids glycine and
cysteine. Furthermore, it is required for production of sphingolipids, phosphatidylserine as
well as porphyrins, and it is incorporated into purine nucleotides and glutathione (Mattaini,
Sullivan and Vander Heiden, 2016). Serine can either be imported via amino acid transporters
or is synthesized de novo. The serine biosynthesis pathway enzymes phosphoglycerate
dehydrogenase (PHGDH), PSAT1 and phosphoserine phosphatase (PSPH) catalyze the
conversion of glycolysis intermediate 3-phosphoglycerate into serine in three steps (Locasale,
2013). Cytosolic serine hydroxylmethyltransferase 1 (SHMT1) and mitochondrial serine
hydroxylmethyltransferase 2 (SHMT2) convert serine into glycine. As part of the folate cycle,
the resulting one-carbon units are transferred to tetrahydrofolate (THF) to form 5,10-
methylTHF.
Subsequently and reversibly, THF can undergo some oxidative and reductive transformations
between THF, 5,10-methylTHF, 10-formylTHF and formate. Since SHMT1 is located to the
cytoplasm and SHMT2 is active in the mitochondria, THF and formate are transported across
Introduction
23
the mitochondrial membrane to close the folate cycle (Ducker and Rabinowitz, 2017). Various
intermediates of this important circuit are required for nucleotide biosynthesis, amino acid
synthesis, protein synthesis, methionine cycle and redox balancing.
The regulation of serine and glycine biosynthesis as well as the folate cycle by YAP1 is not yet
elucidated well. Cancer cells exhibiting inactivating mutations of STK11 (serine/threonine
kinase 11, LKB1) revealed increased gene expression of PSAT1, PSPH and SHMT1/2 (Kottakis
et al., 2016). Another study demonstrated a YAP1 inhibition by the tumor suppressor LKB1,
dependent on its interaction with Hippo kinases MST1/2 and LATS1/2 (Mohseni et al., 2014).
A mRNA correlation analysis indeed revealed a potential association between YAP1 and the
gene encoding the rate-limiting enzyme of de novo serine synthesis, PHGDH, in LKB1-deficient
breast cancer cells (Wu et al., 2017), albeit conclusive data still need to be obtained.
In murine KRAS-driven pancreatic tumor cells, Cre-recombinase-mediated YAP1 ablation has
been shown to downregulate glycolysis genes (e.g., HK2, PGK1) as well as genes regulating the
serine and glutamine metabolism (e.g., GLS1/2, PHGDH, PSAT1, SHMT2) in a Myc-Sox2-p53
dependent manner, resulting in decreased nucleotide and metabolite levels (Murakami et al.,
2019).
Furthermore, the tumor suppressor p73, which is known to interact with YAP1 for
transcriptional gene regulation, directly regulates serine pathway enzymes (Amelio et al.,
2014). Thus, a regulation of serine synthesis together with YAP1 as co-activator is likely, but
not evidenced yet.
1.2.7.4 Hypoxia
Physiological oxygen levels in mammalian organisms range between oxygen partial pressure
pO2 = 8-72 mmHg (1-10% oxygen) (Hammond et al., 2014) and depend on tissue structure,
oxygen diffusion and vascularization (Brahimi-Horn and Pouysségur, 2007). Due to massive
tumor cell proliferation and destructed or disorganized vascularization, solid tumors often
harbor heterogeneous oxygen levels including mild hypoxia (
≤
2% O2) or severe
hypoxia (
≤
0.1% O2) (Hammond et al., 2014). Adjustments required for hypoxic adaption of
the cells are predominantly managed by TFs hypoxia inducible factors (HIF) that all contain
Introduction
24
basic helix-loop-helix-PAS domains to mediate heterodimerization and DNA binding (Wang et
al., 1995). A HIF-1
𝛽
(ARNT) unit binds the oxygen-sensitive HIF-
𝛼
isoforms (HIF-1/2/3
𝛼
) in
order to occupy hypoxia response elements for target-gene transcription initiation. In the
presence of oxygen, HIF-
𝛼
-subunits get hydroxylated at one or two prolyl residues by oxygen-
sensing prolyl hydroxylase domain proteins, creating a binding site for the ubiquitin ligase
complex-protein von-Hippel-Lindau protein. Binding of this tumor suppressor entails the
ubiquitination and subsequent proteasomal degradation of HIF-
𝛼
subunits (Kaelin and
Ratcliffe, 2008). Low oxygen levels inhibit this prolyl hydroxylase domain protein-dependent
cascade and HIF-subunits dimerize, translocate into the nucleus and start gene transcription
to regulate energy metabolism, angiogenesis, autophagy, EMT and tumorigenesis (Brahimi-
Horn and Pouysségur, 2007; Kaelin and Ratcliffe, 2008).
In neuroblastoma, HIF proteins activate the expression of glucose transporter genes and
different glycolysis genes including HK2, ENO1, PDK1, PGK1 and LDHA to meet the energy
requirements for the hypoxic tumor cell (Qing et al., 2010; Kim et al., 2014; Applebaum et al.,
2016). The differentially regulated proteins HIF-1
𝛼
and HIF-2
𝛼
have overlapping as well as
distinct functions in tumorigenesis of neuroblastoma (Holmquist-Mengelbier et al., 2006;
Noguera et al., 2009). HIF-1
𝛼
coordinates the early and acute hypoxic adaption, whereas
HIF-2
𝛼
is responsible for the late and chronic hypoxia (Holmquist-Mengelbier et al., 2006). In
addition, HIF-2
𝛼
protein stabilization is partially observed in oxygenated neuroblastoma cells
(Nilsson et al., 2005; Persson et al., 2020). The hypoxia-initiated gene expression pattern of an
immature and neural-crest like phenotype in neuroblastoma cells (Jogi et al., 2002) seems to
be predominantly orchestrated by HIF-2
𝛼
(Pietras et al., 2009; Mohlin, Hamidian and
Påhlman, 2013).
HIF and YAP1 proteins are both strong regulators of tumor cell metabolism. Recently, the
interaction of both was enlightened in different tumor entities such as hepatocellular
carcinoma (X. Zhang et al., 2018), acute myeloid leukemia (Zhu et al., 2020), pancreatic ductal
adenocarcinoma (Jia et al., 2019) and in colorectal cancer (Sun et al., 2020), but not yet in
neuroblastoma. In addition, the majority of the studies concentrate on HIF-1
𝛼
only. YAP1
stabilizes HIF-1
𝛼
protein to promote hypoxic glycolysis (X. Zhang et al., 2018; Jia et al., 2019;
Sun et al., 2020), and hypoxia in turn activates YAP1 HIF-independently by promoting its
nuclear translocation via inhibition of Hippo kinases (Greenhough et al., 2018; X. Zhang et al.,
Introduction
25
2018; Sun et al., 2020) or mevalonate pathway (Dai et al., 2016). Only one study in colorectal
cancer revealed a HIF-2
𝛼
-mediated increase in YAP1 expression and activity in vitro and in vivo
without elucidating the exact mechanism (Ma et al., 2017).
Scope and objectives
26
2. Scope and objectives
YAP1 has been implicated as relevant for neuroblastoma relapse tumors. At the beginning of
this project, the function of YAP1 has never been studied in the context of neuroblastoma.
Aim of this doctoral thesis was to investigate the role of YAP1 in neuroblastoma progression
based on neuroblastoma cell line models. Aim was to specify the function of this
transcriptional co-activator and determine the potential of YAP1-targeting therapeutics in
neuroblastoma treatment. Therefore, I put particular focus on cellular processes supporting
the development of primary and relapse tumors as well as the formation of metastasis,
including proliferation, apoptosis, migration, drug resistance and metabolic changes.
Materials
27
3. Materials
3.1 Cell lines
Table 1 Cell lines
Designation
Cell origin
Source
CHP134
neuroblastoma
J. H. Schulte
DAOY
medulloblastoma
J. H. Schulte
GI-M-EN
neuroblastoma
J. H. Schulte
HEK293
epithelial
J. H. Schulte
HEK293T
epithelial
F. Buttgereit
HeLa
epithelial
J. H. Schulte
IMR5
neuroblastoma
J. H. Schulte
Kelly
neuroblastoma
DSMZ
LAN-1
neuroblastoma
DSMZ
LAN-5
neuroblastoma
DSMZ
N206
neuroblastoma
J. Molenaar
NB-1643
neuroblastoma
J. H. Schulte
NBL-S
neuroblastoma
J. H. Schulte
NGP
neuroblastoma
F. Speleman
SH-EP
neuroblastoma
J. H. Schulte
SH-EP-TR
neuroblastoma
J. H. Schulte
SH-EP-TR-YAP1S127A
neuroblastoma
K. Ahrens
SH-SY5Y
neuroblastoma
DSMZ
SJ1
neuroblastoma
J. H. Schulte
SK-N-AS
neuroblastoma
J. H. Schulte
SK-N-AS-TR
neuroblastoma
J. H. Schulte
SK-N-AS-TR-YAP1S127A
neuroblastoma
K. Ahrens
SK-N-BE
neuroblastoma
J. H. Schulte
Materials
28
Designation
Cell origin
Source
SK-N-DZ
neuroblastoma
J. H. Schulte
SK-N-FI
neuroblastoma
J. H. Schulte
SK-N-SH
neuroblastoma
J. H. Schulte
TR14
neuroblastoma
J. Molenaar
VH7
fibroblastic
H. Deubzer
3.2 Bacterial strains
Table 2 Bacterial Strains
Designation
Manufacturer
XL Gold ultracompetent Escherichia coli
Agilent Technologies
Top10 Escherichia coli
New England Biolabs
3.3 Cell culture media
Table 3 Cell culture media and reagents
Designation
Manufacturer
Accutase®-Lösung
Merck (Sigma-Aldrich)
Blasticidin
Merck
DMEM (1 g/L glucose, pyruvate, L-glutamine,
phenol red)
Thermo Fisher Scientific (Gibco™)
DMEM (4.5 g/L glucose, pyruvate, L-glutamine,
phenol red)
Thermo Fisher Scientific (Gibco™)
DMSO
Carl Roth
Ethanol 96%
Carl Roth
FCS Superior
Merck (Biochrom)
FCS Tetracycline free
Bio&SELL
Geneticin (G-418) sulfate
Merck (Millipore)
Glutamine
Thermo Fisher Scientific (Gibco™)
Materials
29
Designation
Manufacturer
Lipofectamine 3000
Thermo Fisher Scientific (Invitrogen)
MEM Non-essential amino acids solution 100x
Lonza
Opti-MEM
Thermo Fisher Scientific (Gibco™)
Penicillin-Streptomycin (10,000 U/mL)
Thermo Fisher Scientific (Gibco™)
PBS
Thermo Fisher Scientific (Gibco™)
RPMI 1640 (2 g/L glucose, L-glutamine, phenol red)
Thermo Fisher Scientific (Gibco™)
Tetracycline hydrochloride ≥95%
Merck (Sigma-Aldrich)
Trypan Blue Solution
Thermo Fisher Scientific (Gibco™)
Trypsin/ EDTA 0.05%
Thermo Fisher Scientific (Gibco™)
3.4 Chemicals and enzymes
Table 4 Chemicals
Designation
Manufacturer
2-Propanol 99.8%
Carl Roth
7-AAD
BioLegend
Acetic acid
Carl Roth
Agarose
Biozym
Amphotericin B
Thermo Fisher Scientific (Gibco™ )
Ampicillin sodium salt, ≥97%
Carl Roth
Annexin buffer, 1x
Thermo Fisher Scientific
Bovine serum albumin (BSA) Fraction V
Carl Roth
Buffer P1 (resuspension buffer)
Qiagen
Buffer P2 (lysis buffer)
Qiagen
Buffer P3 (neutralization buffer)
Qiagen
Calcium chloride dihydrate (CaCl2)
Merck (Sigma-Aldrich)
Chloramphenicol
Biomol
chloroform
Merck (Sigma-Aldrich)
Materials
30
Designation
Manufacturer
Chloroquine diphosphate salt
Merck (Sigma-Aldrich)
Crystal Violet
Merck (Sigma-Aldrich)
DEPC-treated H2O
SERVA Eletrophoresis
DNA loading dye (6x Orange)
Thermo Fisher Scientific (Invitrogen)
Doxorubicin HCl
Selleckchem
DTT
Merck (Sigma-Aldrich)
EDTA
Carl Roth
EGTA
Merck (Sigma-Aldrich)
Ethanol
Carl Roth
Ethidiume bromide solution
Carl Roth
Etoposide (VP-16)
Selleckchem
Formaldehyde, 37% min
Carl Roth
GeneRuler 1 kb DNA ladder
Thermo Fisher Scientific
GeneRuler 100 bp DNA ladder
Thermo Fisher Scientific
Glycerol
Merck (Millipore)
Glycine ≥99&
Carl Roth
HEPES 1M in H2O
Merck (Sigma-Aldrich)
Kanamycin sulfate
Carl Roth
Laemmli Protein Sample buffer, 4x
Bio-Rad Laboratories
LB-Agar
Carl Roth
LB-medium
Carl Roth
Magnesium chloride (MgCl2)
Carl Roth
Magnesium sulfate (MgSO4)
Merck (Sigma-Aldrich)
Methanol ROTIPURAN® ≥99.8%
Carl Roth
Milk Power
Carl Roth
Nuclease-free H2O
Thermo Fisher Scientific (Invitrogen)
PAGERuler Prestained Protein ladder
Thermo Fisher Scientific (Invitrogen)
Phosphatase inhibitor tablets, PhosSTOP™
Hoffmann-La Roche
Polybrene
Merck (Sigma-Aldrich)
Polyethylenglycol 3000
Merck (Millipore)
Materials
31
Designation
Manufacturer
Potassium chloride (KCl)
Carl Roth
Potassium phosphate, monobasic (KH2PO4)
Carl Roth
Propidiumiodid
Thermo Fisher Scientific (Invitrogen)
Protease inhibitor cocktail tablets, cOmplete™,
Mini, EDTA-free
Hoffmann-La Roche
rCutSmart buffer™
New England Biolabs
Rotiphorese Gel 30
Carl Roth
SDS pellets
Carl Roth
Sodium chloride (NaCl)
Merck (Sigma-Aldrich)
Sodium phosphate, dibasic (Na2HPO4)
Carl Roth
Sodium pyruvate (Na
Thermo Fisher Scientific (Gibco™ )
Tetramethylethylendiamin (TEMED)
Carl Roth
Toothpicks
DM drug store
Tris base (Trizma® Base)
Carl Roth
Tris-HCl
Carl Roth
Triton X-100
Carl Roth
TRIzol™
Thermo Fisher Scientific
Tryptone
SERVA Electrophoresis
Tween-20
Carl Roth
VectaShield mounting medium
Vector Laboratories
Vincristine sulfate
Selleckchem
Western Lightning Plus-ECL solution
PerkinElmer
Bacto™ Yeast extract
BD Biosciences
𝛽-Mercaptoethanol
Merck (Sigma-Aldrich)
Materials
32
Table 5 Enzymes
Designation
Manufacturer
Acc651
New England Biolabs
AgeI
New England Biolabs
ApaI
New England Biolabs
BamHI
New England Biolabs
Dnase I
New England Biolabs
RNase A
Qiagen
XbaI
New England Biolabs
XhoI
New England Biolabs
3.5 Antibodies
Table 6 Antibodies used for Western Blot
Antigen
Clone
Species
Dilution
conjugate
Manufacturer
HIF-1
𝛼
clone 54
Mouse
1:250
-
BD Bioscience
HIF-2
𝛼
-
Rabbit
1:1000
-
Novus Biologicals
Mouse-IgG
-
Goat
1:5000
HRP
Dianova
Phospho-YAP1 (S127)
EP1675Y
Rabbit
1:1000
-
Abcam
Rabbit-IgG
-
Goat
1:5000
HRP
Dianova
YAP1
EP1674Y
Rabbit
1:500
-
Abcam
𝛽
-Actin
C4
Mouse
1:1000
-
Santa Cruz
Table 7 Antibodies used for Immunostaining
Antigen
Clone
Species
Dilution
conjugate
Manufacturer
Flag M2
F1804
Mouse
1:500
-
Merck (Sigma-Aldrich)
Mouse-IgG
-
Goat
1:500
Alexa Fluor® 488
Thermo Fisher Scientific
Rabbit-IgG
-
Goat
1:500
Alexa Fluor® 488
Thermo Fisher Scientific
YAP1
D8H1X
Rabbit
1:100
-
Cell Signaling Techologies
Materials
33
3.6 Kits
Table 8 Kits
Designation
Manufacturer
APC Annexin V Apoptosis Detection Kit with 7-AAD
Biolegend
APC Annexin V Apoptosis Kit
BioLegend
BCA protein Assay Kit
Santa Cruz
Cell Death Detection ELISAPLUS
Thermo Fisher Scientific
Cell Proliferation ELISA (BrdU), colorimetric
Merck (Sigma-Aldrich)
CellTiter-Glo® Luminscent Cell Viability Assay
Promega
Dead Cell Apoptosis Kit with Annexin V AlexaFluorTM
488 and PI
Thermo Fisher Scientific
FastStart Universal SYBR Green Master (Rox)
Hoffmann-La Roche
Gateway™ LR Clonase™ Enzyme Mix II
Thermo Fisher Scientific (Invitrogen)
LR Clonase Enzyme Mix, Gateway Cloning System
Thermo Fisher Scientific
NucleoBond® Xtra Midi/Maxi Kit
Macherey-Nagel
MycoAlert™ Mycoplasma Detection Kit
Lonza
QIAprep Spin Miniprep kit
Qiagen
RNA 6000 Nano Kit
Agilent Technologies
Rneasy Micro Kit
Qiagen
Rneasy Mini Kit
Qiagen
Transcriptor First Strand cDNA Synthesis Kit
Thermo Fisher Scientific
TruSeq® stranded mRNA Library Prep Kit
Illumina
𝛽
-Galactosidase Staining Kit
Cell Signaling Technology
APC Annexin V Apoptosis Detection Kit with 7-AAD
Biolegend
Materials
34
3.7 Disposables
Table 9 Disposables
Designation
Manufacturer
Amicon Ultra-15, PLTK Ultracel-PL Membrane, 100kDa
Merck (Millipore)
Blotting paper (Whatman), 0.35 mm
Carl Roth
Cell culture Assay Plate 96 well white, flat bottom, sterile
Corning
Cell culture dish (diameter 10 cm, 15 cm), sterile
Corning
Cell culture flask (T75), sterile
Corning
Cell culture multiwell plate CELLSTAR® (6, 12, 24, 96 well), sterile
Corning
Cell scraper
Corning
Centrifuge tube Falcon®, conical, sterile (15 mL, 50 mL)
Corning
Combitips advanced (5 mL, 25 mL)
Eppendorf
Counting chamber Neubauer, 0.1 mm
BRAND
Counting slides for TC10™/TC20™ Cell Couter, Dual chamber
Bio-Rad Laboratories
Cover glass round, 10 mm
VWR International
Dispenser head Cassettes HP T8/D4 PLUS 20PC
Tecan Group
Electroporation Cuvettes GenePulser ®/ MicroPulser™ (0.2 mm,
0.4 mm)
Bio-Rad Laboratories
epT.I.P.S Standard (0.1-2 mL)
Eppendorf
Erlenmeyer flask (250 mL)
Carl Roth
Filter tips, Biosphere® (0.10 µL, 200 µL)
Sarstedt AG
Filter tips, sterile (1 µL, 10 µL, 100 µL, 200 µL, 1250 µL)
Biozym
FrameStar 96-well plates Abi FAST PCR, low profile
4titude
Glass beads, 5mm
Merck (Sigma-Aldrich)
Millicell hanging cell culture insert, PET 8 µm, 12 well
Merck (Millipore)
Nail polish, clear
DM drug store
Parafilm™ M Laboratory Wrapping Film
Bemis Company
Pasteur pipette, glass, 150 mm
Brand
PVDF western blotting membrane
Hoffmann-La Roche
PVDF Western Blotting Membrane, 0.45 µm pore size
Hoffmann-La Roche
Materials
35
Designation
Manufacturer
qPCR Seal, adhesive film
4titude
Reaction tube (0.5 mL, 1.5 mL, 2 mL)
Sarstedt
Reaction tube cryogenic (2 mL), sterile
Sarstedt
Serological pipette (5 mL, 10 mL, 25 mL, 50 mL)
Corning
Stericup-GP (0.22 µm)
Merck (Millipore)
Surphob Reload tips (10 µL, 200 µL, 1250 µL)
Biozym
Syringe Filter ROTILABO®, cellulose acetate (0.22 µm, 0.45 µm)
Carl Roth
Syringe Inject® Solo PP/PE, Luer lock, (2 mL, 5 mL, 10 mL)
B. Braun
Syringe PP Luer (2 mm)
Semadeni
3.8 Laboratory equipment
Table 10 Laboratory equipment
Designation
Manufacturer
Analytical Balance ABS-N
Kern
Biological Safety Cabinet Class II, Safe 2020
Thermo Fisher Scientific
Biophotometer 6131 01964
Eppendorf
C1000 Touch Thermal Cycler
Bio-Rad
Cell Counter TC 20
Bio-Rad
Centrifuge 5424R
Eppendorf
Centrifuge 5427R
Eppendorf
Centrifuge 5810R
Eppendorf
Centrifuge Heraeus™ Laborfuge™ 400
Thermo Fisher Scientific
Centrifuge Megafuge 2.0R
Thermo Fisher Scientific
Centrifuge PerfectSpin Mini
VWR
Centrifuge plates
Thermo Fisher Scientific
Digital Dispenser D300E
Tecan
Epoch™ Microplate Spectrophotometer
BioTek Instruments
Materials
36
Designation
Manufacturer
Flow Cytometer LSR Fortessa X-20
BD Bioscience
Forceps Dumont
Fine Science Tools
Freezer -20°C
Liebherr
Freezer -80°C HERAfreeze
Thermo Fisher Scientific
Freezer -86°C Innova U725-G ULT
Eppendorf
Freezing Container, -1°C/ min
Fisher Scientific
Fusion-FX Imaging System
Vilber Lourmat
Gene Pulser Xcell Electroporation System
Bio-Rad
GloMax® Plate Reader
Promega
Horizontal Mini Gel System PerfectBlue™
VWR
Ice machine
Manitowoc
Incubator Heratherm™ Compact Microbiological Incubator
Thermo Fisher Scientific
Incubator Heracell™ 150i, Kupfer
Thermo Fisher Scientific
Mechnanical Laboratory Agitator, Rotamax/Duomax
Heidolph Instruments
Microscope Axio Vert.A1 Inverse
ZEISS
Microscope BX43
Olympus
Microscope camera
Zeiss
Microwave
Bosch
Mini Trans-Blot Electrophoretic Transfer Cell
Bio-Rad
Mini-PROTEAN Tetra System
Bio-Rad
Multipette® Xstream
Eppendorf
NanodDrop™ 2000
Thermo Fisher Scientific
PCR Cooler 0.2 mL
Eppendorf
PCR Workstation Pro, PEQLAB Biotechnologies
VWR
pH meter FiveEasy
Mettler Toledo
Phoenix RS-TR 5 tube roller
Phoenix Instruments
Pipette Research® Plus Multichannel
Eppendorf
Pipettes, Research® plus (0.1 µL - 1000 µL)
Eppendorf
Pipetus®
Hirschmann
PowerPac Basic Power supply
Bio-Rad
Materials
37
Designation
Manufacturer
Precision Balance EW-N
Kern
Refridgerator 4°C
Liebherr
RH basic Magnetic stirrer
IKA
StepOnePlus Real-Time PCR System
Eppendorf
Syringe, glass (20 µL)
B. Braun
Thermal cycler T100
Bio-Rad
ThermoMixer® comfort
Eppendorf
Trans-Blot Turbo Transfer System
Bio-Rad
Tube Shaker Reax Top
Heidolph Instruments
UV Transilluminator ECX-F20.C
Vilbert Loumat
Vacuum Safety Suction System
Hettich
Waterbath WNB 7
Memmert
3.9 Plasmids
Table 11 Plasmids
Plasmids
Source
pENTR-3xFLag-YAP1-S127A
Addgene, gifted by W. Pu
pENTR1A-2xGlag-YAP1-S127A
Addgene, gifted by X. Chen
pT-Rex-DEST30
J. H. Schulte
pT-Rex-DEST30-YAP1(S127A)-2xFlag
K. Ahrens
pT-Rex-DEST30-YAP1(S127A)-3xFlag
K. Ahrens
pLKO.1-puro
Addgene, gifted by D. Sabatini
psPAX2
F. Buttgereit
pMD2.G
A. G. Henssen
pLKO.1-shRNA-puro-T2A-eGFP
A. Winkler
Materials
38
3.10 Oligonucleotides
Table 12 qPCR Primer
Gene
Sense
Sequence from 5' - 3'
YAP1
forward/reverse
Hs_YAP1_1_SG, QuantiTect Primer Assay, Qiagen
SDHA
forward/reverse
Hs_SDHA_1_SG, QuantiTect Primer Assay, Qiagen
CYR61
forward
CACACCAAGGGGCTGGAATG
CYR61
reverse
CCCGTTTTGGTAGATTCTGG
COL8A1
forward
CAGAAACCAGCCCCAGAGGTGTCAC
COL8A1
reverse
GAAATGGTAAGCAGCACTCCCAGCAG
SKP2
forward
CTGTCTCAAGGGGTGATTGC
SKP2
reverse
TGTACACGAAAAGGGCTGAA
SNAPC
forward
GAATGAAAGTTTGAGTGGAACAGA
SNAPC
reverse
CCAGGCTCTTTGTTCAGTGTT
Table 13 DNA Sequencing Primer
Designation
Sense
Sequence from 5' - 3'
CMV.for
forward
CGCAAATGGGCGGTAGGCGTG
M13 uni (-21)
forward
TGTAAAACGACGGCCAGT
Table 14 siRNAs
Designation
Product Name
Source
YAP1 #1 (Silencer® Select)
s20366
Thermo Fisher Scientific
YAP1 #2 (Silencer® Select)
s20367
Thermo Fisher Scientific
Non-target control #1 (Silencer® Select)
Negative Control #1
Thermo Fisher Scientific
Non-target control #2 (Silencer® Select)
Negative Control #2
Thermo Fisher Scientific
Materials
39
Table 15 shRNA
Designation
sense
Sequence from 5' - 3'
Manufacturer
shRNA
YAP1_1
sense
CCGGAAGCTTTGAGTTCTGACATCC
K. Guan
(Addgene #27368)
loop
CTCGAG
antisense
GGATGTCAGAACTCAAAGCTTTTTTTC
shRNA
YAP1_2
sense
CCGGCTGGTCAGAGATACTTCTTAA
K. Guan
(Addgene #27369)
loop
CTCGAG
antisense
TTAAGAAGTATCTCTGACCAGTTTTTC
shRNA
scramble
sense
CCGGCAGTCGCGTTTGCGACTGGT
D. Sabatini
(Addgene #1864)
loop
CTCGAG
antisense
ACCAGTCGCAAACGCGACTGTTTTT
Methods
40
4. Methods
4.1 Cell culture
4.1.1 Cell lines
Human neuroblastoma cell lines SH-SY5Y, Kelly, LAN-1 and LAN-5 were obtained from German
Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, Germany). NGP were
kindly provided by F. Speleman (Cancer research institute Ghent, Ghent, Belgium). TR14 and
N206 were kindly provided from J. Molenaar (Princess Máxima Center for Pediatric Oncology,
Utrecht, Netherlands). The cell lines SK-N-BE, SK-N-DZ, GI-M-EN, IMR-5, NBL-S, SK-N-FI,
SK-N-SH, SK-N-AS, SH-EP, CHP134, SJ1, NB-1643, DAOY, HeLa and HEK293 were kindly
provided by J.H. Schulte (Pediatric oncology and hematology, Charité Universitätsmedizin
Berlin, Berlin, Germany). H. Deubzer (Pediatric Oncology and Hematology, Charité
Universitätsmedizin Berlin, Berlin, Germany) kindly provided fibroblastic VH7 cells and
F. Buttgereit (Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin,
Berlin, Germany) kindly made HEK293T available. Single clone cell lines of SH-EP and SK-N-AS,
that were already transfected with Tet-repressor (TR) plasmid pcDNATM6/TR, were kindly
provided from J.H. Schulte (Pediatric oncology and hematology, Charité Universitätsmedizin
Berlin, Berlin, Germany). All cell lines were authenticated by short tandem repeat DNA typing
by the IDEXX Bioresearch (Westbrook, ME, USA) and Multiplexion (Heidelberg, Germany).
Furthermore, all cell cultures were tested for the absence of Mycoplasma spp. initially by the
companies and routinely in our laboratory with MycoAlertTM Mycoplasma Detection Kit
(Lonza Group Ltd.). Cell stocks were cryopreserved in liquid nitrogen for our studies (see
section 4.1.5).
4.1.2 Cultivation of neuroblastoma cell lines
Most neuroblastoma cell lines were cultured in a standard medium comprising Roswell Park
Memorial Institute (RPMI)1640 medium supplemented with 1% (v/v)
Methods
41
penicillin-streptomycin (10,000 U/mL) and 10% (v/v) fetal calf serum (FCS). For LAN-1
cultivation, FCS was increased up to 20%. The TR14 and N206 were grown in Dulbecco´s
modified Eagle Medium (DMEM) with 10% FCS, supplemented with 1% penicillin-
streptomycin (10,000 U/mL). The SH-EP-TR and SK-N-AS-TR cells were maintained in standard
medium with additional blasticidin (5 µg/mL) (Thermo Fisher Scientific). SH-EP-TR and
SK-N-AS-TR, that were additionally transfected with pTRex-pDEST30-YAP1(S127A)-2x/3xFlag,
were cultured in selection medium consisting of standard medium with blasticidin (5 µg/mL)
and 600 µg/mL (SK-N-AS-TR-YAP1S127A) or 800 µg/mL (SH-EP-TR-YAP1S127A) geneticin, also
known as G-418 (Biochrom). All cells were incubated in polystyrene cell culture dishes of
different sizes at 37°C, 5% carbon dioxide (CO2) and 21% oxygen (O2) in humidified conditions.
Cells, that were exposed to hypoxic conditions, were incubated in a hypoxia incubator
maintaining 37°C, 1% O2 and 5% CO2. The adjustment of O2 levels was reached by introducing
nitrogen.
4.1.3 Cultivation of non-neuroblastoma cell lines
HEK293, HEK293T and VH7 cells were maintained in DMEM supplemented with 1% penicillin-
streptomycin (10,000 U/mL) and 10% FCS. HeLa cells were cultured in RPMI1640 with the
same additives. The cells were incubated in polystyrene cell culture dishes of different sizes at
37°C, 5% CO2 and 21% O2 in humidified conditions.
4.1.4 Cell passaging
Passaging of adherent cells was performed twice a week. Therefore, the supernatant was
discarded, cells were rinsed once with 1x phosphate buffered saline (PBS) (137 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and Trypsin/ethylenediamineacetic acid (EDTA)
0.05% (Thermo Fisher Scientific) was added with an exposure time of 4-10 min at 37°C. After
detachment of the cells, the reaction was quenched with FCS-containing medium, cells
collected into a falcon and washed with PBS once (300 xg, 5 min). The supernatant was
discarded and a defined volume of fresh prewarmed culture medium was added to the cells.
Collecting 10 µL of the cell suspension, cells were counted with Neubauer chambers or
Methods
42
automated cell counting, respectively (see section 4.1.7). Depending on cell proliferation rate
and cell culture dish size, an appropriate number of cells was transferred into a new culture
vessel containing prewarmed fresh culture medium.
4.1.5 Cell stock preparation
Master cell stocks were prepared for this project which parallelly served for cell line
authentication and as a project cell-line reservoir. Cell freezing was performed at confluency
of about 80%. Cells were detached and washed once with PBS as described earlier. The cell
pellet was resuspended in FCS containing 10% dimethyl sulfoxide (DMSO) (v/v) and 1 mL
aliquots were prepared. Immediately, the cryovials were placed into a freezing container (Mr.
Frosty, Thermo Fisher Scientific) filled with ethanol and stored at -80°C freezer for 24 hours to
ensure a gently freezing with -1°C/min. The frozen vials were placed in a -80°C freezer for
short-term storage or into liquid nitrogen for long-term preservation.
4.1.6 Cell thawing
Cryovials containing cells of interest were placed into 37°C water bath for rapid thawing.
Prewarmed medium was filled into cell culture dishes, the thawed cell suspension was added,
and cells were evenly distributed by gentle movement. After 24 hours, a complete medium
exchange was performed to remove cell toxic DMSO from tissue culture. If selection media
were needed for cell maintenance, those were added at day 2.
4.1.7 Cell counting
The number of viable cells was determined by two different methods. A defined volume of
10 µL of single cell suspension was added by the same volume of trypan blue solution (Thermo
Fisher Scientific), which enters dead or perforated cells and labels them blue. Either for
semiautomated cell counting in the TC20 Automated Cell Counter (Bio-Rad Laboratories) or
cell counting in the Neubauer chamber, the suspension was transferred into appropriate
counting slides. The automated cell counter calculates numbers of live and dead cells, whereas
Methods
43
in the Neubauer chamber bright and unlabeled viable cells in 4 big squares were counted
manually and the total cell number was calculated taking into account the chamber factor 104
and possible dilutions.
4.1.8 Silencing RNA-mediated gene knockdown
The knockdown of YAP1 was accomplished with Silencer®Select RNA Oligonucleotides s20366
(hereafter named siYAP1 #1) and s20367 (hereafter named siYAP1 #2) targeting YAP1. As
scramble controls, we used Silencer®Select Negative Control (NC) #1 and #2. Every silencing
RNA (siRNA) was obtained from Thermo Fisher Scientific and administered at a final
concentration of 40 nM. Transfection of the cells with siRNAs was performed with the `Fast-
forward’ lipid transfection method. For knockdown experiments in 12-well format, either 2 µL
siRNAs [20 µM] or equal volume of medium as transfection control (mock) was pre-mixed with
200 µL reduced medium Opti-MEM (Thermo Fisher Scientific). Afterwards, 2 µL of
Lipofectamine 3000 (Thermo Fisher Scientific) were added and mixed vigorously. After 15 min
incubation, during which membrane-permeable lipid-siRNA-complexes were formed, 200 µL
of the mixture was added into a microtiter plate well and topped with 800 µL of cell suspension
containing a defined cell number. Transfected cells were incubated in standard culture
conditions. For assays performed in 96-well microtiter plates, one tenth of all volumes was
used to gain a final volume of 100 µL per well. After 24 hours, the medium was exchanged
against standard medium. The assay was performed with 104 cells per well in 96-well
microtiter plates or with 1.5x105 cells per well in 12-well microtiter plates.
4.1.9 Lentiviral cell transduction with short hairpin RNA
The cell lines SH-EP, GI-M-EN, SH-SY5Y and IMR-5 were plated 24 hours prior to transduction,
either in 96-well microtiter plates with 103 cells/well for cell viability assay (technical
triplicates) or in 12-well microtiter plates with 105 cells/well for messenger RNA (mRNA)
analysis. Polybrene (Sigma Aldrich) and the virus particles were thawed on ice and
subsequently added to transduction medium for the cells (Table 16) reaching a final
concentration of 0.1 µL virus per 103 cells. Polybrene was titrated in advance for each cell line
Methods
44
and used in different final concentrations: 3 µg/mL (IMR-5), 5 µg/mL (SH-SY5Y, GI-MEN) or 6
µg/mL (SH-EP). For lentiviral transduction, the supernatant from the cells was carefully
removed and replaced with 100 µL (96-well) or 1 mL (12-well) lentivirus-containing
transduction medium. One day post transduction, growth medium was replaced with fresh
transduction medium and cells were incubated in standard cell culture conditions for another
72 hours until harvest or cell viability assay.
Table 16 Components of lentiviral transduction medium
Transduction medium for neuroblastoma cell lines
RPMI 1640 (4.5 g/L Glucose)
10% (v/v) FCS
1% (v/v) penicillin-streptomycin (10,000 U/mL)
1% (v/v) Non-essential amino acids (NEAA) (Biozym Scientific GmbH)
1% (v/v) Glutamine 200 mM (GibcoTM Thermo Fisher Scientific)
4.1.10 Generation and YAP1S127A activation of neuroblastoma cell lines SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A
Analysis of overactive YAP1 was performed in 2 established neuroblastoma cell lines, SH-EP
and SK-N-AS. Therefore, these cell lines were transfected with a tetracycline-inducible (Tet-
ON) expression system for constitutively active YAP1S127A and analyzed in regard to the
function and target gene expression in different approaches.
4.1.10.1 Plasmid transfection of cells with pTRex-DEST30-YAP1(S127A)-2x/3xFlag
The Tet-repressor expressing cell lines SK-N-AS-TR and SH-EP-TR, that originate from single
clone cultures transfected with pcDNATM6/TR, where additionally transfected with either
pT-Rex-DEST30-YAP1(S127A)-2x/3xFlag or with the empty control vector pT-Rex-pDest30.
At 70 – 80% confluency, the cells were harvested with Trypsin/EDTA 0.05% as described
earlier, washed twice with PBS and centrifuged with 300 xg for 5 min. Cell pellets of 1.6x106
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cells were diluted in 500 µL Opti-MEM mixed with 30 µg plasmid DNA, transferred to
appropriate cuvettes and electroporated with a square wave impulse for 20 ms and 200 V in
a GenePulser Xcell (Bio-Rad Laboratories). Immediately after electroporation, cells were
topped with pre-warmed fresh medium and cautiously transferred into a cell culture dish with
pre-warmed fresh standard medium containing 10% FCS and blasticidin. Afterwards, the cells
were cultured in standard tissue culture conditions for 3 days. Successfully transfected cells
started to be selected 72 hours post electroporation by supplementation of the standard cell
culture medium with G-418 (SH-EP-TR-YAP1S127A 800 µg/mL, SK-N-AS-TR-YAP1S127A 700
µg/mL). Selection pressure was perpetuated for all experiments as well as maintenance tissue
culture.
4.1.10.2 Single clone selection of plasmid-transfected cells
Aim of the single clone selection was the reduction of cell heterogeneity of plasmid
transfected cells. Therefore, two 96-well microtiter plates were plated with 0.1 cells/well or
0.5 cells/well per cell line and plasmid. Plates were checked every day for smallest colonies
most likely originating from one cell. About 20 selected clones per cell line and vector were
cultivated up to a 6-well format. After harvesting the cells, half of it was used for detection of
tetracycline-induced YAP1S127A synthesis (western blot) and half was maintained in tissue
culture. Single clone colonies, hereinafter indicated by hash (#), with a strong YAP1 induction
upon tetracycline-treatment and a low background expression of YAP1 were chosen to be
cultivated, cryopreserved and used for the following studies. For the empty vector controls
transfected with pTRex-pDEST30 plasmid backbone, single clones were chosen without any
further tests.
4.1.10.3 Activation of YAP1S127A synthesis with tetracycline
Overexpression of YAP1S127A in pcDNATM6/TR and pT-Rex-DEST30-YAP1S127A-2xFlag/3xFlag
double transfected cells was performed by addition of 4 µg/mL ethanol-diluted tetracycline
(Sigma Aldrich) to the cell culture medium. Tetracycline-treated cells will be declared as +Tet
in this study. With regard to possible side effects of the solvent ethanol, equal volume of
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ethanol was applied to cells of a control group (-Tet) and cultivated and analyzed
simultaneously in all experiments. The tetracycline was used in a highly concentrated
predilution of 15 mg/mL to minimize side-effects of ethanol in all experiments. To consider
possible YAP1-independent effects of tetracycline-treatment on neuroblastoma cell
metabolism, a second control group of cells that only harbors the backbone of the
overexpression plasmid without the cDNA for human YAP1(S127A), hereafter referred to as
LV, was included in most experiments.
4.2 Functional in vitro assays
4.2.1 Cell viability assay
The analysis of cell viability was performed with CellTiter-Glo® Luminescent Cell Viability
Assays (Promega). Measurement principle of this assay is the proportionality of the metabolic
active viable cell number per well to the ATP amounts being released after cell lysis. This assay
was performed with 104 cells/well in light-protective white 96-well flat bottom microtiter
plates added with 100 µL medium per well. Previous YAP1 perturbations were performed in
the same plates, 72 hours prior to cell viability assessment. For cell viability measurement,
freshly mixed reagent for cell lysis and luciferase reaction was added to the cell medium 1:10
according to manufacturer’s protocol and the microtiter plate was shaken in an orbital shaker
for 12 min at RT. The luminescence signal was detected with a GloMax® Multi Detection
System (Promega). All experiments were performed in technical triplicates and the signal of
three control wells containing the equivalent medium without cells was subtracted from each
well. Every single agent added to cell culture such as antibiotics, ethanol or assay reagents
were tested separately and did not affect the luminescence values.
4.2.2 Colorimetric cell proliferation immunoassay (BrdU-ELISA)
The cell proliferation was assessed by a colorimetric Cell Proliferation ELISA (Sigma Aldrich)
assay. The thymine-analog 5-bromo-2-deoxyuridine (BrdU) is incorporated into freshly
synthesized DNA of proliferating cells and can be detected by peroxidase-labeled anti-BrdU
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antibodies in the fixed and subsequently lysed cells. The peroxidase catalyzes the reaction of
added 3,3,5,5-tetramethylbenzidin (TMB) into blue diimin, which can be detected in a
spectrophotometer. The amount of diimin is proportional to the implemented BrdU
molecules.
Per well, 103 cells/well were plated into 96-well microtiter plates, treated according to the
experimental setup with transfection reagents, siRNAs, tetracycline or standard cell culture
medium for 72 hours. Afterwards, the BrdU ELISA assay was performed after manufacturer’s
instructions. Thereby, the optimal BrdU labeling time period for neuroblastoma cells was
tested and determined at 2 hours. Readout was performed exactly 15 min after TMB
application with the Epoch Plate Reader (Bio Tek Instruments) at a wavelength of λ=370 nm
with a reference wavelength of λ=492 nm. Technical triplicates of three independent
experiments were measured and analyzed with appropriate blank and background controls.
4.2.3 Photometric apoptosis immunoassay (DNA-histone ELISA)
The apoptosis rate of YAP1 knockdown cells was examined by relative quantification of
cytoplasmic histone-bound DNA fragments with the Cell Death Detection ELISAPLUS (Thermo
Fisher Scientific). In this sandwich enzyme-linked immunosorbent assay (ELISA) system, DNA-
histone complexes are bound by immobilized anti-histone antibodies and detected by
peroxidase-labeled anti-DNA antibodies, which can be visualized by the violet color of the
oxidized substrate 2,2-azinodi-3-ethylbenzthiazoline sulfonate (ABTS).
For the siRNA mediated knockdown of YAP1, 1.5x105 cells of SK-N-SH, SK-N-BE, SK-N-FI and
IMR-5 were plated in 12-well microtiter plates and transfected ‚fast forward’ with siRNAs (see
section 4.1.8). Supernatants were removed after 72 hours and cells lysed with 400 µL lysis
buffer for 30 min at RT. According to the manufacturer’s instructions, the Cell Death Detection
ELISAPLUS was performed with 20 µL of the cytoplasmic fraction of the cells. Finally, 100 µL of
the ABTS stop solution interrupted the oxidation of the substrate catalyzed by the peroxidase
and the plate was measured at a wavelength of λ=405 nm with Epoch Plate Reader (Bio Tek
Instruments). Appropriate and recommended positive and negative controls accompanied the
technical duplicates. The enrichment factor was calculated as ratio between the siRNA-treated
samples and the untreated negative controls.
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4.2.4 Flow cytometry analysis of apoptotic cells
The detection of apoptotic cells in a cell population can also be carried out via flow cytometry.
Light-emission of extra- or intracellular binding fluorochrome-labeled antibodies or directly
cell-component binding fluorophores can be detected by photomultipliers. Fluorescence
Associated Cell Sorting (FACS) analysis provides information about cell morphology, protein
abundance and other cell characteristics can be obtained on single-cell level.
Here, staining of the cell DNA with intercalating fluorophores such as propidium iodide (PI) or
7-amino-actinomycin D (7-AAD) and labeling of apoptosis-induced cell-surface
phosphatidylserine with fluorochrome-labeled Annexin V was used to distinguish viable,
apoptotic and dead cells.
Determining the influence of a YAP1 knockdown on cell apoptosis, 1.5x105 SK-N-SH cells were
transfected with siRNAs and incubated for 72 hours prior to the FACS analysis. Aiming for the
preservation of intact cell structure, the enzyme-mix Accutase (Sigma Aldrich) was used for
cell detachment. Cells were washed twice in PBS and centrifuged with 300 xg for 5 min. The
pellets were resuspended in 100 µL 1x Annexin buffer (Thermo Fisher Scientific) and incubated
light-protected with a final concentration of 1 µg/mL PI and 5 µL Annexin V Alexa Fluor 488
(Dead Cell Apoptosis Kit with Annexin V Alexa FluorTM 488 and PI, Thermo Fisher Scientific) for
15 min. After addition of 400 µL 1x Annexin buffer, cells fluorescent for PI and
Alexa Fluor™ 488 were detected with BD LSRFortessaTM X20 (BD Biosciences). Here,
FCS-A
SSC-A
FCS-A
FCS-H
SSC-A
SSC-H
Comp:AnnexinV-APC
Comp:7-AAD
Figure 2 Gating strategy for FACS analysis of apoptosis. Cells stained for 7-AAD and Annexin V were analyzed via exclusion
of doublets over signal heights in the forward scatter (FCS-H) and sideward scatter (SSC-H) and, consecutively, measured in
the compensated signals for 7-AAD (Comp:7-AAD) and Annexin V-APC (Comp: Annexin V-APC). 7-AAD and Annexin V double
positive cells were interpreted as dead cells and Annexin V single positive ones as apoptotic cells. Double positive cells
defined the viable population.
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fluorophores were excited by 488 nm laser and detection filters 695/40 and 530/30 were used
to detect PI and Alexa Fluor™ 488, respectively.
For YAP1 activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells, 1x106 cells per
condition were treated with tetracycline or ethanol for 72 hours and processed such as
described for SK-N-SH. Staining was performed with 5 µL 7-AAD and 5 µL Annexin-V-APC from
APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) after manufacturer’s
instructions and also detected with BD LSRFortessaTM X20. Red laser (640 nm) and 670/14
detection filter were used for detection of APC.
In order to analyze the proportion of GPF-positive SH-EP cells transduced with YAP1-targeting
shRNAs, 104 cells were lentivirally transduced with 0-10 µL virus (see section 4.1.9) and
harvested after 72 hours in ice-cold PBS. Dead cells were marked by addition of 5 µL 7-AAD
solution of APC Annexin V Apoptosis Detection Kit. Fluorescence of 7-AAD and GFP was excited
with 488nm laser and detected with the detection filters 695/40 and 530/30, respectively.
Data analysis with the software FlowJo V10 (Becton, Dickinson & Company) started with
exclusion of cell doublets and debris in the mean of signal height and signal area in the
sideward-scatter and forward-scatter to guarantee a single-cell analysis (Figure 2). For the
purpose of apoptosis detection, double positive cells for PI/7-AAD and Annexin V were
declared as dead and Annexin V (Alexa Fluor 488™/APC) single positive cells were assigned as
apoptotic cells.
4.2.5 Cell cycle analysis
Actively dividing cells run through a four-phase cell cycle: a DNA-synthesis (S)-phase, when
DNA is doubled, a mitosis (M)-phase for division of the cells and two gap phases (G1, G2). DNA
content is different in those phases and can be detected with an DNA-intercalating
fluorophore such as PI or 7-AAD.
Here, the influence of a YAP1 perturbation in neuroblastoma cells on their cell cycle was
analyzed. Therefore, a YAP1 knockdown was performed in one cell line SK-N-SH with 1x105
cells/condition. 72 hours post transfection, cells where gently harvested with enzyme-mix
Accutase and centrifuged with 300 xg for 5 min at 4°C together with the medium containing
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already detached dead cells. The cell pellet was rinsed with ice-cold PBS twice and afterwards
cells were fixed by dropwise addition of 200 µL ice-cold 70% ethanol while vortexing. After an
incubation for 30 min on ice, residues of ethanol were removed by washing cells with PBS
twice. Addition of 30 µL RNase A [100 µg/mL] (Qiagen) ensured the deletion of interfering
RNAs. After 30 sec incubation, 300 µL PI-staining solution (50 µg/mL PI in PBS) was added.
The activation of YAP1S127A protein in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A was
performed by addition of 4 µg/mL tetracycline on 5x105 cells, 72 hours prior to cell cycle
analysis. Empty vector clones and ethanol controls were treated contemporaneously. All cells
were processed and stained such as described for SK-N-SH.
The DNA content was detected via FACS analysis with BD LSRFortessaTM X20. Data was
analyzed with FlowJo V10 by its DNA/cell cycle analysis task after exclusion of doublets and
debris (see section 4.2.4).
4.2.6 Wound healing assay
The wound healing assay provides information about motility of cells. Neuroblastoma cells
SK-N-SH, SK-N-BE, SK-N-FI and IMR-5 were seeded with 1.5x105 cells/ well into 12-well
microtiter plates and transfected with siRNAs. After 72 hours, the standard culture medium
was exchanged to a starvation medium (RPMI1640, 2% FCS, 1% penicillin-streptomycin
(10,000 U/mL)). Next day, the initial cell-free region (wound) was inflicted with a 100 µL pipet
tip through the center of the cell monolayer. A PBS rinse ensured the removement of detached
cells to prevent a re-attachment. The cells were topped up with starvation medium and
pictures of the initial scratch (0 hours) were taken with the 5x objective of Olympus
microscope BX43 (Olympus) with camera attachment (Zeiss). Pictures of the cell-free region
were taken after 14 hours, 24 hours and 48 hours.
The wound healing assay was also performed with YAP1-activated SH-EP-TR-YAP1S127A cells
and empty vector controls. Therefore, 1.5x105 cells/well were plated into 6-well microtiter
plates and treated for 48 hours with tetracycline or ethanol. After reaching a confluency of
about 70%, the standard culture medium was also exchanged against a starvation medium
with 2% FCS only, one night prior to the initial “wound” inflict. The scratch was performed as
described earlier and pictures of initial cell-free regions were taken like described above.
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Data analysis was performed with ImageJ32 1.49v (National Institutes of Health, NIH). Wound
edge distances were measured at 3 different points per wound in 3 independent experiments
with technical duplicates and is depicted as a fold change of distances at given timepoints
compared to the initial scratch.
4.2.7 Transwell migration assay
Migration of cells through a transwell membrane requires migratory potential together with
therefore required cell skeleton rearrangements. In comparison to the wound healing assay,
it diminishes effects of cell proliferation and factor release upon cell damage through wound
initiation.
YAP1 was pre-activated in Tet-ON YAP1-inducible cells 72 hours prior to the transwell
migration assay. Therefore, 1x105 SH-EP-TR-YAP1S127A and 1x106 SK-N-AS-TR-YAP1S127A as well
as empty vector controls were plated into 10 cm dishes and treated with 4 µg/mL tetracycline
or equal volumes of ethanol. Cells reached a confluency not higher than 80% and were
harvested and counted using the semiautomatic cell counter (see section 4.1.7).
The assay was performed with 12-well transwell polyethylene terephthalate (PET)
membranes, that are 11 µm thick with a pore size of 8.0 µm and were placed into a standard
12-well microtiter plate. The bottom of the well was filled with 750 µL of 10% FCS-containing
clone-specific medium including either tetracycline for YAP1-activated cells or ethanol as
control. The transwell membrane was placed into the chamber and 250 µL cell suspension
containing 1x105 cells were added carefully into the upper part of the Boyden chamber. The
cell suspension was prepared with the same medium like in the bottom part, but without FCS,
so that active cell migration was triggered by the serum. Subsequently, cells were incubated
in standard culture conditions for 20 hours (SH-EP-TR-YAP1S127A) or 24 hours (SK-N-AS-TR-
YAP1S127A).
To visualize the migrated cells, they were fixed and stained with crystal violet. Therefore, the
transwell inlet was removed from the chamber and inverted to remove media and detached
cells. After washing the membrane twice by dipping into PBS-filled glass trays, it was placed
into a new 12-well microtiter plate and all cells on both sides were fixed with 4% (v/v)
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formaldehyde solution (Carl Roth) for 2 min at RT. Again, the membrane was washed twice in
PBS and transferred into a 12-well filled with 0.2% (w/v) crystal violet (Sigma Aldrich) staining
solution (in 10% (v/v) ethanol) for 6 min at RT. Cells were rinsed twice in PBS and gently
scraped off from the top of the membrane with a cotton swab, so that just the migrated cells
on the bottom still stuck to the membrane.
Per condition and membrane, five pictures of the membrane bottom (one: center, four: sides)
were taken with a light microscope with camera attachment and quantitatively analyzed with
ImageJ 32 software by measurement of the blue areas populated by migrated cells (regions of
interest, ROI).
4.2.8 Colony formation assay
Colony formation assay determines the cell division potential of single cells by displaying the
number and size of colonies formed out of these single cells. Per well and condition, 500 single
cells of YAP1-inducible SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A clones as well as their
empty vector control cells were seeded into a 6-well microtiter plate. YAP1S127A
overexpression was induced by tetracycline treatment and medium of control cells was
supplemented with the solvent ethanol. Medium of all cells was exchanged twice a week.
After 6 days (SH-EP) or 8 days (SK-N-AS), medium was discarded, and cells were washed with
PBS once. Cells were covered with 1.5 mL of 4% (v/v) formaldehyde solution and fixed for 15
min at RT on a slow-moving rocking shaker. Cells were washed with distilled water once and
stained with a 1% (w/v) crystal violet solution (in 10% (v/v) ethanol) for 20 min at RT on the
rocking shaker. Staining solution was discarded afterwards and cells were washed three times
with distilled water and air dried. Pictures were taken with a photocopy machine. For assay
analysis, blue spots were quantified with ImageJ32 software by measurement of ROI. Data
were collected in 4 independent experiments.
4.2.9 Serum starvation of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
The YAP1-inducible cell lines SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A were seeded into
white flat bottom 96-well microtiter plates with 1x104 cells/well and preincubated with
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4 µg/mL tetracycline or the same volume of ethanol. The medium was exchanged 24 hours
later, and cells were re-covered with RPMI1640 medium containing Blasticidin (5 µg/mL), 1%
penicillin-streptomycin (10,000 U/mL) and given volume of FCS, added with either 4 µg/mL
tetracycline for YAP1 activation or the same volume of ethanol as solvent control. For both
groups, three different serum concentrations were used: standard 10% as control as well as
2% and 0% as serum starvation media. Cells were incubated for another 72 hours in standard
tissue culture conditions and CellTiter-Glo® Assay was performed afterwards as described in
section 4.2.1.. All conditions were measured in technical triplicates and in three independent
experiments.
4.2.10 Detection of senescence via β-galactosidase staining
Senescence is the irreversible growth arrest of cells and can be detected rapidly and simply by
the indirect detection of the pH-dependent senescence-associated β-galactosidase (SA-β-Gal).
The substrate 5-bromo-4-chlor-3-indolyl β-D-galactopyranoside (X-Gal) can be hydrolyzed by
the SA-β-Gal of senescent cells and turns into blue color, which eventually can be visualized
via light microscopy (Dimri et al., 1995). Here, 1x103 SH-EP-YAP1S127A and 1x104 SK-N-AS-
YAP1S127A were seeded into 6-well microtiter plates in the standard YAP1S127A-clone media and
concurrently treated with tetracycline or ethanol as control. Medium together with treatment
agents was changed twice a week and after a total period of 14 days, the Senescence
β-galactosidase Staining Kit (Cell Signaling Technology) was used in accordance with the
manufacturer’s protocol for detection of YAP1-induced cell senescence. After fixation of the
cells for 13 min and washing twice with PBS, cells were incubated in 1 mL/well β-galactosidase
staining solution overnight in the dry incubator with plates sealed with parafilm. Pictures were
taken with the 20x or 40x objective of an Olympus microscope BX43 with camera attachment
next day.
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4.2.11 Measurement of pH value, glucose and lactate concentration in supernatants of in
vitro cell cultures
The analysis of the pH value, glucose and lactate concentration in growth media of YAP1-
activated or YAP1 silenced cells was performed by utilizing a blood gas analysis machine, that
is normally used for patient blood analysis. By the kind permission of Katrin Zeilinger (Berlin-
Brandenburg School for Regenerative Therapies, BSRT), the aspired data were collected at a
blood gas analysis machine ABL 800 BASIC (ABL).
Supernatant of siRNA transfected neuroblastoma cell lines SK-N-SH, SK-N-BE, IMR-5 and
SK-N-FI or YAP1-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells and appropriate
controls was collected 72 hours after YAP1 perturbation, immediately frozen on dry ice and
stored at -80°C until analysis. Directly before the measurement of pH value, lactate
concentration and glucose levels, samples of three independent replicates per condition
where thawed on ice and transferred to the machine according to manual instructions.
4.3 Molecular biology methods
4.3.1 Gateway® Cloning of pDEST30-YAP1(S127A)-2xFlag/-3xFlag for overexpression of
constitutively active YAP1
Figure 3 Gateway cloning strategy. The target cDNA is transferred from an attL-site carrying entry-clone into the attR-site
of a destination vector via the fast LR Clonase reaction. The ccdB gene, which is located in the attR site within the original
destination plasmid, additionally prevents the growth of empty vector transformed bacteria cells and thereby functions as a
selection marker.
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The entry plasmids pENTR-3xFLAG-YAP1-S127A (Addgene #42239, gifted by W. Pu) and
pENTR1A-2xFlag-YAP1-S127A (Addgene #46050, gifted by X. Chen) are entry-clones (Figure 3)
that carry a modified human YAP1 cDNA and were obtained from Addgene. The cDNA encodes
the protein YAP1 with an exchange of serine to alanine at position 127 to prevent the
phosphorylation of the serine127, which in turn leads to nuclear translocation provoking the
constitutive activation of the protein. The target cDNA was cloned into the gene-expression
vector pT-Rex-DEST30 with the LR Clonase Enzyme-Mix II from Gateway cloning system
(Thermo Fisher Scientific) according to manufacturer´s specifications. Here, 300 ng of each
plasmid was mixed with 6 µL of 1x TE buffer and 2 µL of LR clonase enzyme mix and incubated
for 1.75 hours at RT. Addition of 2 µL proteinase K for 10 min at 37°C terminated the reaction.
A volume of 50 µL electro competent Escherichia Coli (E.Coli) cells were transformed with 1 µL
of clonase-DNA-mix by use of electroporation.
After plasmid DNA extraction from small-scale cultures, 1 µg of the plasmids was incubated in
rCutSmart buffer™ with 1U of the restriction enzymes XhoI and ApaI (NEB) for 1 hour and
checked with agarose gel electrophoresis for the presence of expected DNA band patterns.
The exact insertion and direction of the YAP1 cDNA was verified by plasmid DNA sequencing
(Eurofins Genomics GmbH, Ebersberg, Germany) with primer CMV.for and M13uni(-21) and
sequence alignment with BLASTN (National Center of Biotechnology Information, NCBI).
Extraction of the plasmid DNA from large-scale bacteria cultivation (see section 4.3.7) was
performed with NucleoBond Maxi-Kit (Machery&Nagel) according to manufacturer’s
instructions. Prior to transfection of neuroblastoma cell lines, DNA sequences were validated
via DNA sequencing and sequence analysis via BLASTN.
4.3.2 Cloning of shRNA vectors for lentiviral transduction
Lentiviruses for shRNA delivery were composed of the 3rd generation transfer plasmid
pLKO.1-puro backbone (scramble shRNA was a gift from David Sabatini), packaging vector
psPAX2 (kindly provided by F. Buttgereit, Rheumatology and Clinical Immunology, Charité
Universitätsmedizin Berlin, Berlin, Germany) and VSV-G envelope-expressing plasmid pMD2.G
kindly provided by A. G. Henssen (Experimental and Clinical Research Center, ECRC, Berlin,
Germany). The pLKO.1-scramble-shRNA-vector (Addgene, #1864) was modified into a pLKO.1-
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shRNA-puro-T2A-eGFP, in which shRNA is place holder for scramble shRNA or YAP1-targeting
shRNA. Therefore, in the pLKO.1-scramble-shRNA vector the puromycin resistance gene was
replaced by a puromycin-T2A-eGFP cassette, which was generated by overlap extension PCR
using puromycin and T2A-eGFP cassettes as templates. The exchange was performed by
BamHI and Acc651 restriction and subsequent ligation of that PCR product and the pLKO.1-
scramble-shRNA vector. In order to insert desired shRNAs without using EcoRI, which was
present in the new cassette, an AgeI-XBaI-EcoRI-linker was introduced into the pLKO.1-
puromycin-T2A-eGFP between U6 promoter and central polypurine tract/ central termination
sequence (cPPT/CTS) site. Reverse complementary binding oligonucleotides containing
shRNAs and the already cut forms of the restriction enzyme sites AgeI (ACCGGT) and XbaI
(TCTAGT) were ordered and ligated into the AgeI and XbaI restricted vector. The sequence of
scramble shRNA was used from the original scramble shRNA clone, whereas shRNAs for YAP1
knockdown were chosen from plasmids gifted by Kunliang Guan (shYAP1_1:Addgene plasmid
#27368; shYAP1_2: Addgene plasmid #27369)(Zhao et al., 2008). Plasmid maps can be
obtained from the appendix.
4.3.3 Preparation of chemically competent bacteria cells
For preparation of chemically competent cells, 50 mL Lysogeny Broth (LB)-medium
supplemented with 34 μg/mL chloramphenicol and 10 mM MgCl2 was inoculated with XL10-
Gold ultracompetent E. coli cells (Agilent Technologies, Inc.) and gently shaken overnight at
37°C. Next morning, 0.5 mL of the overnight culture were added to 300 mL LB-medium
supplemented with chloramphenicol and MgCl2 and gently shaken for 3-4 hours at 37°C, until
the bacteria cell suspension reached an optical density of OD of 0.5 at 600 nm wavelength.
OD was measured repeatedly in a Biophotometer (Eppendorf). The bacteria culture was
harvested into six 50 mL reaction tubes and placed on ice for 15 min. Afterwards, cell
suspension was spun down at 4°C with 590 xg for 15 min. The supernatant was discarded, cell
pellets were resuspended in 1.5 mL transformation buffer (10 mM MgCL2, 10 mM MgSO4, 5%
(v/v) DMSO, 10% (m/v) PEG 3000 in LB-medium) and aliquots were stored at -80°C. The
preparation of chemically competent E. coli was kindly provided by Annika Winkler.
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4.3.4 Transformation of E. coli cells
E. coli were transformed with plasmids for shRNA-mediated YAP1 knockdown via heat-shock
transformation. The ligation mix of shRNA oligonucleotides and pLKO.1-puromycin-T2A-eGFP
vectors was added with 200 µL of the chemically competent E. coli cells and incubated for
30 min on ice. In order to transform the cells by heat shock, they were incubated at 42°C for
90 s followed by incubation on ice for 2 min. Then, 800 µL super optimal broth with catabolite
repression (SOC) medium (2% (m/v) tryptone, 0.5% (m/v) yeast extract, 10 mM NaCl, 10 mM
MgCl2, 10 mM MgSO4, 20mM glucose) were added per reaction and, to allow recovery, cells
were gently shaken at 37°C for 1 h. The cell suspension was centrifuged at 2350 xg for 2 min
and supernatant was discarded. The cells were resuspended in left over supernatant and
equally spread onto ampicillin-supplemented (100 µg/mL) LB-agar plates (1% (w/v) NaCl, 1%
(w/v) tryptone, 0.5% (w/v) yeast extract, 1.5% (w/v) Bacto Agar) using glass beads. Agar plates
were incubated at 37°C overnight.
The transformation of E. coli cells with the Tet-ON YAP1 overexpression plasmids was realized
via electroporation. After inactivation of the LR clonase enzyme mix with Proteinase K for
10 min at 37°C, 50 µL of electro competent E. coli Top10 (NEB) were transformed with 1 µL of
the plasmid mix via electroporation and incubated for 1 hour at 37°C in super optimal broth
(SOC) medium (0.5% (w/v) yeast extract, 2% (w/v) tryptone, NaCl 10mM, KCl 2.5 mM, MgCl2
10 mM, MgSO4 10 mM). For overnight incubation at 37°C, 50 µL of the suspension were plated
on LB-agar plates supplemented with 100 µg/mL ampicillin.
After cell transformation, single colonies were transferred into 3 mL LB-medium with a sterile
toothpick and incubated in an orbital shaker for at least 2 hours and DNA was extracted (see
section 4.3.6). In addition, all chosen clones were parallelly transferred onto a replica LB-agar
plate. After insert validation by agarose gel electrophoresis and subsequent DNA sequencing,
clones carrying the correct inserts were picked from the replica LB-agar plate in order to
prepare large scale bacteria culture of 100 mL volume.
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4.3.5 Agarose gel electrophoresis
Plasmid DNA was cut with restriction enzymes and DNA fragment sizes were checked in
agarose gel electrophoresis for an expected pattern. For 2% agarose gels, 2 g agarose powder
in 100 ml 1xTAE-buffer (Tris-base 40 mM, acetic acid 20 mM, EDTA 1 mM) were heated,
ethidium bromide was added, and the gel was hand casted with appropriate combs. For gel
electrophoresis, samples were probed with 2 μL DNA loading dye Orange G (6x; Thermo Fisher
Scientific), the gel was placed into a 1xTAE-buffer-filled gel chamber (Horizontal mini gel
electrophoresis system, VWR) and samples as well as 5 µL molecular mass standard GeneRuler
1000bp (Thermo Fisher Scientific) were run at 90 V for about 90 min.
4.3.6 Small scale plasmid DNA extraction
The plasmid DNA extraction from small-scale cultures of transformed E. coli was performed
with buffers from the QIAprep Spin Miniprep kit (Qiagen) without usage of the separation
columns. Cell comprised in 1.5 mL of the overnight bacteria culture were pelleted in a 1.5 mL
reaction tube with 2350 xg for 2 min at RT. The pellet was resuspended in 200 µL of buffer P1
and placed on ice. 200 µl of buffer P2 were added and the sample was mixed by inversion,
immediately. The reaction was completed by addition of 200 µL of buffer P3, followed by
repeated inversion and centrifugation for 10 min at 4°C at full speed to pellet the debris.
Meanwhile, 800 µL of isopropanol (Carl Roth) were placed into a new 1.5 mL reaction tube
and 600 µL of the sample supernatant were added and hold on ice for 10 min. After
centrifugation for 30 min at 4°C and full speed, supernatant was discarded, and the pellet was
resuspended in 500 µL 70% ethanol. Afterwards, DNA was pelleted again with high speed at
4°C for 10 min and supernatant was discarded. Removal of residual supernatant was ensured
by second spin down and the DNA-pellet was air-dried under a fume hood. Finally, the pellet
was resuspended in 30 µL 0.1% TE-buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and stored at
4°C. Plasmid DNA from small-scale DNA extractions was used for DNA sequencing or
retransformations of E. coli.
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4.3.7 Large scale plasmid DNA extraction
Plasmid isolation from large-scale bacteria cultures was performed with the column based
NucleoBond® Xtra Midi/Maxi Kit (Macherey-Nagel) according to manufacturer’s instructions.
All vectors used and extracted for this project (backbones of pENTR1A, pENTR3C, pTRex-
pDEST30, psPAX2, pMD2.G and pLKO.1) were high-copy plasmids. Therefore, we used the low
amount of buffer for large-scale plasmid DNA extraction, as recommended by Macherey-
Nagel. The DNA precipitation was performed following the NucleoBond® Xtra Maxi protocol.
The final DNA pellet was resuspended in 0.5 mL 0.1% TE-buffer.
4.3.8 Production and titration of lentiviral particles
4.3.8.1 Calcium-phosphate precipitation transfection of HEK293T cells for lentivirus
production
The human embryonic kidney cell line HEK293T contains the SV40 T-antigen and thus is able
to replicate vectors carrying the SV40 origin of replication such as the above-mentioned
plasmids. For lentivirus production, 5x106 HEK293T cells were seeded into 10 cm diameter cell
culture dishes 24 hours prior to transfection. In a 1.5 mL reaction tube, 10 µg of psPAX2, 10 µg
pMD2.G and 20 µg pLKO.1-puro-shSCR or pLKO.1-puro-shYAP1 DNA were mixed and topped
up to 540 µL volume with 2.5 mM HEPES buffer (Sigma Aldrich). Afterwards, 60 µL 2.5 M
calcium chloride (CaCl2) were added. A volume of 600 µL of 2x HeBS solution (50 mM HEPES,
280 mM NaCl, 1.5 mM Na2HPO4) was preloaded into a 15 mL reaction tube. The DNA/CaCl2
solution was added dropwise to the HeBS buffer. The mixture was vortexed vigorously in
between. Afterwards, the DNA/CaCl2/HeBS solution was incubated for 30 min at RT for the
calcium-phosphate (CaPO4)/DNA precipitate formation. Meanwhile, the medium of pre-
cultured HEK293T cells was removed and 12 mL of fresh transfection medium (Table 17)
including chloroquine were administered.
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Table 17 Components of transfection medium for HEK293T cells
Transfection medium
DMEM (1 g/L glucose)
2% (v/v) L-glutamine
2% (v/v) HEPES buffer 0.5 M
0.1% (v/v) chloroquine 25mM
10% (v/v) FCS
1% (v/v) penicillin-streptomycin (10,000 U/mL)
1% (v/v) Amphotericin B
1% (v/v) sodium pyruvate
The solution containing the DNA/CaPO4 precipitates was added dropwise and the cells were
incubated overnight in standard culture conditions. After 16 hours, supernatant was
discarded, cells were washed once with 5 mL 1xHeBS buffer and incubated with 2 mL of
glycerol (Merck Millipore) solution (15% glycerol in HeBS) for exactly 2 min. Glycerol solution
was discarded afterwards, cells were rinsed with 5 mL 1xHeBS and 12 mL fresh growth
medium (Table 18) was added for incubation in standard culture conditions. Transfected cells
were handled very carefully due to increased tendency to detach.
Table 18 Components of HEK293T post transfection growth medium
Growth medium for transfected HEK293T cells
DMEM (4.5 g/L Glucose)
10% (v/v) FCS
1% (v/v) penicillin-streptomycin (10,000 U/mL)
1% (v/v) Non-essential amino acids (NEAA)
1% (v/v) L-glutamine
After 24 hours, the lentivirus-containing supernatants were harvested and after filtration
through 0.45 µm pore size membrane filters to get rid of cells and debris, virus was collected
in 15 mL Amicon® Ultra Centrifugal Filters (Merck). The samples were centrifuged for 30 min
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at maximum speed and 4°C to concentrate the virus in about 200 µL volume. The virus was
aliquoted and stored immediately at -80°C.
4.3.8.2 Titration of lentiviral particles in neuroblastoma SH-EP cells
Transduction efficiency of produced lentiviral particles was tested and optimized in SH-EP
cells. Therefore, the above-mentioned transduction protocol (see section 4.1.9) was
conducted and cells were analyzed via microscopy and FACS analysis as described above.
Distinction between living and dead cells was conducted by addition of 5 µL 7-AAD
(BioLegend).
Working with 1 µL virus per 104 cells has been shown to gain the best ratio between GFP-
positive and dead cells after 72 hours (Figure 4).
Figure 4 Transduction efficiency of lentivirus carrying shRNA targeting YAP1 in neuroblastoma cells. (A) GFP-positive SH-
EP cells at day 3 after lentiviral transduction with 1 µL virus solution (B) FACS-analysis of SH-EP cells, 72 hours post
transduction with 0 – 10 µL of lentivirus carrying shRNAs against YAP1 (green) or scramble shRNA (black). Best ratio between
positive and living cells was reached by application of 1 µL virus volume (black arrow) transducing 104 cells.
4.3.9 RNA extraction
Cellular RNA was isolated and transcribed into cDNA for the analysis of endogenous and
differential gene expression of human cells. Aiming for RNA-sequencing of YAP1S127A-
expressing SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A and their corresponding controls,
RNA was purified with TRIzolTM. All other gene expression analyses via qPCR were carried out
with column-based extracted RNA.
GFP-posi)veSH-EP
0
0,1
0,5
1
5
10
0
25
50
75
100
µl conc. virus / 104cells
GFP-positive
single cells [%]
scramble
shYAP1_1
shYAP1_2
scramble dead
shYAP1_1 dead
shYAP1_2 dead
A B
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4.3.9.1 Column-based RNA extraction
Dependent on the available cell number, isolation of RNA was performed with RNeasy Mini
Kit (Qiagen) or RNeasy Micro Kit (Qiagen), respectively. Cells were cultivated and treated in
tissue culture dishes or microtiter plates (12-well, 6-well) and washed once with PBS prior to
RNA isolation. Cells were scraped off the dish or well, collected into a 1.5 mL reaction tube
and centrifuged at RT for 5 min with 300 xg. The cell pellet was frozen on dry ice immediately
and stored at -80°C until thawing on ice was necessary for further procedures. According to
the protocol provided by Qiagen, cells were lysed in a recommended volume of RLT-buffer
containing 1% 14.3 M β-mercaptoethanol (v/v). After binding of the RNAs in the silica column,
80 µL DNase I (Qiagen), that was diluted 1:8 in RDD-buffer, was added for 15 min and
afterwards, the column was rinsed twice with RPE buffer. The RNA was eluted in 10 µL
diethylpyrocarbonate (DEPC)-treated deionized water (SERVA Electrophoresis) into pre-
cooled reaction tubes and placed on ice. The RNA was quantified immediately after extraction
(see section 4.3.10.).
4.3.9.2 Guanidinium thiocyanate-phenol-chloroform RNA extraction
For transcriptome analysis via RNA sequencing, a huge amount of high-quality RNA is
necessary. For this purpose, 1x106 cells of SH-EP-TR-YAP1S127A and 2x106 cells of
SK-N-AS-TR-YAP1S127A cells were plated on 15 cm diameter cell culture dishes and treated with
tetracycline or ethanol for 72 hours. Cells were detached with a cell scraper, rinsed off the
plate and transferred into a reaction tube for centrifugation at 4°C for 5 min at 300 xg. Cell
pellets were resuspended in PBS, distributed equally to three 1.5 mL tubes and after a second
centrifugation, pellets were frozen on dry ice and stored at -80°C. One aliquot of each
condition was lysed in 1 mL guanidinium thiocyanate-phenol (TRIzol™) reagent (Thermo Fisher
Scientific) and rested at desk for 5 min for complete dissociation of nucleoprotein-complexes.
Addition of 200 µL chloroform was followed by vigorous handshaking for 15 sec and another
3 min rest awaiting phase separation of upper RNA-containing phase, the middle layer
containing protein and DNA and the third organic phenol-chloroform phase at the bottom.
After centrifugation at 4°C for 15 min with 15.800 xg, about 500 µL of the top layer were
transferred into a new reaction tube and RNA extraction was repeated. To precipitate the
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isolated RNA, 500 µL isopropanol were added, vortexed and incubated for 10 min at RT. After
centrifugation at 4°C for 10 min with 18.500 xg, the supernatant was discarded, and the pellet
was rinsed with 1 mL 75% ethanol. After pelleting the RNA with 8.700 xg at 4°C for 5 min,
supernatant was discarded, RNA pellet was air dried for 20 min and finally resuspended in
30 µL nuclease-free water. RNA was quantified with NanoDrop2000 directly after purification
and stored at -80°C until assessment of RNA integrity number (RIN) with Bioanalyzer 2100
Expert (Agilent) and transcription into cDNA.
4.3.10 Nucleic acid quantification
The concentration of nucleic acids was quantified spectrophotometrically with a NanoDrop
2000 (Thermo Fisher Scientific) directly after extraction. Machine calibration with the
nucleic-acid containing substrate (DEPC-water / nuclease-free water / TE-buffer) was
performed before measurement. The NanoDrop 2000c software calculates RNA or DNA
concentration with respect to light absorbance at wavelength λ =260 nm and extinction
coefficients of ε=40 ng-cm/µL (RNA) or ε=50 ng-cm/µL (dsDNA). Purity of nucleic acids was
determined by ratio of absorbance A260/280. Pure RNA and DNA results at A260/280 = 2.0
and A260/280 = 1.8, respectively.
4.3.11 RNA quantification and RIN determination
Aiming for high-quality RNA for next generation sequencing, integrity and final concentration
of the RNA was determined with Bioanalyzer 2100 (Agilent Technologies) using the RNA 6000
Nano Kit (Agilent Technologies). Samples and standards were thawed on ice, denatured for
2 min at 70°C and placed on ice again. RNAs were pre-diluted in nuclease-free water to achieve
assay-wise needed RNA-concentrations between 25-500 ng/µL. According to the Agilent
Technologies RNA 6000 Nano Kit Guide 01/2017, an RNA Bioanalyzer Chip was loaded with
the gel-dye mix, marker, RNA standard and RNA samples and run in the Bioanalyzer
instrument. All RNA samples of three independent experiments fulfilled the RNA-sequencing
criteria of RIN ≥ 9.5.
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4.3.12 Reverse transcription
Complementary DNA was transcribed from isolated RNA with the Transcriptor First Strand
cDNA Synthesis Kit from Roche. A maximum of 1000 ng total RNA was added by 2 µl 600 µM
random hexamer primer and filled with nuclease-free water to end up with 11 µL volume. The
mix was denatured for 10 min at 60°C in a Thermocycler (Thermo Fisher) and cooled down to
4°C for the addition of reaction buffer, RNase inhibitors [40 U/µL], dNTPs [10 mM each] and
the reverse transcriptase [20 U/µL] according to manufacturer’s instructions. Synthesis of
cDNA was carried out for 1 hour at 50°C. After inactivation of the enzyme activity for 5 min at
85°C, the synthesized cDNA samples were tempered to 4°C and stored at -20°C.
4.3.13 Quantitative real-time PCR
Gene expression levels were determined by quantitative real-time polymerase chain reaction
(qPCR) in a StepOnePLus Real-Time PCR System (Applied Biosystems). RNA was extracted,
processed (see sections 4.3.9 to 4.3.12) and cDNA was pre-diluted 1:10 in nuclease-free water.
A qPCR-mix containing SYBR-green master mix of Fast Start Universal SYBR Green MasterMix
Kit (Hoffmann-La Roche) as well as gene-specific forward and reverse primer and nuclease-
free water (Thermo Fisher Scientific) was prepared as master mix and 18.5 µl together with
1.5 µL of cDNA were transferred into wells of a 96-well PCR-plate (Table 19).
Table 19 Components of qPCR master mix
Component
Volume 1x
Fast Start Universal SYBR Green MasterMix
10 µL
Gene-specific forward and reverse Primer-Mix [10 µM each]
or QuantiTect Primer Assay Mix [10x]
2 µL
Nuclease-free water
6.5 µL
cDNA
1.5 µL
To enable a semi-quantitative mRNA expression analysis, expression of the housekeeping
gene succinate-dehydrogenase complex, subunit A (SDHA), that was shown to be a reliable
control gene in neuroblastoma (Fischer, Skowron and Berthold, 2005), was analyzed in all
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samples. A non-template control for each primer pair was added to demonstrate absence of
any contaminants. The specificity of amplicons was proven by melting curve analysis. The
threshold cycle value (Ct) was stated for every gene and sample with the StepOne software
(Applied Biosystems). Expression of endogenous YAP1 in human cell lines was calculated as
2-ΔCt. Differential expression of genes was determined as 2-ΔΔCt for treated and control cells
(Livak and Schmittgen, 2001). All samples were measured in technical duplicates and
independent biological triplicates if not indicated otherwise.
Table 20 Temperature profile SYBRgreen-based qPCR
Stage
Temperature
Time
Holding
50°C
2 min
95°C
10 min
Cycling
95°C
15 sec
40x
60°C
1 min
Melting Curve
95°C
15 sec
60°C
1 min
95°C (+0.3°C)
15 sec
4.3.14 RNA sequencing and data analysis
The RNA sequencing data analysis was kindly provided by Dr. Filippos Klironomos. The mRNA
library preparation according to Illumina TruSeq stranded mRNA (Illumina) protocol and the
RNA sequencing was performed by Berlin Institute of Health (BIH) Core Facility GENOMICS
((Charité Universitätsmedizin Berlin & Max Delbrück Centre for Molecular Medicine in the
Helmholtz Association). Three biological replicates of each condition were sequenced in an
Illumina NextSeq 500 sequencer. The RNA-sequencing data were aligned to the human
genome assembly GRCh38 with GENCODE v27 annotation using the STAR aligner (Dobin et al.,
2013). The number of reads per gene was determined with the tool FeatureCounts (Liao,
Smyth and Shi, 2014) and normalized to the library size. A variance-stabilization
transformation by DESeq2 package was applied on these and a principal component analysis
was performed. Significantly differentially expressed genes were defined as those with an
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absolute fold change mRNA abundance of
≥
1.5 in YAP1-activated cells compared to ethanol
controls of the same cell line and a Benjamin-Hochberg corrected p-value < 0.05, using the
DESeq2 (Love, Huber and Anders, 2014). A GO enrichment analysis was separately performed
using the topGO tool package from Bioconductor for significantly upregulated or
downregulated genes in biological processes of YAP1-induced SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells compared to their ethanol-treated controls, using all genes with
base means
≥
10 as background and a cut-off level with p<10-4. Additionally, a GO enrichment
analysis was performed for commonly significantly upregulated or downregulated genes with
results produced at a cut-off level of p < 10-3. Further, commonly significantly upregulated and
downregulated genes enriched in the C2 collection of genes of the Molecular Signature
Database were analyzed with clusterProfiler package from Bioconductor and enriched gene
sets were identified with a cut-off level of FDR-adjusted p < 0.05. An analysis of putative
transcription factor binding sites for HIF and TEAD proteins in promoter regions of YAP1-
mediated significantly upregulated genes in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
cells by utilization of TFBSTools Bioconductor package along with JASPAR2018 database.
Unique 1 kb promoters of annotated genes were identified and putative binding sites for TEAD
and HIF proteins with at least 90% similarity with the corresponding known motif were
determined among all promoters. To estimate significance for the TF:gene binding sites, all
genes that were not significantly upregulated and expressed with base mean
≥
1000, were
included as control, and p-value was equal to the probability to find the number of TF:gene
binding sites or more in the control set.
4.4 Biochemical methods
4.4.1 Protein isolation of whole-cell lysates
Western blot analysis was performed from whole-cell lysates. The growth medium of cell
cultures was discarded, and the adhesive cells were rinsed with PBS once. After addition of
Trypsin/EDTA 0.05%, cells were incubated at 37°C for 5-15 min and the Trypsin enzyme activity
was stopped by application of 10% FCS-supplemented medium in a 1:4 ratio. The solution was
collected into a 15 ml or 50 ml reaction tube and centrifuged with 300 xg for 5 min at 4°C. The
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cell pellet was washed once with cold PBS to remove residual FCS. After a second PBS wash
and subsequent centrifugation, supernatant was discarded, and the cell pellet was frozen
at -80°C until lysis. For this purpose, cells were thawed on ice, which resulted in a damage of
the cell membranes. Additionally, cells were resuspended in an appropriate volume of lysis
buffer (15 mM HEPES, 150 mM NaCl, 10 mM EGTA, 2% Triton X-100) including protease and
phosphatase inhibitor cocktails (Hoffmann-La Roche) and incubated on ice in an orbital shaker
to further lyse the cells including the nuclei. Separation of cell debris and proteins was
performed via maximum speed centrifugation at 4°C for 30 min. The upper protein fraction
was transferred and aliquoted into 0.5 mL reaction tubes and one sample was used directly
for protein concentration measurement. Additional volumes were either stored at -80°C or
put to use for western blot analysis directly. For detection of the proteins HIF-1α or HIF-2α,
1 mM dithiothreitol (DTT) was added.
4.4.2 Measurement of protein concentration with bicinchonic acid assay
Protein concentration of whole-cell lysates was detected with the Colorimetric bicinchonic
acid (BCA) protein assay kit (Santa Cruz). Principle of that firstly 1985 by Smith et al. described
method is the reaction of Cu1+ ions with two BCA-molecules resulting in purple-colored
complexes, which can be detected photometrically at wavelength 562 nm (Smith et al., 1985).
The rate-limiting amount of Cu1+ ions depends on the number of Cu2+ ion-reducing amino acids
cysteine, tryptophan and tyrosine residues and correlates to protein quantity in the whole-
cell lysates.
The BCA assay was performed according to the manufacturers protocol. Bovine serum
albumin (BSA) from 0 µg/mL to 2000 µg/mL was used to generate standard curves. Depending
on expected protein concentrations, the whole-cell lysates were applied directly or pre-
diluted in distilled water up to 10-fold. After addition of 200 µL BCA reaction mix to 25 µL
sample volume, the experiment samples, BSA standards and blank controls (lysis buffer) were
collectively incubated on one 96-well flat bottom microtiter plate for 30 min at 37°C. Signal
read out was realized with an Epoch plate reader at wavelength
𝜆
=562 nm at RT. In the mean
of BSA standard curve and taking into account possible predilutions, protein concentrations
were calculated with the software Gen5 (Bio Tek Instruments).
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4.4.3 Protein separation by SDS-PAGE
Prior to immunoblots, proteins of whole-cell lysates were separated by their molecular weight
with sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) as described by
U. Laemmli (Laemmli, 1970). To ensure protein fractionation based only on size, proteins were
denaturated and SDS was added to all buffers for masking the net electric charge. The matrix
was built of acrylamide and bisacrylamide in two different concentrations (see Table 21) to
enforce protein accumulation at the separation gel for a simultaneous migration start towards
the cathode.
Table 21 SDS-PAGE separation and stacking gel composition for a single gel
Reagent
10% (v/v) Separation gel
5% (v/v) Stacking gel
Polyacrylamide-Mix 30% (v/v)
10 mL
1.3 mL
Deionized water
11.9 mL
1 mL
1.5 M Tris base buffer, pH 8.8
7.5 mL
-
1 M Tris base buffer, pH 6.8
-
5.5 mL
20% SDS
150 µL
40 µL
10% APS
300 µL
80 µL
TEMED
12 µL
8 µL
A total amount of 10 µg protein per sample was mixed with 4x Laemmli protein sample buffer
(Bio-Rad), denatured for 10 min at 95°C, spun down and hold on ice until loading. As molecular
weight marker, 3 µl of prestained protein ladder (PageRuler, Thermo Fisher Scientific) were
loaded on each gel additionally. Electrophoresis was performed in a mini-PROTEAN Tetra
electrophoresis cell (Biorad) together with the power supply from Biorad. Proteins were
separated for about 1.5 hours in an electric field with 80-110 V voltage in SDS-running buffer
(25 mM Tris base, 192 mM glycine, 0.1% SDS (v/v)).
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4.4.4 Western Blot
Proteins separated via SDS-PAGE were transferred onto a hydrophobic polyvinylidene
difluoride (PVDF) membrane, dependent on the size of proteins of interest, in two different
ways.
4.4.4.1 Semi-dry blot
The PVDF membrane was activated in methanol for 1 min, washed in distilled water and
equilibrated in transfer buffer (25 mM Tris base, 192 mM glycine, 20% (v/v) methanol),
subsequently. Ten sheets of 0.35 mm blotting paper (Carl Roth) were equilibrated in transfer
buffer and afterwards, PVDF membrane and the SDS-PAGE gel were stacked in between and
transferred into a cassette of Trans-Blot Turbo Transfer (Biorad) for semi-dry blotting at 25
volts and 1.0 A for 30 min.
4.4.4.2 Wet Blot
Transfer of high-molecular weight proteins HIF-1α and HIF-2α onto a PVDF membrane was
performed overnight in a wet blot tank. Therefore, two sponges, ten blotting paper as well as
the activated PVDF membrane were soaked with transfer buffer and stacked into a Mini Trans-
Blot® Cell (Biorad). At 4°C and under gentle stirring, the proteins were transferred overnight
at 30 V.
4.4.4.3 Protein detection
Preventing unspecific antibody binding, membranes were blocked with 5% (w/v) low-fat milk
or, if phosphorylated proteins were detected, 5% BSA (w/v) in Tris-buffered saline (Tris base
10 mM, NaCl 150 mM, pH 7.4) with 0.05% (v/v) Tween-20 (TBS-T) for 60 min at RT. After three
times washing with TBS-T for 10 min each, membranes were probed with primary antibody in
different dilutions overnight at 4°C. Next day, the membrane was washed three times (10 min
each) in TBS-T again to remove the primary antibody. The horse-radish peroxidase (HRP)-
conjugated secondary antibody was chosen according to the antibody species and pre-diluted
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in TBS-T 1:10,000. Probing of the membrane with the secondary antibody was performed at
RT for 60 min. Prior to the detection of the labeled proteins, the membrane was washed three
times in TBS-T to remove residual antibodies, placed into the Fusion-FX imaging system (Vilber
Lourmat) and 1 mL Western Lightning Plus-ECL solution (Perkin Elmer) was spaced out evenly
over the membrane for protein detection. For detection of further proteins (such as protein
loading control β-Actin), membrane was washed three times in TBS-T and protocol was
executed again starting at incubation with a primary antibody.
4.4.5 Immunofluorescent staining of YAP1 and Flag-Tag
In the YAP1S127A overexpression plasmids, an N-terminal 2x or 3xFlag-Tag was added enable
detection of the construct independently from endogenous YAP1 protein. Aiming for a further
validation of the Tet-ON overexpression system, YAP1 and Flag-Tag was immunostained in
SH-EP-TR-YAP1S127A cells. Cells were cultured in tetracycline-supplemented medium to induce
YAP1 expression. Control cells were treated with equal volumes of ethanol. For staining
purposes, cells were grown on glass plates in 24-well microtiter plates. After 72 hours, cells
were washed once with PBS and fixed with 200 µL of 4% Formaldehyde for 10 min at RT. Cells
were rinsed three times with PBS and penetrated with 0.1% (v/v) Triton X-100 in PBS for 5 min.
After washing the cells three times for 5 min with PBS, the glass slides with adherent cells
were placed into a humidified chamber for further staining procedure. Unspecific antibody
binding was reduced by incubation of the cells in 5% (v/v) FCS in PBS for 30 min. The primary
antibody rabbit anti-YAP1 (Cell Signaling Technologies) or the mouse anti-Flag (Sigma Aldrich)
was diluted in 1% (v/w) PBS/BSA, applied to the cells and incubated overnight in the
humidified chamber at 4°C. Next morning, the primary antibody was discarded, and cells were
washed three times with PBS for 15 min each. Secondary antibodies goat
anti-rabbit-Alexa Fluor™ 488 (Thermo Fisher Scientific) for detection of YAP1 and goat
anti-mouse-Alexa Fluor™ 488 (Thermo Fisher Scientific) for recognition of Flag-Tag were
applied 1:500 in 1% PBS/BSA to the cells and incubated for 30 min at RT. A staining control
with secondary antibody only was included into the setup in order to record unspecific
antibody binding in the cells or in the extracellular matrix. After removal of residual antibodies
by rinsing the cells three times with PBS for 15 min, the slides were incubated in distilled water
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for 2 min to rinse salts away. After 2 min incubation with 100% ethanol aiming for a
dehydration of the cells, the slides were air dried at RT in darkness, sealed with one drop of
VectaShield mounting medium (Vector laboratories) and covered by a cover slip, which was
fixed with clear nail polish (DM drug store). Pictures of cells were taken with an Olympus
microscope BX43 (Olympus) with camera attachment (Zeiss).
4.5 Statistics
Statistical analysis was predominantly realized with GraphPad Prism Software version 6
(GraphPad Software). Data are presented as means ± standard deviation (SD) from technical
and biological replicates. Normal distribution was tested with Shapiro-Wilk test online at
StatistikGuru Version 1.96 (Hemmerich, 2018). Data that did not pass the normality test were
analyzed with unpaired Mann-Whitney U test. Other statistical tests than that are indicated
in figure descriptions. All p-values under p ≤ 0.05 were considered as significant with
significance levels set as * 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01 and *** p ≤ 0.001. The sample
sizes vary between experiments and are indicated in respective figure legends.
4.6 Software
Plasmid maps were received from Addgene (https://addgene.org). Vector maps were created
with SnapGene® software and SnapGene® viewer v3.1.4 (GSL Biotech LLC). Gene and RNA
sequences were taken from and compared to UCSC genome browser
(https://genome.ucsc.edu) and Ensembl genome browser (https://www.ensembl.org).
Primers for qPCR were designed with Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0).
Expression analysis and Ct threshold determination was conducted with StepOne software
(Applied biosystems) and Microsoft Excel software (Microsoft). Distances of wound healing
edges and ROIs of migrated cells were measured with imaging software Fiji ImageJ32 (v1.48,
NIH). Flow cytometry data arose from BD FACSDiva™ software (BD Biosciences). Further
analysis was performed with FlowJo-X software (BD Biosciences,v10.0.7r2 ). In order to find
YAP1-regulated pathways and cellular processes, gene-set enrichment analyses were
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performed on the basis of biological processes listed in the Gene Ontology (GO) database and
gene encompassed in the C2 collection of genes of the Molecular Signatures database MSigDB
(https://www.gsea-msigdb.org/gsea/index.jsp). Venn diagrams of upregulated and
downregulated genes in YAP1-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
have been designed with BioVenn (Hulsen, de Vlieg and Alkema, 2008). Statistical analysis and
data visualization was realized with GraphPad Prism Software v6 (GraphPad Prism).
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5. Results
Aim of this project was to analyze the contribution of YAP1 to neuroblastoma pathogenesis.
To gain insights into its oncogenic role, YAP1 was perturbed in neuroblastoma cell lines
possessing various endogenous YAP1 levels. Subsequently, the cells have been subjected to
transcriptomic and functional profiling. Establishment of a genetical YAP1 silencing model and,
in a second approach, a tetracycline-inducible overexpression system for YAP1S127A was
followed by transcriptome sequencing and in vitro assays to highlight possible contribution of
YAP1 to cellular processes such as proliferation, apoptosis, migration, metabolism and drug
resistance development.
5.1 Endogenous YAP1 expression in neuroblastoma cell lines and tumor
tissues
A panel of 19 human neuroblastoma cell lines (SK-N-BE, LAN-1, IMR-5, NB-1643, LAN-5, NBL-S,
SK-N-AS, SJ1, SK-N-FI, SH-EP, Kelly, SK-N-SH, IMR-32, CHP-134, NGP, TR14, N206, GI-M-EN,
SH-SY5Y) was cultivated in standard tissue culture conditions and analyzed for endogenous
YAP1 mRNA and protein levels. YAP1 protein was detected with western blot technique in 17
neuroblastoma cell lines and in four YAP1-positive human control cell lines (epithelial HeLa
and HEK293, fibroblastic VH7, medulloblastic DAOY), together with the structural protein
β-actin as loading control (Figure 5).
Figure 5 Various YAP1 protein synthesis levels in neuroblastoma cell lines. Representative immunoblot of YAP1, detected
in 10 µg whole cell lysates of 17 neuroblastoma cell lines and four YAP1-positive control cell lines (Hela, VH7, DAOY, HEK293)
with the corresponding loading control β-Actin. Representative immunoblots of 3 biological replicates.
SK-N-SH
IMR-32
CHP-134
NGP
TR14
N206
HeLa
VH7
DAOY
HEK293
YAP1
β-Actin
SK-N-BE
Lan-1
IMR-5
NB-1643
Lan-5
NBL-S
SK-N-AS
SJ1
SK-N-FI
SH-EP
Kelly
Neuroblastoma cell lines Control cell lines
Results
74
The quantitative real time-PCR (qPCR) analysis of the same cell lysates revealed congruent
patterns of YAP1 mRNA relative to SDHA expression (Figure 6). Neuroblastoma cell lines
exhibited highly variable endogenous YAP1 expression levels, ranging from high to not
detectable signals. In order to figure out, whether this heterogeneity of YAP1 expression levels
in cell line models mirrors the clinical neuroblastoma phenotype, transcriptome data sets of
neuroblastoma tumor tissues were obtained from published data sets (Hiyama et al., 2004;
Łastowska et al., 2007; Bourdeaut et al., 2009; Molenaar et al., 2012) and expression of YAP1
(as represented by microarray oligonucleotide probe identifier 224894_at) was determined
(Figure 7). The YAP1 transcript levels range between ten log2 levels, which confirmed the
variability of neuroblastoma tumors with regard to YAP1 expression.
SK-N-FI
SK-N-SH
SH-EP
GI-ME-N
IMR-32
SK-N-AS
IMR-5
TR14
LAN-1
SK-N-BE
CHP-134
SH-SY5Y
SJ1
KELLY
N206
LAN-5
NB-1643
NBL-S
NGP
0.00
0.05
0.10
0.15
0.5
1.0
1.5
Relative YAP1 mRNA expression
Figure 6 YAP1 expression levels
are variable in 19 neuroblastoma
cell lines. Gene expression of
YAP1 in neuroblastoma cells is
depicted as means ± SD, relative
to Succinate dehydrogenase
complex subunit A (SDHA), n=3.
Figure 7 Neuroblastoma tumors exhibit various YAP1
expression levels. The YAP1 expression is mapped in four
independent mRNA expression data sets that comprise
neuroblastoma tumors from patients of different age,
gender, INSS stages and with tumor exhibiting different
chromosomal aberrations (MYCN-amplification, 1p/11q-
deletions, 17q-gain, ALK mutations). YAP1 expression
values were obtained from published data (Hiyama et al.,
2004; Lastowska et al., 2007; Bourdeaut et al., 2009,
Molenaar et al., 2012).
DeLattre - 34
Hiyama - 51
Lastowska - 30
Versteeg - 88
0
1
2
3
4
5
6
7
8
9
10
YAP1 expression log2
Neuroblastoma tumors
Results
75
In a transcriptome dataset comprising 649 neuroblastomas (Kocak et al., 2013), the event-free
survival (EFS) probability of patients was visualized with R2: Genomics Analysis and
Visualization Platform (R2 Database: Neuroblastoma Kocak-649, 2021) as a function of high
(cut-off: last quartile) and low YAP1 expression levels (Figure 8). Additionally, corresponding
YAP1 gene expression values are presented next to it. In INSS stage 1/2 tumors and INSS stage
4 tumors, the EFS probability is independent of YAP1 gene expression levels. However, in INSS
stage 3 and 4s neuroblastoma, high YAP1 levels correlate negatively with the EFS probability.
Aiming for the identification of possible correlations between YAP1 and neuroblastoma-
specific, frequent genetic alterations, clinical data and recurrent genetic aberrations were
compiled for cell lines that were predominantly used in this project (Table 22) (Donti et al.,
1988; Thiele, 1998; De Brouwer et al., 2010; Harenza et al., 2017; ATCC, 2021). The loss or
deletion of chromosome 11q affects YAP1 expression to some extent. Cytogenic abnormalities
other than 11q deletion/loss had no effect.
Figure 8 Event-free survival probability of neuroblastoma patients negatively correlates with YAP1 mRNA expression
levels in INSS stage 3 and 4s tumors. Gene expression data of 649 neuroblastomas (Kocak et al., 2013) revealed lower EFS
probability for patients suffering from INSS stage 3 and stage 4s neuroblastomas with high YAP1 gene expression compared
to those with lower YAP1 levels. Corresponding YAP1 expression values are depicted next to the Kaplan-Meyer curves.
649 Neuroblastoma – Kocak et al., 2013
YAP1 high (n=49)
YAP1 low (n=147)
Stage 1/2
YAP1 high (n=17)
YAP1 low (n=54)
Stage 3
YAP1 high (n=37)
YAP1 low (n=111)
Stage 4
YAP1 high (n=16)
YAP1 low (n=45)
Stage 4s
low
high
0
200
400
600
800
1000
1200
1400
YAP1 expression
low
high
0
200
400
600
800
1000
1200
1400
1600
1800
2000
YAP1 expression
low
high
0
200
400
600
800
1000
1200
1400
YAP1 expression
low
high
0
200
400
600
800
1000
1200
1400
YAP1 expression
Results
76
Table 22 Characteristics of ten neuroblastoma cell lines used in this project1.
1 Age=month, m=male, f=female, BM=bone marrow, abdom.=abdominal, metast.=metastasis, N/A=not available,
amp=amplified, NA=non-amplified, wt=wildtype, LOH=loss of heterozygosity, del=deletion, t=translocation,
INSS=international neuroblastoma staging system.
5.2 Neuroblastoma cell density has no effect on YAP1 in vitro
Cancer cells generally bypass the restrictions of contact inhibition (Abercrombie, 1979).
Nevertheless, since the Hippo-YAP1 signaling directly regulates cell contact inhibition (see
section 1.2.2), the effect of neuroblastoma cell density on YAP1 expression and protein
abundance was examined. Ten cell lines (SK-N-FI, SK-N-SH, GI-M-EN, SK-N-AS, LAN-1, SK-N-BE,
IMR-5, CHP134, KELLY and NB-1643) were cultivated in low and high cell density for 24 hours
(Figure 9 A). YAP1 mRNA expression (Figure 9 B) as well as protein levels of YAP1 and
inactivated, at serine 127 phosphorylated pYAP1(S127) were detected (Figure 9 C).
Transcriptional levels of YAP1 were stable in sparsely seeded cells compared to cells cultured
in high density. Immunoblot analysis also showed no alteration of YAP1 protein levels by cell-
cell contact of high-density cultures. In addition, the pYAP1(S127) signals were distributed
equally between both groups. In SK-N-SH, a cell line with high endogenous YAP1 levels, strong
signals for pYAP1(S127) were detected. However, a regulation of YAP1 activity through
inhibiting S127-phoshorylation by cell-cell contact could not be shown. In conclusion, physical
cell-cell contact did not alter the expression or the activity of YAP1 in neuroblastoma cells.
Cell line
Sex
Age
Tumor site
INSS
MYCN
ALK
1p
11q
17q
SK-N-FI
m
11
BM metast.
4
NA
wt
wt
wt
gain
SK-N-SH
f
48
BM metast.
4
NA
ALKF1174L
wt
wt
gain
SH-EP
f
48
BM metast.
4
NA
ALKF1174L
wt
wt
gain
GI-M-EN
f
24
BM metast.
4
NA
wt
del
t
gain
IMR-5
m
13
Abdom. metast.
4
amp
wt
gain+LOH
loss
gain
SK-N-AS
f
96
BM metast.
4
NA
wt
del
del
gain
SK-N-BE
m
20
BM metast.
4
Amp
wt
del
wt
gain
SH-SY5Y
f
48
BM metast.
4
NA
ALKF1174L
wt
loss
gain
Kelly
f
12
N/A
N/A
amp
ALKF1174L
del, t
del
gain
NBL-S
m
42
Adren. prim.
3
NA
wt
wt
loss
wt
Decreasing YAP1 levels
Results
77
5.3 Inhibition of YAP1 protein synthesis via RNA interference
The targeted deregulation of genes in vitro and in vivo is a strong tool in the toolbox of cancer
researchers to learn about protein functions and decipher affected pathways in a tissue-
specific environment. RNA interference is one possible method to reduce the expression and
prevent the translation of one target gene in a cell. Small RNA structures delivered into the
Lowconfluency
SK-N-BE
LAN-1
GI-M-EN
NB-1643
Highconfluency
pYAP1(S127)
YAP1
β-Ac/n
SK-N-FI SK-N-SH GI-ME-N SK-N-AS LAN-1 SK-N-BE IMR-5 CHP-134 KELLY NB-1643
ConfluencyLHLHLHLHLHLHLHLHLHLH
Figure 9 Cell-cell contact has no effect on YAP1 levels and YAP1 protein phosphorylation. Ten neuroblastoma cell lines
were cultured in low (L) or high (H) cell density for 24 hours (SK-N-BE, LAN-1, GI-M-EN, NB-1643, SK-N-FI, SK-N-SH, SK-N-AS,
IMR-5, CHP-134, KELLY). (A) Representative microscopy pictures of SK-N-BE, LAN-1, GI-M-EN and NB-1643 are displayed.
(B) Gene expression levels of YAP1 were detected by qPCR and are presented as means ± SD, n=2. Statistical analysis was
performed with the Wilcoxon Test. (C) Immunoblot of YAP1, phosphorylated YAP1 (pYAP1(S127)) and β–Actin (loading
control).
SK-N-FI
SK-N-SH
GI-M-EN
SK-N-AS
LAN-1
SK-N-BE
IMR-5
CHP134
KELLY
NB-1643
10
-4
10
-3
10
-2
10
-1
10
0
10
1
Relative YAP1 mRNA expression
50% confluency
100% confluency
A
B
C
Results
78
cell lead to mRNA degradation via RISC-complex mediated processing, and the cell response
in turn teaches us about the function of this gene/protein in this explicit cell type.
Aiming for the analysis of YAP1 function in neuroblastoma cells, a transient knockdown of
YAP1 with small interfering RNA (siRNA) and a short hairpin RNA (shRNA)-mediated approach
were established.
5.3.1 Small interfering RNA-mediated YAP1 knockdown
Neuroblastoma cell lines were “fast forward” lipid-transfected with two specific siRNAs and
two unspecific scramble control siRNAs, cultured up to 72 hours and harvested at indicated
time points for analysis. A mock control consisting of cells treated with transfection reagents
only was included into qPCR analysis. As expected, YAP1 mRNA expression in siYAP1-treated
cells was depressed after 24 hours (Figure 10 A), whereas protein was no longer measurable
after 72 hours (Figure 10 B). Consequently, it turned out to be 72 hours after transfection that
YAP1 mRNA as well as protein levels were reduced up to 100%. Next, YAP1 was transiently
silenced in five neuroblastoma cell lines with variable endogenous YAP1 levels (SK-N-SH,
SH-EP, SK-N-BE, SK-N-FI, IMR-5) and, subsequently, a qPCR of YAP1 and SDHA was performed.
The relative YAP1 mRNA expression was calculated as ratio of the YAP1 expression to the
SDHA expression. In all tested cell lines, the YAP1 expression could be significantly reduced
compared to scramble controls (Figure 10 C). To validate the functional consequence upon
knockdown of transcriptional co-activator YAP1, the expression of its target genes CYR61,
COL8A1, SKP2 and SNAPC was analyzed in one representative cell line SH-EP. An almost
complete inhibition of YAP1 translation resulted in downregulated YAP1 target genes,
although only COL8A1 was statistically significantly repressed (Figure 10 D).
Taken together, a robust siRNA-based YAP1 knockdown protocol with 81-98% knockdown
efficiency was established and validated in different neuroblastoma cell lines.
Results
79
NC#1
Time[hrs]612244872
YAP1
β-Ac:n
NC#2
siYAP1#1
siYAP1#2
YAP1
β-Ac:n
YAP1
β-Ac:n
YAP1
β-Ac:n
024 48 72
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time [h]
Relative YAP1 mRNA expression
SK-N-SH
mock
NC #1
NC #2
siYAP1 #1
siYAP1 #2
A
B
Figure 10 Establishment of a siRNA-mediated YAP1 knockdown in neuroblastoma cells. (A) Transcriptional levels and (B)
protein signals of YAP1 were measured at different time points (0, 6, 12, 24, 48 and 72 hours) after transfection of SK-N-SH
with two YAP1 targeting siRNAs (siYAP1 #1, #2) and two non-target control siRNAs (NC #1, #2), extended by a transfection
reagent control (mock) for qPCR analysis. (C) The YAP1 mRNA expression was analyzed by qPCR after a transient YAP1
knockdown in five cell lines with various endogenous YAP1 levels (SH-EP, SK-N-SH, SK-N-FI, SK-N-BE and IMR-5), 72 hours
after siRNA transfection and is displayed in relation to SDHA expression. (D) Transcript levels of YAP1 and its target genes
CYR61, COL8A1, SKP2 and SNAPC are reduced 72 hours after YAP1 knockdown in SH-EP. Data are normalized to non-target
controls and presented in relation to SDHA expression. Bars represent means ± SD of 3 independent replicates. Statistical
significance was assessed using the Mann-Whitney U test; * 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01.
0
-20
-40
-60
-80
-100
Relative gene expression [%]
YAP1
CYR61
COL8A1
SKP2
SNAPC
*
*
NC #1 + #2
siYAP1 #1
siYAP1 #2
0.0
0.5
1.0
1.5
2.0
2.5
Relative YAP1
mRNA expression
SH-EP
**
NC #1 + #2
siYAP1 #1
siYAP1 #2
0
1
2
3
4
5
Relative YAP1
mRNA expression
SK-N-SH
*
NC #1 + #2
siYAP1 #1
siYAP1 #2
0.00
0.25
0.50
0.75
1.00
SK-N-FI
Relative YAP1
mRNA expression
**
*
NC #1 + #2
siYAP1 #1
siYAP1 #2
0.00
0.05
0.10
0.15
0.20
Relative YAP1
mRNA expression
SK-N-BE
*
NC #1 + #2
siYAP1 #1
siYAP1 #2
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
IMR-5
Relative YAP1
mRNA expression
D
C
[h]
Results
80
5.3.2 Short hairpin RNA-mediated YAP1 knockdown
Aiming for validation of the siRNA-mediated YAP1 knockdown experiments, two vectors
carrying different shRNAs against YAP1 and a scramble control vector were generated for
lentiviral transduction of cells, all extended by a green fluorescent protein (GFP) linked to
puromycin resistance cassette via T2A linker. The cloning strategy and vector generation for
shRNA-mediated knockdown of YAP1 was kindly provided by Annika Winkler.
Based on GFP-fluorescence, the produced virus was titrated in the neuroblastoma cell line
SH-EP and subsequently applied with 1 µL virus concentrate to transduce 104 cells of SH-EP,
GI-M-EN, IMR-5 and SH-SY5Y. In order to prove a technically successful transduction, the
neuroblastoma cells were checked for GFP-based green fluorescence (Figure 11 A). After
harvesting the cells 72 hours post transduction, gene expression analysis via qPCR was
performed to assess the knockdown efficiency in each cell line. The qPCR data (Figure 11 B)
demonstrate, that YAP1 gene expression is inhibited by lentivirus-mediated shRNA
SH-EP
GI-M-EN
IMR-5
SH-SY5Y
Figure 11 Insufficient YAP1 knockdown was achieved by shRNA delivery. (A) Representative GFP-fluorescing SH-EP, GI-M-
EN, IMR-5 and SH-SY5Y cells are shown 72hours after lentiviral transduction with shRNAs. (B) Bars represent fold changes of
YAP1 gene expression (mean ± SD) relative to scramble control, 72hours after lentiviral transduction of YAP1 high-expressing
SH-EP and GI-M-EN and YAP1 low-expressing IMR-5 and SH-SY5Y with scramble shRNA and two different target shRNAs, n=2.
scramble
shYAP1_1
shYAP1_2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Gene expression fold change
SH-EP
scramble
shYAP1_1
shYAP1_2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
GI-M-EN
Gene expression fold change
scramble
shYAP1_1
shYAP1_2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
IMR-5
Gene expression fold change
scramble
shYAP1_1
shYAP1_2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SH-SY5Y
Gene expression fold change
A
B
Results
81
expression, but not as successful as with siRNA-mediated knockdown. Depending on the cell
line, lentiviral transduction with shRNAs directed against YAP1 reduced the gene expression
by only 20-90%. Thus, siRNA-mediated YAP1 knockdown was favored for further approaches
to ensure the lowest possible residual protein amounts.
5.4 Impaired cell proliferation and viability upon YAP1 knockdown
YAP1 is frequently described to regulate proliferation and organ growth in many tissue types
(see section 1.2.3) (Camargo et al., 2007; Zhao et al., 2007; von Gise et al., 2012). Therefore,
the impact of YAP1 knockdown on the cell viability was investigated in a panel of nine
neuroblastoma cell lines of variable endogenous YAP1 levels. A transient knockdown with two
siRNAs directed against YAP1 and a scramble control was performed. Cell viability was
assessed 72 hours after transfection via an ATP-based luminescence assay (Figure 12).
In four out of the nine cell lines the cell viability was significantly reduced by up to one third
upon YAP1 knockdown. The YAP1 high-expressing cell lines GI-M-EN, SH-EP and SK-N-SH were
included in this group. Another three cell lines showed the same tendency, whereas the cell
viability of two cell lines, IMR-5 and NBL-S, was not affected by YAP1 knockdown. Since the
ATP-based cell viability assay is not sufficient to differentiate between cell proliferation and
SH-EP
GI-M-EN
SK-N-SH
SK-N-BE
SK-N-FI
SK-N-DZ
Kelly
NBL-S
IMR-5
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Cell viability,
normalized to mock
siYAP1 #1
siYAP1 #2
scramble
**
**
***
*
**
Figure 12 The cell viability of neuroblastoma cells is reduced upon YAP1 knockdown. An ATP-based cell viability assay was
performed with neuroblastoma cell lines 72 hours after YAP1 siRNA transfection. Bars represent means ± SD of three (SH-EP,
SK-N-SH, SK-N-BE, SK-N-FI, IMR-5) or five (GI-M-EN, SK-N-DZ, Kelly, NBL-S) biological replicates, each measured in technical
triplicates. Statistical significance was assessed with Mann-Whitney U test; * 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01;
*** p ≤ 0.001.
Results
82
metabolic changes within the cells, a proliferation-ELISA assay that is based on BrdU-
incorporation into proliferating cells, was additionally conducted. Therefore, five cell lines
were transfected with YAP1-targeting and scramble control siRNAs for 72 hours followed by
the proliferation-ELISA (Figure 13). A solvent control sample lacking RNA molecules (mock)
was included and used as reference. The knockdown of YAP1 significantly inhibited the
proliferative capacity of SK-N-SH, SK-N-BE and SH-EP cells. The cell growth of IMR-5 was
unaffected and SK-N-FI cells showed a tendency of inhibited proliferation upon YAP1 silencing.
Simultaneous semiautomated counting of viable SH-EP, SK-N-SH and IMR-5 cells validated the
BrdU-ELISA results.
The reduction of cell proliferation in YAP1 silenced neuroblastoma cells could be reasoned by
i) an apoptotic signaling with subsequent cell death or ii) a cell cycle regulation, both reported
to be affected by YAP1 (Mizuno et al., 2012; Wu et al., 2018). In order to analyze whether a
YAP1 knockdown leads to increased apoptosis and cell death in neuroblastoma cells, four cell
lines were analyzed with a cell death ELISA assay detecting cytoplasmic histone-bound DNA
72 hours after transient YAP1 knockdown (Figure 14 A). In addition, apoptotic and dead cells
were assessed in YAP1 high-expressing SK-N-SH cells in a FACS-based approach utilizing
PI/Annexin V staining to detect double-positive dead cells and Annexin V single-positive
apoptotic cells (Figure 14 B). Compared to scramble control, neither the ratio of dead cells nor
the proportion of apoptotic cells were altered upon YAP1 knockdown. Both assays revealed
that YAP1 knockdown does not affect neuroblastoma cell apoptosis. Cell cycle profiles of
SK-N-SH cells were achieved in a FACS-based assay measuring DNA amounts based on
SK-N-SH
SK-N-BE
SH-EP
IMR-5
SK-N-FI
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Proliferation,
normalized to mock
siYAP1 #1
siYAP1 #2
*
*
** *
scramble
Figure 13 Cell proliferation is impaired upon
YAP1 knockdown. A BrdU-ELISA assay
detecting cell proliferation was conducted
with five neuroblastoma cell lines, 72 hours
post siRNA transfection. Bars represent means
± SD of n=4 with technical triplicates each.
Statistical significance was assessed using the
Mann-Whitney U test; * 0.01 < p ≤ 0.05;
** 0.001 < p ≤ 0.01.
Results
83
PI-intercalation (Figure 14 C). A 1.1-fold, albeit not statistically significant, accumulation of
YAP1 knockdown cells in the G0/G1 cell phase was detected compared to scramble controls.
Taken together, the cell viability and proliferation of neuroblastoma cell lines was diminished
upon a YAP1 knockdown via limited deregulation of the cell cycle, although that effect only
reduced, but missed to kill the neuroblastoma cell population. Preferably, cells exhibiting high
endogenous YAP1 expression levels are more vulnerable to YAP1 silencing than the ones
possessing low YAP1 expression.
scramble
siYAP1 #1
siYAP1 #2
0
20
40
60
80
100
Cell cycle distribution [%]
SK-N-SH
subG
G0/G1
S
G2/M
Dead cells
Apoptotic cells
0
1
2
3
4
5
Apoptotic / dead cells [%]
SK-N-SH
scramble
siYAP1 #1
siYAP1 #2
SK-N-BE
SK-N-SH
SK-N-FI
IMR-5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Enrichment factor
scramble
siYAP1 #1
siYAP1 #2
C
Figure 14 Cell cycle, but not apoptosis is marginally affected by YAP1 knockdown. (A) Cell death ELISA of four
neuroblastoma cell lines and (B) additional PI/Annexin V staining of SK-N-SH, 72 hours after siRNA transfection, reveals no
alteration of apoptosis upon YAP1 knockdown. (C) FACS-based cell cycle analysis of the same SK-N-SH stained with PI,
revealed a 1.1-fold higher proportion of cells in the G0/G1 phase. Bars represent means ± SD of n=3 independent
experiments. No statistical significance was assessed using the Mann-Whitney U test.
A
B
scramble siYAP1 #1 siYAP1 #2
mock
scramble
shYAP1_1
shYAP1_2
0
1×106
2×106
3×106
4×106
5×106
6×106
Cell viability
[RLU]
GI-M-EN
mock
scramble
shYAP1_1
shYAP1_2
0
1×10
7
2×10
7
3×10
7
4×10
7
Cell viability
[RLU]
SH-EP
mock
scramble
shYAP1_1
shYAP1_2
0
2×10
6
4×10
6
6×10
6
8×10
6
Cell viability
[RLU]
IMR-5
mock
scramble
shYAP1_1
shYAP1_2
0.0
5.0×10
5
1.0×10
6
1.5×10
6
Cell viability
[RLU]
SH-SY5Y
Figure 15 Cell viability of neuroblastoma cells with shRNA-mediated YAP1 knockdown. Cells were lentivirally transduced
with scramble and YAP1 shRNA and cell viability was assessed four days post transduction. Bars represent means ± SD of 3
independent experiments with technical triplicates. RLU = relative luminescence units. No statistical significance was
assessed using the Mann-Whitney U test.
Results
84
Aiming for a validation of these results, viability of cells transduced with shRNAs against YAP1
was assessed with the same assay.
Although YAP1 knockdown was not as sufficient as accomplished by siRNA interference, four
neuroblastoma cell lines were transduced lentivirally for one day, followed by a cell viability
assay after another 72 hours of cultivation. Experimental setup included one scramble shRNA
and a mock control comprising cells and all transduction solvents except shRNAs. The relative
luminescence units [RLU] were unaltered upon shRNA-mediated YAP1 knockdown and thus,
the cell viability was not affected (Figure 15). This finding was contrary to the results of cells
being transfected with siRNAs targeting YAP1.
5.5 Reduced YAP1 levels hampered migratory potential of neuroblastoma
cells
To gain insights into a possible interrelation between YAP1 and cell motility, in vitro wound-
healing assays were performed in four neuroblastoma cell lines over time. Therefore, siRNAs
directed against YAP1 and non-target control siRNAs were applied to SK-N-SH, SK-N-BE, IMR-5
and SK-N-FI, a cell-free region was inflicted on the mono cell layer and gap closure was
documented over 48 hours (Figure 16 A). The cell migration potential of the cells was deduced
from measured “wound”-edge distances, that are given as fold change of the initial scratch
(Figure 16 B).
The cell migration of SK-N-SH and SK-N-BE cells was significantly reduced over the time period
of 48 hours upon YAP1 knockdown, and IMR-5 and SK-N-FI cells showed a tendency to
inhibited cell motility. The re-population of the cell-free area by SK-N-SH cells was nearly
complete after 48 hours, whereas in the other cell lines the general re-population of the cell
free region was slower. In conclusion, the migration potential of neuroblastoma cells was
found to depend on YAP1 levels.
Results
85
Figure 16 YAP1 knockdown hampers cell motility of neuroblastoma cells. In vitro wound-healing assay of SK-N-SH,
SK-N-BE, IMR-5 and SK-N-FI cells transfected with scramble siRNA and YAP1 targeting siRNA #2 for 0, 14, 24 and 48 hours
revealed retarded re-population of the cell-free region by YAP1 silenced cells. (A) Shown are representative images of
SK-N-SH cells, that were transfected with siRNA directed against YAP1 and a scramble siRNA for 72 hours, and that
migrated into the cell free region (outlined) of cell monolayer at indicated time points after “wound” infliction. (B) Fold
change distances of “wound” edges at different time points (14h, 24h, 48h) after the initial scratch (0h) show reduced
mobility of cells treated with siRNA directed against YAP1, compared to scramble control. Results represent mean ± SD,
n=3 with technical duplicates each. P-values were calculated using 2way ANOVA; * 0.01 < p < 0.05.
0 h
14 h
24 h
48 h
0.00
0.25
0.50
0.75
1.00
1.25
Time [h]
Foldchange
scratch edge distance
SK-N-SH
scramble
siRNA YAP1
*
0 h
14 h
24 h
48 h
0.00
0.25
0.50
0.75
1.00
1.25
Time [h]
Foldchange
scratch edge distance
SK-N-BE
scramble
siRNA YAP1
*
A
B
0 h
14 h
24 h
48 h
0.00
0.25
0.50
0.75
1.00
1.25
Time [h]
Foldchange
scratch edge distance
IMR-5
p=0.14
scramble siRNA YAP1
0 h
14 h
24 h
48 h
0.00
0.25
0.50
0.75
1.00
1.25
Time [h]
Foldchange
scratch edge distance
SK-N-FI
p=0.12
050 100 150
0
50
100
150
Data 1
Fold change
scratch edge distance
050 100 150
0
50
100
150
Data 1
Fold change
scratch edge distance
050 100 150
0
50
100
150
Data 1
Fold change
scratch edge distance
050 100 150
0
50
100
150
Data 1
Fold change
scratch edge distance
0 h
14 h
24 h
48 h
Results
86
5.6 Influence of YAP1 knockdown on extracellular glucose and lactate
concentrations
The interplay of Hippo-YAP pathway and cellular glucose metabolism was described in several
publications over the past five years (Enzo et al., 2015; Cox et al., 2016, 2018; Hu et al., 2017;
Peng et al., 2017). To address this mutual influence in a first approach, extracellular glucose
and lactate concentrations were quantified in the cell culture medium of YAP1-perturbed
cells. The four cell lines SK-N-SH, SK-N-BE, SK-N-FI and IMR-5 were treated with either siRNAs
directed against YAP1 or with scramble control siRNAs for 72 hours and concentrations of
lactate and glucose as well as the pH value were measured in the supernatant of each
condition utilizing a blood gas analysis machine. The pH values were unaffected in all cell lines
in either scramble or YAP1 targeting siRNA samples (Figure 17 A). Furthermore, the silencing
of YAP1 revealed no alterations in extracellular glucose and lactate levels (Figure 17 B+C).
Figure 17 Unaltered pH values, lactate and
glucose levels in the supernatant of
neuroblastoma cells after YAP1 knockdown.
Blood gas analysis of cell culture media 72 hours
after YAP1 targeting siRNA transfection revealed
no alterations in extracellular (A) pH values, (B)
lactate or (C) glucose concentrations in the
supernatants of the cell lines SK-N-SH, SK-N-BE,
SK-N-FI and IMR-5. Bars represent means ± SD of
three independent experiments. No statistical
significance was assessed by using Mann-
Whitney U test.
A
B
C
SK-N-SH
SK-N-BE
IMR-5
SK-N-FI
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
pH value
scramble
siYAP1 #1
siYAP1 #2
SK-N-SH
SK-N-BE
IMR-5
SK-N-FI
0
25
50
75
100
125
150
Lactate [mg/dl]
SK-N-SH
SK-N-BE
IMR-5
SK-N-FI
0
25
50
75
100
125
150
175
200
Glucose [mg/dl]
scramble
siYAP1 #1
siYAP1 #2
Results
87
5.7 Establishment of a tetracycline-inducible overexpression system for
constitutively active YAP1S127A in neuroblastoma cell lines SH-EP and
SK-N-AS
In neuroblastoma relapse tumors, YAP1 signaling pathway was found to be overactivated
(Schramm et al., 2015). Additionally, well-established neuroblastoma cell lines from different
tumors, various tumor sites and patient-derived primary tumor samples show a wide range of
YAP1 expression levels. To determine the benefits high YAP1 activity confers on
neuroblastoma progression and relapse development, a tetracycline-inducible, so called
Tet-ON, overexpression system was established. Tetracycline treatment of single clone
cultures of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells caused an overexpression of
genetically modified human YAP1 protein (NM_001130145) with a serine to alanine exchange
that prevents a phosphorylation and subsequent protein inactivation. In a time-resolved
experiment, whole-cell lysates were harvested every 24 hours for three days and, via
immunoblot analysis, YAP1 was found to be synthesized at highest levels after 72 hours
tetracycline treatment (Figure 18). Therefore, all overexpression experiments were performed
for at least 72 hours to ensure an appropriate YAP1 activation.
Active endogenous YAP1 and YAP1S127A is translocated into the nucleus to act as a co-
transcription factor. Tetracycline-induced YAP1S127A-activated SH-EP-TR-YAP1S127A #10 as well
as ethanol-treated controls were immunostained for YAP1 and Flag-tag to display the nuclear
accumulation of Flag-tagged YAP1S127A. The technical controls were included to prove the
Figure 18 Experimental validation of a
successful tetracycline-induced YAP1
activation in SH-EP-TR-YAP1S127A cells.
Treatment of SH-EP-TR-YAP1S127A cells
with 4 µg / mL tetracycline (+Tet)
activated YAP1S127A protein already after
24 hours, and high levels were reached in
both YAP1S127A clones (#10, #14) after 72
hours. A supplemental control was
ethanol-treated in equal volumes (-Tet).
The empty vector control (LV)
constructed with Tet-repressor and a
target vector lacking the target cDNA,
showed no YAP1 induction upon
tetracycline application. The signal of
β-Actin was included as loading control.
Results
88
absence of unspecific binding of secondary antibodies anti-mouse-Alexa Fluor® 488 (Figure 19
A,B) and anti-rabbit-Alexa Fluor® 488 (Figure 19 C,D) to cells or ECM. As expected, the
N-terminally attached Flag-tag was detectable in and around the nuclei of tetracycline-treated
cells (Figure 19 G,H), but not in the ethanol controls (Figure 19 E,F). Positive signals of YAP1
protein composed of inactive phosphorylated YAP1, active YAP1 and YAP1S127A, was detected
in ethanol-treated controls (Figure 19 I,J) and tetracycline treated SH-EP-TR-YAP1S127A cells
(Figure 19 K,L). Here, a higher level of positive signals was found in the nuclei of
YAP1S127A-induced cells compared to the control cells, because overexpressed YAP1S127A
successfully accumulated in the nucleus. In summary, immunostaining of YAP1 protein and
N-terminal Flag-tag of transgenic YAP1S127A in SH-EP-TR-YAP1S127A #10 cells validated a reliable
activation of the exogenous YAP1 on the one hand and a translocation of YAP1S127A into the
nuclei on the other.
Next, the function of YAP1S127A as a transcriptional co-activator was examined. Tetracycline-
induced mRNA induction of YAP1 and its target genes CYR61, COL8A1, SKP2 and SNAPC were
Figure 19 Immunofluorescent staining of YAP1 and Flag-Tag demonstrates activated YAP1S127A
predominantly in and around the cell nuclei. (A-D) First row of pictures shows the lack of unspecific antibody
binding by secondary antibodies anti-mouse-Alexa Fluor® 488 and anti-rabbit-Alexa Fluor® 488. Detection of
the Flag-tag linked to YAP1S127A revealed lack of signal in (E, F) ethanol-treated SH-EP-TR-YAP1S127A (-Tet)
control cells compared to (G,H) tetracycline-induced YAP1S127A-expressing SH-EP-TR-YAP1S127A cells (+Tet), in
which Flag is detected, predominantly in and around the nuclei of the cells. YAP1 antibody staining revealed
positive signals in (I-L) all cells. Note the stronger signals and predominantly nuclear staining in (K, L)
SH-EP-TR-YAP1S127A (+Tet) cells overexpressing active YAP1S127A compared to (I,J) (Tet -) controls. All sections
are visualized in brightfield and green fluorescent channel.
-Tet
-Tet
-Tet
+Tet
+Tet
Flag Flag
YAP1 YAP1
2° a-mouse -Tet 2° a-rabbit
A
B
C
D
E
F
G
H
I
J
K
L
Results
89
assessed in both cell lines SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A. The qPCR data
revealed a 11.3 to 16.7-fold YAP1 mRNA increase in the YAP1S127A-expressing clones of
SK-N-AS-TR-YAP1S127A and a 4.4 to 13.6-fold induction in SH-EP-TR-YAP1S127A cells (Figure
20 A). Empty vector controls showed no YAP1 mRNA alteration upon the tetracycline
treatment. The gene expression of the chosen YAP1 target genes CYR61, COL8A1, SKP2 and
SNAPC was elevated in all YAP1-activated cells compared to ethanol-treated controls, ranging
between 1.3 to 9.2-fold induction (Figure 20 B). Thus, the inserted YAP1S127A cDNA was
translated into a fully functional enzyme and thereby was able to induce the expression of
typical target genes in both cell lines, SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A.
The availability of the inducible YAP1 overexpression system enabled a valuable control
experiment to prove specificity of siRNAs directed against YAP1 and, to the same extent, the
function of YAP1S127A. The decreased cell viability upon YAP1 knockdown in
SH-EP-TR-YAP1S127A cells should be able to be reversed by the induction of constitutively active
YAP1S127A. Thus, a cell viability assay was performed 72 hours post siRNA transfection with a
scramble control and a siRNA targeting YAP1 in SH-EP-TR-YAP1S127A cells, which were in
parallel treated with either ethanol as control or tetracycline to activate YAP1S127A (Figure 21).
**
CYR61
Col8A1
SKP2
SNAPC
1
2
4
8
16
Gene expression fold change
log2
YAP1 target genes
SK-N-AS-TR-YAP1
S127A
SH-EP-TR-YAP1
S127A
LV
#2
#9
0
3
6
9
12
15
18
YAP1 fold change expression
SK-N-AS-TR-YAP1
S127A
LV
#10
#17
0
3
6
9
12
15
18
YAP1 fold change expression
SH-EP-TR-YAP1
S127A
A
B
Figure 20 The expression of YAP1 and its target genes could be induced by tetracycline-treatment of SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A cells. (A) YAP1 expression is shown in empty vector controls (LV) and YAP1S127A-inducible clones
(#) of the two genetically modified cell lines SH-EP and SK-N-AS, n=2 (B) Expression of YAP1 target genes (CYR61, COL8A1,
SKP2 and SNAPC) was assessed in SH-EP-TR-YAP1S127A #10 and SK-N-AS-TR-YAP1S127A #2 after YAP1 induction. Bars represent
expression enrichment of YAP1-activated cells over solvent controls (means ±SD), in ratio to SDHA, n=3. The p-values were
calculated with Mann-Whitney U test; ** 0.001 < p ≤ 0.01.
Results
90
Previous cell viability assays showed a reduced cell viability in neuroblastoma cells upon YAP1
knockdown. Similarly, the cell viability of ethanol-treated SH-EP-TR-YAP1S127A could be
diminished via RNA interference. However, this effect could be rescued by inducing YAP1S127A
activation via tetracycline treatment of the same cells. This attempt validated the specificity
of siRNAs and in the same degree the functional activity of overexpressed YAP1S127A.
5.8 Transcriptome analysis of YAP1-activated neuroblastoma cells
YAP1 is a transcriptional co-activator and thus, induces target gene expression in cooperation
with various transcription factors. In order to analyze those target genes and affected signaling
pathways induced by active YAP1 in neuroblastoma cells, the transcriptome of 72 hours
YAP1S127A-activated SH-EP-TR-YAP1S127A #10 and SK-N-AS-TR-YAP1S127A #2 together with
ethanol-treated controls was sequenced. Total RNA of the cells was extracted guanidine
isothiocyanate-phenol-chloroform-based and high RNA quality (RIN
≥
9.5) was assessed via
high-resolution automated electrophoresis (Bioanalyzer).
The qPCR-based detection of YAP1 mRNA levels verified a robust tetracycline-induced gene
expression (Figure 22 A). Additionally, whole cell lysates isolated from a different aliquot of
the same samples were analyzed for YAP1 protein abundance to verify the induction of
YAP1S127A on translational level (Figure 22 B).
Figure 21 The reduced cell viability upon YAP1
knockdown could be reversed by additional YAP1S127A
activation in SH-EP-TR-YAP1S127A cells. YAP1-inducible
SH-EP-TR-YAP1S127A #10 cells were treated with siRNAs
(scramble, siYAP1 #2) and ethanol (-Tet) or tetracycline
(+Tet) for 72 hours. Cell viability data of n=2 with
technical triplicates is presented in relation to scramble
controls.
scramble -Tet
siYAP1 -Tet
scramble +Tet
siYAP1 +Tet
0.6
0.8
1.0
1.2
1.4
Relative cell viability
SH-EP-TR-YAP1S127A
Results
91
The RNA library preparation according to the Illumina TruSeq stranded mRNA protocol and
the paired-end sequencing with Illumina NextSeq 500 was performed by BIH Core Facility
Genomics (Charité Universitätsmedizin Berlin & Max Delbrück Centre for Molecular Medicine
in the Helmholtz Association). The analysis of the RNA sequencing data was kindly provided
by Dr. Filippos Klironomos.
The RNA sequencing reads were aligned to human genome assembly GRCh38 and the number
of reads per gene (transcriptome feature definition according to GENCODE v27) was
determined. Genes with a fold change expression of +/- 1.5-fold in YAP1-activated cells
compared to ethanol-treated control cells were defined as differentially expressed genes.
Fold changes of YAP1 and its target genes CYR61, COL8A1, SKP2 and SNAPC in YAP1S127A-
activated cells were compared between the RNA-sequencing data and qPCR data (Figure
22 C). Overall, the observed fold-change values highly correlated according to Pearson
correlation coefficient r=0.9998 and r=0.9765 for cell lines SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A, respectively.
SH-EP-TR-YAP1
S127A
YAP1
β-Ac0n
Tetracycline - + - + - +
Biol.Repl.n= 1 1 2 2 3 3
Tetracycline - + - + - +
Biol.Repl.n= 1 1 2 2 3 3
SK-N-AS-TR-YAP1
S127A
YAP1
β-Ac?n
A
B
SH-EP-TR-YAP1S127A
SK-N-AS-TR-YAP1S127A
0
10
20
30
40
50
YAP1mRNAexpression
foldchange
Figure 22 YAP1 activation in RNA sequencing samples. (A) Transcriptional YAP1 levels in YAP1-induced SH-EP-TR-YAP1S127A
#10 and SK-N-AS-TR-YAP1S127A #2 are shown with bars representing means ± SD of relative YAP1 enrichment in activated cells
over ethanol control, n=3. (B) Corresponding immunoblots of YAP1 and loading control β-Actin are depicted in three
independent replicates (1-3), activated with tetracycline (+) or treated with solvent ethanol (-) for 72 hours. (C) Comparison
of qPCR and transcriptome data of YAP1 and its target genes CYR61, COL8A1, SKP2 and SNAPC revealed a high correlation
(R2=coefficient of determination) between both datasets. Data are presented as means of gene-enrichment of YAP1-
activated cells over ethanol-treated controls. FKPM=fragments per kilobase million.
124816 32 64
1
2
4
8
16
32
64
qRT-PCR fold change log2
FPKM fold change log2
SK-N-AS-TR-YAP1
S127A
SH-EP-TR-YAP1
S127A
R
2=0.8565
p<0.0001
R2=0.9960
p<0.0001
C
050 100 150
0
50
100
150
Data 1
qPCR fold change log
2
Results
92
A principal component analysis of the RNA-sequencing data separated the two cell lines
perfectly at the first principal component (Figure 23 A, x-axis). The second principal
component strongly separated YAP1-overexpressing (tetracycline-induced) versus non-
induced (ethanol-treated) samples (Figure 23 A, y-axis), indicating a strong impact of YAP1
expression on the expression of other genes. Exemplarily, the MA plot of
SK-N-AS-TR-YAP1S127A (Figure 23 B) shows genes defined as significantly up- or downregulated
by a fold change of 1.5 or higher. Upon YAP1 activation, 846 and 525 genes were significantly
upregulated in SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A, respectively (Figure 23 C). Only
185 common genes were significantly upregulated in both. Diminished expression levels have
SH-EP-TR-YAP1
S127A
SK-N-AS-TR-YAP1
S127A
Tet–
Tet+
Upregulatedgenes
525 185 846
Downregulatedgenes
740 75 741
SH-EP-TR-YAP1
S127A
SK-N-AS-TR-YAP1
S127A
0.1 10.0 1000.0 100000.0
−10
−5
0
5
10
Mean counts
Treatment Tet vs Control
log
2
fold change
YAP1
COL12A1
PAPPA
DBH
THBS1
TMOD1
AC136621.1
MFAP5
MARCH4
PLEKHA6
PSG5
KALRN
PODXL
MATN2
CPA4
AXL
NPPB
ANXA3
AP001803.2
AHNAK
TMEM108−AS1
SHD
UPK1B
SLC8A2
AP000942.2
CLIC5
RHBG
RGS5
CHGA
COL1A1
MEST
KRT8
AC046195.1
GNAS
TRPA1
PRSS12
Figure 23 Genes are differentially expressed upon YAP1S127A activation in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
cells. (A) A principal component analysis separated the RNA-sequencing data perfectly well according to cell line identity at
the first principal and treatment with YAP1-activating tetracycline (+Tet) or solvent control ethanol (-Tet) at principal
component 2. Variances between biological replicates were small. (B) A MA-plot of SK-N-AS-TR-YAP1S127A exemplarily shows
differentially expressed genes (red dots), defined by a 1.5-fold or higher upregulation or downregulation (C) The quantity of
significantly up- and downregulated genes reveal a minimal overlap of genes regulated similarly in both cell lines.
A
B
C
Results
93
been detected for almost the same number of genes (740 and 741 genes) in both cell lines.
Only 75 genes were downregulated in both SH-EP-TR-YAP1S217A and SK-N-AS-TR-YAP1S127A. All
gene names with corresponding p-values can be found in the supplementary section. The
relatively low quantity of bilaterally YAP1-regulated genes prompted us to analyze i) genes
affected similarly in both cell lines and ii) gene sets affected exclusively in one cell line only.
This approach could enlighten general regulatory mechanisms of YAP1 in neuroblastoma, but
also considers cell-type specific prerequisites.
5.9 Gene ontology enrichment analysis of YAP1-activated neuroblastoma
cells
In order to find pathways affected by YAP1 induction in SH-EP-TR-YAP1S127A (Table 23) and
SK-N-AS-TR-YAP1S127A (Table 24) cells, the list of differentially expressed genes were tested for
significantly enriched Gene Ontology (GO) terms. Here, all genes with base mean≥10 were
used as background and a cut-off level with p<0.0001 was applied to define these significantly
enriched GO terms. Regarding these conditions, 47 induced biological processes and 19
inhibited ones were listed in SK-N-AS-TR-YAP1S127A. The analysis of genes that were
upregulated or downregulated in SH-EP-TR-YAP1S127A cells only revealed 16 GO terms in total.
Numerous migration-associated genes were upregulated in SK-N-AS-TR-YAP1S127A cells upon
YAP1 activation, whereas genes regulating the de novo serine biosynthesis were induced in
SH-EP-TR-YAP1S127A cells.
Table 23 Gene Ontology analysis of up- and downregulated genes in YAP1-induced SH-EP-TR-YAP1S127A 1.
GO terms upregulated genes
Gene no.
p-value
GO terms downregulated genes
Gene no.
p-value
cell-matrix adhesion
25
3.84-05
immune response
106
1.9E-05
positive regulation of protein kinase B signaling
19
5.3E-08
cell-cell signaling
91
2.1E-05
response to glucocorticoid
16
9.2E-06
proteolysis
68
2.5E-05
positive regulation of angiogenesis
16
6.4E-05
inflammatory response
47
1.1E-07
intrins. apoptot. signaling pathway in response to ER stress
12
9.5E-06
extracellular matrix organization
45
3.7E-06
serine family amino acid biosynthetic process
7
3.9E-06
regulation of receptor activity
33
3.0E-06
L-serine metabolic process
6
1.2E-06
extracellular matrix disassembly
19
4.4E-09
CD4-positive. alpha-beta T cell cytokine production
5
3.3E-05
collagen catabolic process
14
3.2E-08
Results
94
Table 24 Gene Ontology analysis of up- and downregulated genes in YAP1-induced SK-N-AS-TR-YAP1S127A 1.
GO terms upregulated genes
Gene
no.
p-value
GO terms downregulated genes
Gene
no.
p-value
signal transduction
383
5.2E-05
nervous system development
187
1.8E-05
regulation of cell migration
111
3.6E-06
G-protein coupled receptor signaling pathway
66
3.5E-05
cell-cell-adhesion
83
6.7E-06
chemical synaptic transmission
62
1.2E-06
negative regulation of cell proliferation
75
6.1E-08
axonogenesis
50
2.2E-05
angiogenesis
72
2.5E-09
response to drug
33
5.3E-06
wound healing
70
2.9E-07
axon guidance
30
1.1E-06
extracellular matrix organization
69
9.7E-15
Homophil. cell adhes. via plasma membr. adh. mol.
18
1.1E-05
positive regulation of cell migration
63
1.4E-05
excitatory postsynaptic potential
16
1.5E-05
regulation of receptor activity
59
2.6E-11
palate development
13
4.1E-05
positive regulation of cell adhesion
55
8.5E-05
sympathetic nervous system development
10
1.7E-09
leukocyte migration
46
1.7E-08
response to nicotine
10
7.1E-07
skin development
42
2.3E-05
neural nucleus development
9
2.5E-06
cell-matrix adhesion
41
1.1E-07
cell fate determination
8
1.3E-05
muscle contraction
38
1.3E-05
cell differentiation in hindbrain
7
4.2E-06
negative regulation of cell migration
37
2.7E-05
response to pain
7
2.4E-05
response to hypoxia
33
9.4E-07
nose development
6
3.9E-05
lung development
31
2.1E-08
cerebellar cortex formation
6
9.0E-05
response to peptide hormone
29
2.5E-05
startle response
6
9.0E-05
positive regulation of epithelial cell migration
26
2.2E-05
regulation of synapse maturation
5
5.0E-05
positive regulation of cell-substrate adhesion
25
1.0E-08
keratinocyte differentiation
25
5.7E-07
female pregnancy
24
5.2E-06
positive regulation of angiogenesis
23
3.2E-07
positive regulation of protein kinase B signaling
22
1.1E-07
tissue remodeling
22
5.9E-05
viral entry into host cell
19
5.6E-06
extracellular matrix disassembly
18
2.5E-07
collagen catabolic process
17
1.2E-08
phosphatidylinositol phosphorylation
17
2.7E-06
Regul. of plasma membrane bounded cell projection assembly
17
6.6E-05
positive regulation of endothelial cell migration
16
9.5E-06
negative regulation of angiogenesis
16
2.9E-05
response to calcium ion
15
3.0E-05
positive regulation of smooth muscle cell proliferation
14
5.3E-06
positive regulation of inflammatory response
14
4.6E-05
vasculogenesis
13
5.7E-05
collagen fibril organization
11
1.5E-05
mammary gland morphogenesis
11
3.5E-05
regulation of gastrulation
9
1.5E-05
cornification
9
7.1E-05
microvillus assembly
7
2.3E-05
negative regulation of chondrocyte differentiation
7
3.7E-05
peptide cross-linking
7
5.7E-05
branching involved in salivary gland morphogenesis
7
8.5E-05
regulation of odontogenesis of dentin-containing tooth
6
9.1E-06
glomerular mesangium development
6
3.6E-05
negative regulation of muscle adaption
6
3.6E-05
1 migration-associated genes (green) and serine/glycine synthesis-associated genes (brown)
5.10 Effect of YAP1 induction on tumor cell growth
The first part of this work has shown, that YAP1 knockdown can cause inhibition of
proliferation and cell vitality in neuroblastoma cells. In turn it was questioned, if
neuroblastoma cells could benefit from YAP1 activation with regard to cell division, cell growth
and apoptosis regulation, which would end up with space-occupying tumor growth and tumor
progression in a full-featured biological system.
Results
95
The cell viability of bulk SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells, either YAP-
induced by tetracycline treatment (+Tet) or only treated with the solvent ethanol as non-
induced control (-Tet), was assessed with CellTiter-Glo® assay (Figure 24 A) and found to be
unaffected by YAP1S127A overexpression. After single clone selection of both cell lines,
YAP1-induced and ethanol-treated control cells were counted manually, and viable cell
numbers were found to be unaltered (Figure 24 B). Additionally, an ELISA-based BrdU
proliferation assay was performed with inducible clones as well as empty vector controls of
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells (Figure 25). The unaffected cell
proliferation upon YAP1 activation was in line with previous cell viability data. The application
of tetracycline minimally reduced the signal in all three SH-EP-TR-YAP1S127A clones, but this
was also true for the empty vector control and thereby not a YAP1S127A-specific effect.
SH-EP-TR-YAP1
S127A
-Tet
+Tet
0.0
5.0×106
1.0×107
1.5×107
2.0×107
2.5×107
3.0×107
3.5×107
Cell viablity [RLU]
Figure 24 Cell viability and cell numbers are not affected in YAP1-activated cells. (A) Results of an ATP-based cell viability
assay in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A bulk cells, 72 hours after YAP1 activation, are shown. Data
represent means ± SD of three independent experiments, each measured in technical triplicates. (B) Cell numbers of
tetracycline-treated (+Tet) SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A clones and empty vector controls (LV) are
represented relative to ethanol treated (-Tet) cell numbers, means ± SD of three independent experiments, each
measured in technical triplicates. Data were assessed as statistically not significant by using Mann-Whitney U test. RLU =
relative luminescence units.
LV
#10
#17
0.0
0.5
1.0
1.5
SH-EP-TR-YAP1
S127A
Cell numbers,
relative to EtOH control
LV
#2
#9
0.0
0.5
1.0
1.5
SK-N-AS-TR-YAP1S127A
Cell numbers,
relative to EtOH control
A
B
EV
#1
#2
0.0
0.5
1.0
1.5
Cell numbers
SK-N-AS-YAP
S127A
Cell numbers,
relative to Tet - control
EV
#1
#2
0.0
0.5
1.0
1.5
Cell numbers
SK-N-AS-YAP
S127A
Cell numbers,
relative to Tet - control
SK-N-AS-TR-YAP1S127A
-Tet
+Tet
0.0
5.0×106
1.0×107
1.5×107
2.0×107
2.5×107
3.0×107
3.5×107
Cell viablity [RLU]
Results
96
In parallel, flow cytometric PI/Annexin V apoptosis assay and cell cycle analysis was performed
with 72 hours YAP1-activated cell lines. Apoptotic Annexin V positive cells ranged between
2-4% of the whole population over all conditions (Figure 26 A). Dead cells, that were identified
via positive signals for both PI and Annexin V, accounted for 1-2% of the cell population and
were also not affected by YAP1 activation. The cell cycle of SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A, assessed by the total DNA-content of the cells, revealed no regulation
of cell cycle phases upon YAP1 overexpression (Figure 26 B). SH-EP-TR-YAP1S127A cells
harbored 14-25% of all single cells in the DNA-synthesis, so called S-phase, and around 4-11%
were detected in the transition phase G2/M. SK-N-AS-TR-YAP1S127A cells displayed a similar
distribution with a few more cells persisting in the G2/M phase (12-17%).
LV #10 #14 #17
0.0
0.5
1.0
1.5
Absorbance (A
370nm
- A
492nm
)
SH-EP-TR-YAP1
S127A
- Tet
+ Tet
LV #4 #9
0.0
0.2
0.4
0.6
0.8
Absorbance (A
370nm
- A
492nm
)
SK-N-AS-TR-YAP1
S127A
- Tet
+ Tet
Figure 25 Cell proliferation is not
altered in YAP1-activated cells.
BrdU-ELISA of different
SH-EP-TR-YAP1S127A and SK-N-AS-
TR-YAP1S127A clones (#) and
empty vector controls (LV) after
72 hours treatment with
tetracycline (+Tet) or solvent
ethanol (-Tet). Bars represent
means ± SD of
n=3 (SK-N-AS-TR-YAP1S127A) and
n=5 (SH-EP-TR-YAP1S127A) with
technical triplicates each. Data
were assessed as statistically not
significant by using Mann-
Whitney U test.
Results
97
LV - Tet
LV + Tet
# 10 - Tet
# 10 + Tet
# 14 - Tet
# 14 + Tet
0
2
4
6
8
Apoptotic / dead cells [%]
SH-EP-TR-YAP1S127A
Dead cells
Apoptotic cells
LV - Tet
LV + Tet
# 2 - Tet
# 2 + Tet
# 9 - Tet
# 9 + Tet
0
2
4
6
8
Apoptotic / dead cells [%]
SK-N-AS-TR-YAP1
S127A
Dead cells
Apoptotic cells
LV - Tet
LV + Tet
#10 - Tet
#10 + Tet
#14 - Tet
#14 + Tet
0
20
40
60
80
100
Cell cycle distribution [%]
SH-EP-TR-YAP1
S127A
Sub G1
G0/G1
S
G2/M
LV - Tet
LV + Tet
#2 - Tet
#2 + Tet
#9 - Tet
#9 + Tet
0
20
40
60
80
100
Cell cycle distribution [%]
SK-N-AS-TR-YAP1S127A
subG
G0/G1
S
G2/M
Figure 26 Apoptosis and cell cycle analysis revealed no effect of YAP1 activation in neuroblastoma cells. Flow
cytometry analysis of YAP1-activated (+Tet) and ethanol-treated (-Tet) control cells of SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A clones revealed (A) constant proportions of PI /Annexin V double-positive dead cells and Annexin
V single-positive apoptotic cells. (B) YAP1 activation did not affect the distribution of the cell cycle phases, calculated by
cellular DNA-content. Bar plots represent means ± SD of n=3. Data were assessed as statistically not significant by using
Mann-Whitney U test.
A
B
Results
98
Not only the upregulation of the cell proliferation rate by an oncogene, but also the potential
of cells to undergo unlimited cell divisions contributes to tumor progression and maintenance.
Especially after first line tumor treatment diminishing the main tumor body by surgery or
chemotherapy, recurrent tumor masses develop from minimal residual disease and single
outgrowing cancer cells. Therefore, YAP1-activated cells were cultured starting at single cell
level and colonies that formed after 6-8 days were stained with crystal violet to explore, if
YAP1 activation alters the clonogenic potential of neuroblastoma cells.
The areas populated by colonies of YAP1-activated SH-EP-TR-YAP1S127A (Figure 27 A) cells
equal the ones of solvent control cells and empty vector controls. In SK-N-AS-TR-YAP1S127A
(Figure 27 B), tetracycline application increased the number of growing colonies in empty
vector controls 1.6-fold. An approximately 2.2-fold higher colony formation was observed in
ethanol-treated SK-N-AS-TR-YAP1S127A clone #2 compared to tetracycline-induced cells and no
effect was found in clone #9 cells.
LV
LV
#2
#2
#9
#9
0.0
5.0×10
2
1.0×10
3
1.5×10
3
2.0×10
3
2.5×10
3
3.0×10
3
3.5×10
3
4.0×10
3
Colony formation (ROI [px2])
SK-N-AS-TR-YAP1
S127A
- Tet
+ Tet
LV
LV
#10
#10
#17
#17
0.0
2.5×10
4
5.0×10
4
7.5×10
4
1.0×10
5
1.3×10
5
1.5×10
5
Colony formation (ROI [px
2
])
SH-EP-TR-YAP1
S127A
- Tet
+ Tet
LV #10 #17
EtOH
Tet
EtOH
Tet
LV #2 #9
Figure 27 Clonogenicity of
neuroblastoma cells is not affected
upon YAP1 activation. Clonogenic
assay was performed with YAP1-
activated cells (+Tet) and ethanol
treated (-Tet) controls of (A)
SH-EP-TR-YAP1S127A and (B)
SK-N-AS-TR-YAP1S127A clones (#) and
empty vector controls (LV). Colonies
were fixed and stained with crystal
violet after 6 (SH-EP-TR-YAP1S127A)
or 8 (SK-N-AS-TR-YAP1S127A) days.
The boxes represent total ROIs of
blue colonies per well, n=4. On the
right-handed side, representative
growth areas populated by colonies
of control and YAP1-activated cells
are shown. Data were assessed as
statistically not significant by using
paired Wilcoxon test.
A
B
- Tet
+ Tet
- Tet
+ Tet
Results
99
In early 2019, a publication from He et al. described active YAP1 as a potential positive
regulator of cell senescence via a YAP1-LATS feedback loop in human ovarian surface epithelial
cells (He et al., 2019). Thus, a senescence-associated β-galactosidase activity assay was
performed 14 days after YAP1 activation in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
(Figure 28). In all conditions, senescent cells upon YAP1 activation could not be identified.
Additionally, senescence-accompanying morphological changes could not be observed.
Taken together, two neuroblastoma cell lines, genetically modified to be inducible for
constitutively active YAP1S127A, were analyzed in the manner of cell proliferation, viability,
apoptosis, clonogenicity as well as senescence. Tumorigenic growth of neuroblastoma cell
lines was not affected by induced high active YAP1 levels. Also, transcriptome data of
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells did not reveal induction of cell cycle-
regulating or proliferation-promoting gene sets after YAP1 activation, which perfectly
complements our findings.
5.11 Enhanced cell migration in YAP1-activated neuroblastoma cells
Besides regulating cell cycle and proliferation, YAP1 is also known to promote cell migration
and thereby contribute to manifestation of metastasis in various tumor entities such as
melanoma, mammary carcinoma, lung cancer, ovarian cancer, hepatocellular carcinoma and
SH-EP-TR-YAP1
S127A
-Tet +Tet
#10#10
#17#17
SK-N-AS-TR-YAP1
S127A
-Tet +Tet
#2#2
#9#9
Figure 28 YAP1 overexpression does not induce senescence. Representative pictures of tetracycline-induced (+Tet)
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells as well as ethanol-treated controls (-Tet) are shown. The cells were
treated with X-Gal to detect senescence-associated
𝜷
-galactosidase 14 days after YAP1 activation, but no cell senescence
could be detected, n=3.
Results
100
gastric cancer (Lamar et al., 2012; Lau et al., 2014; Xia et al., 2014; Liu et al., 2016; Qiao et al.,
2017; Shi et al., 2018). Aiming for the elucidation of YAP1 function on migratory capacities of
neuroblastoma cells, cell migration and migration-related gene expression changes were
analyzed in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells.
In a first approach, the quick and cost-effective wound-healing assay was performed with the
SH-EP-TR-YAP1S127A clones and empty vector controls (Figure 29 A) as described in section 5.5.
The SH-EP-TR-YAP1S127A cells demonstrated enhanced, albeit not necessarily statistically
significant, migration into the cell free region upon YAP1 activation compared to the solvent
and empty vector controls (Figure 29 B).
After demonstrating a tendency of increased cell motility of YAP1-activated
SH-EP-TR-YAP1S127A cells, the more conclusive transwell migration assay was conducted with
both tetracycline-inducible cell lines, SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A. Cells
were seeded into the transwell and after 20 hours (SH-EP-TR-YAP1S127A) or 24 hours
(SK-N-AS-TR-YAP1S127A), migrated cells settled on the bottom site of the membrane were
fixated and visualized with crystal violet staining. The cells migrated non-homogeneously
through the membrane, resulting in areas with very low cell numbers as well as areas with an
uncountable cell layer (Figure 30 A). Thus, blue stained, migrated cells were detected as a
0h
24h
-Tet +Tet
LV
LV
#10
#10
#14
#14
#17
#17
0.0
0.2
0.4
0.6
0.8
Scratch distance (foldchange)
SH-EP-TR-YAP1
S127A
- Tet
+ Tet
*
Figure 29 Cell motility is elevated in YAP1-activated SH-EP-TR-YAP1S127A cells. In vitro wound-healing assay of
SH-EP-TR-YAP1S127A cells treated with tetracycline (+Tet) or ethanol (-Tet) was performed and distances of “wound” edges
were measured 24 hours after the initial scratch (A) Representative images are shown of SH-EP-TR-YAP1S127A #10 cells
migrated into the cell free region. (B) Distances of wound edges are given as fold change of the initial scratch (0h). Results
represent mean ± SD, n=3. Mann-Whitney U test was used to assess statistical significance; p* 0.01 < p ≤ 0.05.
A
B
050 100 150
0
50
100
150
Data 1
Fold change
scratch edge distance
Results
101
„region of interest“ (ROI) in 5 recurrent spots on every membrane, followed by comparison of
these ROIs between the two groups of tetracycline-induced YAP1-activated cells and the
ethanol treated solvent controls (Figure 30 B). As expected, the migration of empty vector
controls in both cell lines was not affected by the treatment with tetracyline. The SH-EP-TR-
YAP1S127A #17 cells showed a significantly higher migration capacity with induced high YAP1
levels, whereas #10 cells revealed no significant change upon YAP1 induction. In both
SK-N-AS-TR-YAP1S127A clones, a moderate, albeit not statistically significant (due to high
standard deviations of the measurements), increase of migratory cells was detected upon
YAP1 overexpression.
As described above, genes that were upregulated upon YAP1 activation included a significant
number of genes associated with cell-motility related GO terms (see section 5.9). Genes
affecting cytoskeletal remodeling (actins, tyrosine kinases) and interactions between cells and
surrounding ECM (collagens, laminins, fibronectin, integrins, growth factors) were
upregulated in both cell lines (Figure 31).
low mediumlow mediumhigh high
LV #10 #17
0
2×10
4
4×10
4
6×10
4
8×10
4
ROI (px2)
SH-EP-TR-YAP1S127A
- Tet
+ Tet
*
LV #4 #9
0.0
5.0×104
1.0×105
1.5×105
2.0×105
ROI (px
2
)
SK-N-AS-TR-YAP1
S127A
- Tet
+ Tet
p=0.1
Figure 30 Neuroblastoma cell migration is increased upon YAP1 activation. Transwell migration assay of SH-EP-TR-YAP1S127A
and SK-N-AS-TR-YAP1S127A (A) Representative pictures of low up to high numbers of SH-EP-TR-YAP1S127A cells migrated
through the transwell membrane. (B) Analysis of the area (ROI) populated by the migrated cells. Analysis included three
independent experiments with empty vector controls (LV), ethanol controls (-Tet) and tetracycline (+Tet) induced YAP1-
activated cells. Statistical analysis was performed with Mann-Whitney-Test; * 0.01 < p ≤ 0.05.
A
B
Results
102
TWIST2
SLUG
SPARC
FN1
LAMC1
LAMC2
COL1A1
COL3A1
COL4A1
COL4A4
COL4A5
COL5A2
COL8A1
COL12A1
COL13A1
COL15A1
COL17A1
COL18A1
ACTN1
ACTN4
NEXN
DLC1
PDGFRB
ABL2
2
0
2
5
2
10
2
15
2
20
Gene expression, normalized counts [log
2
]
EMT Extracellular matrix assembly Cytoskeletal rearrangements
##
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
°
#
#
#
#
####
#
THBS1
AMOTL2
ITGA2
ITGA3
ITGA6
ITGB2
ITGB3
ITGB5
ITGB8
ITGBL1
KLF4
L1CAM
SEMA3E
PRKCE
DDR2
FGF1
FGF2
FGF5
FGFR1
FGFR4
TGFBR1
TGFBR2
TGFB2
CAMK2D
ADGRG1
2
0
2
5
2
10
2
15
2
20
Gene expression, normalized counts [log
2
]
Cell - cell / cell - ECM interactions
#
#
##
#
#
##
#
#
°
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
##
°
MMP2
MMP3
MMP7
MMP9
MMP10
MMP11
MMP13
MMP16
MMP17
MMP19
MMP24
MMP28
ADAM8
ADAM9
ADAM12
ADAM19
ADAMTS1
ADAMTS12
ADAMTS15
ADAMTS16
SERPINE1
SERPINF2
CTSH
CTSL
CTSS
PLAU
20
25
210
215
220
Gene expression, normalized counts [log
2
]
Proteases
SH-EP-TR-YAP1S127A Tet - SH-EP-TR-YAP1S127A Tet + SK-N-AS-TR-YAP1S127A Tet - SK-N-AS-TR-YAP1S127A Tet +
#
#
°
#
#
°
#
#
##
°
#
#
°
°°
°°
#
°
°
#
°
°
#
°
#
°
#
Figure 31 Migration-associated genes are upregulated in YAP1-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
cells. Significantly upregulated (hash) or downregulated (circle) genes (p<0.0001) that are included in migration-related GO
terms of transcriptome data analysis of YAP1-activated (+Tet) or ethanol control (-Tet) SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells are presented as normalized counts. EMT = epithelial-mesenchymal transition; ECM = extracellular
matrix.
1
0
1
2
3
4
5
Data 1
SK-N-AS-TR-YAP1
S127A
Tet -
SK-N-AS-TR-YAP1
S127A
-Tet SK-N-AS-TR-YAP1
S127A
+Tet SH-EP-TR-YAP1
S127A
-Tet SH-EP-TR-YAP1
S127A
+Tet
Results
103
Genes that promote the EMT (TWIST2, SLUG, SPARC) were only induced in
SK-N-AS-TR-YAP1S127A and not in SH-EP-TR-YAP1S127A cells. Proteases such as matrix
metalloproteases (MMP), cathepsins (CTS), a disintegrin and metalloproteinases (ADAM) and
ADAM with thrombospondin motifs (ADAMTS) were upregulated in YAP1-activated
SK-N-AS-TR-YAP1S127A and mainly unaffected or even downregulated in SH-EP-TR-YAP1S127A.
Taken together, transcriptome data from two YAP1-inducible neuroblastoma cell lines
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A revealed a long list of significantly affected
genes positively regulating the migratory capacitiy of these cells upon overexpression of
constitutively active YAP1S127A. Experimental data from migration assays verified a higher
potential of YAP1-activated neuroblastoma cells to close wounds and migrate actively towards
an attractant in both cell lines. Interestingly, genes related to EMT and matrix-degradation
were exclusively induced in YAP1-activated SK-N-AS-TR-YAP1S127A cells.
5.12 YAP1 activation induces a mesenchymal/neural crest cell-like phenotype
in SK-N-AS-TR-YAP1S127A
In addition to recurrent genetic aberrations, distinct cell populations were identified in
neuroblastoma cells. One model assumes at least two phenotypes that co-exist within a tumor
and are defined by gene expression profiles, one of which entailed YAP1 (see section 1.2.6).
In order to investigate, whether YAP1 substantially contributes to one phenotype, mRNA
-6
-4
-2
0
2
4
6
log2 fold change
SH-EP-TR-YAP1
S127A
MES ADRN
-6
-4
-2
0
2
4
6
log2 fold change
SK-N-AS-TR-YAP1
S127A
MES ADRN
Figure 32 Assignment of YAP1-induced differentially expressed genes to MES and ADRN gene signatures. Differentially
expressed genes in YAP1-induced SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A, that are included in the published gene
expression signatures of 485 mesenchymal genes (MES) or 369 adrenergic genes (ADRN) (van Groningen, 2017), are depicted
as gene expression fold changes to solvent control. Means for differentially expressed signature genes (orange dotted line)
revealed an induction of MES genes in SK-N-AS-TR-YAP1S127A accompanied by a decrease of ADRN signature.
SH-EP-TR-YAP1S127A cells exhibited a minor and more balanced distribution of both gene expression profiles in the
differentially expressed genes.
Results
104
signatures described by Van Groningen et al. (Van Groningen et al., 2017) were compared to
the transcriptome data of YAP1-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
(Figure 32). In SK-N-AS-TR-YAP1S127A, 177 of 485 genes assigned as MES-genes were
significantly upregulated upon YAP1 activation, whereas 88 of 369 ADRN-genes were
significantly downregulated, indicating an identity shift towards the mesenchymal phenotype
upon YAP1 activation. In contrast, tetracycline-induced YAP1S127A expression in
SH-EP-TR-YAP1S127A revealed 29 significantly upregulated MES genes and 17 decreased ADRN-
genes only (find a detailed list in the supplementary section).
In order to strengthen the assumption of a YAP1-mediated phenotype switch in
neuroblastoma cells, another gene expression data set was compared to the transcriptome
data of SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A cells. Boeva et al. defined the “core
regulatory circuitries” (CRCs) (Boeva et al., 2017) that consist of TF genes that are specifically
upregulated in the two cell phenotypes of neuroblastoma (Figure 33). The gene expression
values of TFs comprised in the CRC of noradrenergic cells were reduced in YAP1-activated
SK-N-AS-TR-YAP1S127A compared to ethanol control cells, whereas simultaneously the
mesenchymal TF module was strongly upregulated. YAP1-induced SH-EP-TR-YAP1S127A cells
showed a different transcriptional regulation lacking a clear preference for a CRC.
Taken together, the analysis of data sets originated from neuroblastoma tumors and cell lines
as well as two genetically modified YAP1-overexpressing cell lines revealed, that an activation
of the transcriptional co-activator YAP1 in neuroblastoma cells is able to induce genes, that
are assigned as mesenchymal, in a cell-context dependent manner.
Figure 33 RNA expression of transcription factors
defines neuroblastoma subpopulations in YAP1-
activated cells. Transcription factors of core
regulatory circuitries, that are described to define an
adrenergic (grey) or mesenchymal/ neural crest cell
(NCC)-like (green) subpopulation within
neuroblastoma tumors, are affected by YAP1
induction in SH-EP-TR-YAP1S127A and SK-N-AS-TR-
YAP1S127A cells. In SK-N-AS-TR-YAP1S127A, TFs
determining the mesenchymal cells were significantly
upregulated, whereas adrenergic module was
strongly inhibited. SH-EP-TR-YAP1S127A cells behaved
marginally opposite upon YAP1 activation. Data
extracted from RNA sequencing analysis are
presented as log2 fold change expression from three
independent experiments.
21-1 0
Colorkey
Log2foldchange
SH-EP-TR-
YAP1
S127A
SK-N-AS-TR-
YAP1
S127A
1,0604 -1,3570 PHOX2A
1,6441 -1,1126 PHOX2B
0,4690 -1,0411 GATA3
0,8842 0,0000 HAND1
0,2064 -0,1274 HAND2
0,0000 -1,0066 ISL1
-0,4211 -0,3874 KLF7
0,3344 2,7368 RUNX1
-0,2499 1,9306 RUNX2
1,4312 2,1440 FOSL1
-0,1457 1,7966 FOSL2
0,1049 0,6808 TBX18
-0,1290 1,1833 NR3C1
-0,2664 0,9507 IRF1
-0,2372 0,4278 IRF2
-0,0893 0,7700 GLIS3
-0,4425 -0,3924 PRRX1
noradrenergic NCC-like
SH-EP-TR-
YA P 1
S127A
SK-N-AS-TR-
YA P 1
S127A
1.0604 -1.3570
PHOX2A
Noradrenergic
1.6441 -1.1126
PHOX2B
0.4690 -1.0411
GATA3
0.8842 0.0000
HAND1
0.2064 -0.2174
HAND2
0.0000 -1.0066
ISL1
-0.4211 -0.3874
KLF7
0.3344 2.7368
RUNX1
Mesenchymal / NCC-like
-0.2499 1.9306
RUNX2
1.4312 2.1440
FOSL1
-0.1457 1.7966
FOSL2
0.1049 0.6808
TBX18
-0.1290 1.1833
NR3C1
-0.2664 0.9507
IRF1
0.0237 0.4278
IRF2
-0.0893 0.7700
GLIS3
-0.4425 -0.3924
PRRX1
Results
105
5.13 Induction of genes regulating serine de novo synthesis, folate cycle and
cysteine synthesis in YAP1-activated SH-EP-TR-YAP1S127A
Gene-set enrichment analysis of YAP1-induced SH-EP-TR-YAP1S127A cells revealed two
upregulated gene sets subjecting the serine family amino acid biosynthesis (Table 23). A more
detailed analysis of genes that were included in these GO terms revealed a significant
PHGDH
PSAT1
PSPH
SHMT1
SHMT2
MTHFD1
MTHFD1L
MTHFD2
MTHFD2L
MTHFR
CBS
CTH
20
25
210
215
220
Gene expression, normalized counts [log2]
Serine
synthesis
Folate cycle Cysteine
synthesis
#
#
#
#
#
#
°
#
#
Figure 34 Genes regulating
serine/cysteine synthesis and the folate
cycle are upregulated in YAP1-activated
SH-EP-TR-YAP1S127A. (A) Significantly
upregulated (hash) or downregulated
(circle) genes (p<0.0001) of YAP1-activated
(+Tet) or ethanol control (-Tet)
SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells, presented as
normalized counts from RNA-sequencing
data. (B) Schematic illustration of pathway
interaction: enzyme (boxes) and
intermediates of serine and cysteine
synthesis as well as folate cycle are shown.
Significantly up- and downregulated genes
in SH-EP-TR-YAP1S127A are indicated as blue
boxes. THF = tetrahydrofolate, 5,10-meTHF
= 5,10-methyleneTHF, 5-meTHF = 5-
methylTHF, PHGDH = phosphoglycerate
dehydrogenase, PSAT = phosphoserine
aminotransferase, PSPS = phosphoserine
phosphatase, CTH = cystathionine γ-lyase,
CBS = cystathionine β-synthase, SHMT1/2 =
serine hydroxy methyltransferase 1/2,
MTHFD1/2(L) = methyleneTHF
dehydrogenase 1/2 (like), MTHFR =
methyleneTHF reductase, MTFMT =
mitochondrial methionyl-tRNA formyl
transferase, ALDH1L2 = Aldehyde
dehydrogenase 1 family member L2, TYMS
= thymidylate synthetase. (Figure adapted
from Ducker and Rabinowitz, 2017)
A
B
SK-N-AS-TR-YAP1S127A -Tet
SK-N-AS-TR-YAP1S127A +Tet
SH-EP-TR-YAP1S127A -Tet
SH-EP-TR-YAP1S127A +Tet
SK-N-AS-TR-YAP1S127A -Tet
SK-N-AS-TR-YAP1S127A +Tet
SH-EP-TR-YAP1S127A -Tet
SH-EP-TR-YAP1S127A +Tet
5-meTHF
PHGDH
Glucose
3-Phosphoglycerate
3-Phosphohydroxypyruvate
PSAT
Phosphoserine
Serine Serine
THF
5,10-meTHF
5,10-meTHF
10-formylTHF 10-formylTHF
Formate
PSPH
SHMT2SHMT1
MTHFD1L
MTHFD1
MTHFD1 MTHFD2/L
Glycine
Glycine
ALDH1L2
MTFMT
formylMet
MTHFR
Cytosol Mitochondria
Serine
Cystathionine
Cysteine
CBS
CTH
Serine
biosynthesis
Folate
cycle
Transsulfuration
pathway
Homocysteine
Methionine
cycle
TYMS
Thymidine
synthesis
NADH/NADPH
NAD+/NADP+
NADP+
NADPH
NADPH
NADP+
ALDH1L1
CO2
NADP+
NADPH
CO2
Purine
synthesis
NAD+
NADH
Glu
!KG
Upreg. in SH-EP-TR-YAP1S127A
Downreg. in SH-EP-TR-YAP1S127A
Results
106
upregulation of serine de novo synthesis pathway genes PHGDH, PSAT1 and PSPH (Figure
34 A). Two pathways that are dependent on intracellular serine levels were also upregulated
on transcriptional level: on the one hand, CBS and CTH were induced, which stepwise convert
serine and homocysteine to cysteine in the transsulfuration pathway, and on the other hand
mRNAs of proteins regulating the mitochondrial part of the folate cycle, SHMT2, MTHFDL1
and MTHFD2, were upregulated (Figure 34 B). None of these gene expression changes could
be detected in SK-N-AS-TR-YAP1S127A cells.
5.14 YAP1 enhances amino acid metabolism in neuroblastoma cells
In SH-EP-TR-YAP1S127A cells, an activation of YAP1 induced the expression of genes regulating
the de novo serine synthesis, the folate cycle and the transsulfuration pathway, which all
participate in the generation of amino acids serine, glycine and cysteine (Figure 35).
Additionally, it was investigated if YAP1 i) regulates gene expression of amino acid
transporters and ii) supports additional metabolic amino acid pathways.
ASCT1 (SLC1A4)
ASCT2(SLC1A5)
SNAT1 (SLC38A1)
SNAT2 (SLC38A2)
SNAT3 (SLC38A3)
SNAT4 (SLC38A4)
GlyT1 (SLC6A9)
CAT1 (SLC7A1)
LAT1 (SLC7A5)
LAT2 (SLC7A8)
LAT3 (SLC43A1)
LAT4 (SLC43A2)
XCT (SLC7A11)
EAAT1 (SLC1A3)
4F2HC (SLC3A2)
GLS1
GLS2
GLUD1
GOT1
GOT2
GPT1
GPT2
PSAT1
2
0
2
5
2
10
2
15
Gene expression, normalized counts [log
2
]
SK-N-AS-TR-YAP1
S127A
Tet - SK-N-AS-TR-YAP1
S127A
Tet + SH-EP-TR-YAP1
S127A
Tet - SH-EP-TR-YAP1
S127A
Tet +
#
##
#
#
#
##
#
°°
##
#
#
#
#
#
Amino acid transporter Amino acid synthesis
from glutamine
°
#
Figure 35 YAP1 induces the upregulation of amino acid transporter genes in neuroblastoma cells. Gene expression of
amino acid transporters and amino acid synthesis from glutamine, with significantly upregulated (hash) or
downregulated (circle) genes (p<0.0001) in YAP1-activated (+Tet) or ethanol control (-Tet) SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells are presented as normalized counts from RNA-sequencing data.
1
0
1
2
3
4
5
Data 1
SK-N-AS-TR-YAP1S127A Tet -
SK-N-AS-TR-YAP1S127A -Tet SK-N-AS-TR-YAP1S127A +Tet SH-EP-TR-YAP1S127A -Tet SH-EP-TR-YAP1S127A +Tet
Results
107
Analyzing transcriptome data of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells, the
activation of YAP1 was found to induce expression for amino acid transporters genes in both
cell lines, albeit the effect was stronger in SH-EP-TR-YAP1S127A cells. The sodium-dependent
alanine and serine antiporter SLC1A4 and SLC1A5 (ASCT1/2) and glutamine transporter
SLC38A1 (SNAT1) were upregulated in SH-EP-TR-YAP1S127A exclusively (Kandasamy et al.,
2018). SLC6A9, which is upregulated in both cell lines upon YAP1 activation and known as
GlyT1, is a sodium- and chloride dependent glycine transporter (Kandasamy et al., 2018).
SLC7A5 (LAT1), also upregulated in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A, transports
large neutral amino acids across the cell membrane such as glutamine (Kandasamy et al.,
2018). SLC3A2 encodes for the heavy chain of CD98 and functions as heterodimer with
different amino acid transporters, which determine the predominant substrates (Kandasamy
et al., 2018). SLC7A11, also known as xCT, functions as sodium-dependent antiporter for
cysteine and glutamate (Kandasamy et al., 2018). Both are upregulated in SH-EP-TR-YAP1S127A
as well as in SK-N-AS-TR-YAP1S127A cells.
The search for pathways and proteins that supply the cell with further amino acids revealed
enhanced transcription of genes affecting glutamine metabolism, GOT1 and GPT2, in YAP1-
activated SH-EP-TR-YAP1S127A cells. The cytoplasmic GOT1 (glutamate oxaloacetate
transaminase 1) catalyzes the biosynthesis of glutamate from aspartate and cysteine
(GeneCards: GOT1, 2021), whereas the mitochondrial GPT2 (glutamate pyruvate
transaminase 2) generates pyruvate and glutamate by catalyzing the reversible alanine to 2-
oxoglutarate transamination (GeneCards: GPT2, 2021).
Taken together, many transporters for amino acids including serine, glutamine and cysteine,
were upregulated in high level YAP1-expressing neuroblastoma cell lines. In addition,
transcripts of glutamate promoting proteins were induced in SH-EP-TR-YAP1S127A cells upon
YAP1 activation.
Results
108
5.15 Active YAP1 reduces the sensitivity to chemotherapy-induced cytotoxicity
in neuroblastoma cells
In order to analyze a possible YAP1-mediated protection of neuroblastoma cells against
chemotherapeutic-drug toxicity, three chemotherapeutic agents, that are included in the
standard neuroblastoma-therapy strategy (see section 1.1.4), were administered to the YAP1-
activated neuroblastoma cells.
YAP1S127A-expression was induced in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A clones to
analyze the cell viability of YAP1-activated neuroblastoma cell lines treated with a
chemotherapeutic agent. A solvent control group with ethanol treatment was included.
Twenty-four hours after activation, the cells were additionally treated with etoposide,
doxorubicin or vincristine in concentration ranges of 0.1 nM - 150 µM (optimal concentration
ranges were obtained previously), depending on cell line and agent. After 72 hours of
incubation, cell viability was measured based on ATP-levels. Data are presented as fold change
of the chemotherapeutically untreated (0 µM) samples. In SH-EP-TR-YAP1S127A #10 (Figure 36
A) and both SK-N-AS-TR-YAP1S127A cell lines (Figure 36 B), the chemotherapeutically treated
cells showed significantly higher cell viability in the dynamic range when YAP1 was activated.
In SH-EP-TR-YAP1S127A #10, YAP1 increased the vitality up to 3.5-fold in a distinct range of
etoposide, doxorubicin and vincristine concentration. In higher concentrations, the
chemotherapeutic agents minimized the cell viability. Of note, no cell viability differences
between YAP1-activated and control cells could be detected in SH-EP-TR-YAP1S127A #14.
In both chemotherapeutically drug-treated clonal cell lines of SK-N-AS-TR-YAP1S127A, the
viability increased in YAP1-induced cells compared to ethanol controls, although the effect
was not as prominent as in SH-EP-TR-YAP1S127A cells. Etoposide as well as vincristine were not
sufficient to completely diminish the viability of SK-N-AS-TR-YAP1S127A cells, even at really high
and clinically no longer relevant concentrations up to 150 µM. Doxorubicin impaired the cell
vitality of YAP1-active and control cells, but, nevertheless, complete signal loss of YAP1-
induced cells only occurred upon application of 50 µM doxorubicin. In comparison,
measurable viability of ethanol treated cells could be suppressed with 2.05 µM of the
substance.
Results
109
0.00065
0.001
0.005
0.01
0.05
0.1
1
10
50
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Etoposide [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#10
**p=0.0039
0.00065
0.003162
0.001
0.01
0.05
0.1
0.5
1
10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
SH-EP-TR-YAP1
S127A
#10
Doxorubicin [µM]
Cell viability,
foldchange 0µM
**p=0.0039
0.0001
0.0002
0.0006
0.002
0.008
0.02
0.046
0.1
0.2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Vincristine [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#10
**p=0.0039
0.0001
0.0002
0.0006
0.002
0.008
0.02
0.046
0.1
0.2
0.0
0.5
1.0
1.5
Vincristine [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#14
p=0.8203
Tet -
Tet +
0.00065
0.001
0.005
0.01
0.05
0.1
1
10
50
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Etoposide [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#14
p=0.0547
0.00065
0.003162
0.001
0.01
0.05
0.1
0.5
1
10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Doxorubicin [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#14
p=0.1641
0.0001
0.0002
0.0006
0.002
0.008
0.02
0.046
0.1
0.2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Vincristine [µM]
Cell viability,
foldchange 0µM
SH-EP-TR-YAP1
S127A
#14
p=0.8203
Tet -
Tet +
0.1
0.5
1.
5
10
30
70
100
150
0.0
0.5
1.0
1.5
Etoposide [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1
S127A
#2
**p=0.0039
0.01
0.029
0.084
0.24
0.71
2.05
5.95
17.24
50
0.0
0.5
1.0
1.5
Doxorubicin [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1
S127A
#2
**p=0.0039
0.0013
0.01
0.1
1
5
10
50
100
150
0.0
0.5
1.0
1.5
Vincristine [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1S127A #2
**p=0.0039
0.1
0.5
1.
5
10
30
70
100
150
0.0
0.5
1.0
1.5
Etoposide [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1S127A #9
**p=0.0078
0.01
0.029
0.084
0.24
0.71
2.05
5.95
17.24
50
0.0
0.5
1.0
1.5
Doxorubicin [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1S127A #9
*p=0.017
0.0013
0.01
0.1
1
5
10
50
100
150
0.0
0.5
1.0
1.5
Vincristine [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1S127A #9
**p=0.0039
0.0013
0.01
0.1
1
5
10
50
100
150
0.0
0.5
1.0
1.5
Vincristine [µM]
Cell viability,
foldchange 0µM
SK-N-AS-TR-YAP1
S127A
#9
**p=0.0039
Tet -
Tet +
Figure 36 Cell viability of YAP1-activated cells under treatment with etoposide, doxorubicin and vincristine. Two YAP1-
inducible cell lines were treated with different concentrations of etoposide, doxorubicin and vincristine. (A)
SH-EP-TR-YAP1S127A #10 revealed significantly higher cell viability under treatment with all three agents when YAP1S127A was
activated (+Tet) compared to solvent control (-Tet). On the contrary, no difference was detected in clone #14. (B) Both cell
lines of SK-N-AS-TR-YAP1S127A were more viable upon doxorubicin, etoposide or vincristine treatment, when YAP1S127A was
overexpressed. Bars represent mean enrichments over the untreated controls (± SD), n=3. P-values were calculated with
Wilcoxon matched-pairs signed rank test, p*0.01 < p < 0.05, **0.001 < p < 0.01.
A
B
- Tet
+Tet
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
1
0
1
2
3
4
5
Data 2
SK-N-AS-TR-YAP1
S127A
Tet -
Cell viability,
fold change 0 µM
- Tet
+Tet
Results
110
Remarkably, only three out of four cell lines demonstrated variable cell viability upon
tetracycline-mediated YAP1 induction. The fourth cell line, SH-EP-TR-YAPS127A #14, raised our
attention. It exhibited the same cell line background and the same generation procedure as
SH-EP-TR-YAPS127A #10, which is why similar results from both clones were expected. Analysis
of the transcriptional YAP1 levels by qPCR in all experiment samples revealed the same YAP1
expression in #14 as in the empty vector control (Figure 37). The YAP1 overexpression in was
not inducible anymore in SH-EP-TR-YAP1S127A #14 and thereby, this dataset was disregarded.
Taken together, YAP1 mediates a reduced sensitivity to chemotherapy-induced cytotoxicity of
doxorubicin, etoposide and vincristine in SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A cells,
in vitro, albeit this effect was only observed for distinct drug-concentration ranges.
Figure 37 Failed activation of YAP1 in
SH-EP-TR-YAP1S127A clone #14. YAP1 gene
expression level in YAP1-induced
SH-EP-TR-YAP1S127A and SK-N-AS-YAP1S127A
cells, treated and harvested simultaneously
to chemotherapeutically treated cells (see
Figure 33). No overexpression of YAP1 in #14
could be detected via qPCR. Bars represent
expression enrichment of YAP1-activated
cells over solvent controls (means ±SD), in
ratio to SDHA.
LV
#2
#9
0
3
6
9
12
15
18
YAP1 mRNA expression
fold change
SK-N-AS-TR-YAP1
S127A
LV
#10
#14
0
1
2
3
4
5
6
7
8
SH-EP-TR-YAP1S127A
YAP1 mRNA expression
fold change
Results
111
5.16 YAP1 enables neuroblastoma cells to survive growth factor limitations
YAP1 was described to promote cell growth by the acquisition of nutrients (Hansen et al.,
2015). Thus, high levels of YAP1 might enable neuroblastoma cells to overcome the limitation
of growth signals and biomolecules. In vitro, this nourishment shortening was mimicked by
serum starvation of the YAP1-induced SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells as
well as ethanol treated controls. The cell viability was measured 72 hours after the onset of
serum starvation. The cell viability of SH-EP-TR-YAP1S127A empty vector and YAP1-inducible
clones was comparable in standard culture conditions with 10% serum (Figure 38 A). In the
same condition, YAP1-induced SK-N-AS-TR-YAP1S127A cells showed higher cell viability
compared to solvent and empty vector controls (Figure 38 B). The cell viability decreased with
reduced serum concentrations in empty vector cells and ethanol controls. Tetracycline-
induced YAP1 activation partially reversed this effect. Cultured in 2% serum-containing media,
LV
LV
#10
#10
#14
#14
LV
LV
#10
#10
#14
#14
LV
LV
#10
#10
#14
#14
0
1×107
2×107
3×107
4×107
Cell viability
[relative luminescence units]
SH-EP-TR-YAP1
S127A
10% FCS 2% FCS Tet -
Tet +
0% FCS
p=0.1 p=0.1
LV
LV
#2
#2
#9
#9
LV
LV
#2
#2
#9
#9
LV
LV
#2
#2
#9
#9
0
1×10
7
2×10
7
3×10
7
4×10
7
Cell viability
[relative luminescence units]
SK-N-AS-TR-YAP1
S127A
10% FCS 2% FCS 0% FCS
p=0.1
p=0.1
*p=0.06
Tet -
Tet +
Figure 38 YAP1 activation
increased the cell viability of
serum-starved neuroblastoma
cells. Ethanol-treated control (-
Tet) and YAP1-activated (+Tet)
(A) SH-EP-TR-YAP1S127A and (B)
SK-N-AS-TR-YAPS127A cells were
cultured in 10%, 2% and 0%
serum-containing media for
72 hours. In 2% serum-
containing medium, YAP1-
activated cells show higher
viability than control cells. Bars
represent means ± SD of three
independent experiments.
Statistical significance was
assessed by using Mann-
Whitney U test;
p* 0.01 < p ≤ 0.05. FCS=fetal
calf serum.
- Tet
+Tet
- Tet
+Tet
A
A
Results
112
YAP1-activated cells showed higher cell viabilities than all control cells. Cultivation of
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells without any serum diminished this effect.
Next it was investigated whether the comparatively higher cell-viability measurements of YAP-
induced, serum-starved cells coincidence with an enhanced cell-proliferation rate. A BrdU-
based proliferation ELISA assay was performed with YAP1-induced and ethanol-treated
control cells of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A, that were exposed to different
serum concentrations for 72 hours. Proliferation of SH-EP-TR-YAP1S127A (Figure 39 A) as well
as SK-N-AS-TR-YAP1S127A cells (Figure 39 B) was not found to be altered upon YAP1 induction,
even not in serum-reduced conditions. Thus, the increased cell viability ofYAP1-activated cells
cultured in serum-reduced conditions was not caused by altered proliferation rates.
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SH-EP-TR LV
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SH-EP-TR-YAP1S127A #10
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SH-EP-TR-YAP1
S127A
#14
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SH-EP-TR-YAP1S127A #14
Tet -
Tet +
10 5210
0
25
50
75
100
125
FCS [%]
Proliferation %,
normalized to control condition
SK-N-AS-TR LV
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SK-N-AS-TR-YAP1
S127A
#2
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SK-N-AS-TR-YAP1
S127A
#9
Tet -
Tet +
10 5210
0
25
50
75
100
125
[%] FCS
Proliferation,
normalized to 10% FCS Tet -
SK-N-AS-TR-YAP1
S127A
#9
Tet -
Tet +
Figure 39 YAP1 activation has no effect on cell proliferation of serum-starving cells. Empty vector controls (LV) and clones
(#) of YAP1-activated (+Tet) or ethanol treated (-Tet) SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells were cultured in
different FCS-concentrations (10%, 5%, 2%, 1% and w/o FCS) and analyzed in BrdU-ELISA for their proliferation after
72 hours incubation time. Bars represent means ± SD of n=3 experiments, measured in technical triplicates. No statistically
significant differences were detected, tested with unpaired multiple t-tests assuming different SD, corrected with Holm-
Sidak method.
A
B
- Tet
+Tet
- Tet
+Tet
Results
113
5.17 YAP1 initiates a glycolytic switch in neuroblastoma cells
In tissue culture, a color change of the pH-sensitive phenol-red containing medium was
observed upon YAP1 activation in neuroblastoma cells (Figure 40 A). This effect was very
prominent in SH-EP-TR-YAP1S127A cells. Thus, the glucose and lactate concentrations as well as
pH values were measured in the supernatants of SH-EP-TR-YAP1S127A cells 72 hours after
tetracycline-mediated YAP1 induction. In contrast to stable pH values in empty vector
controls, the extracellular pH decreased upon YAP1 activation (Figure 40 B). Corresponding
extracellular glucose concentrations (Figure 40 C) declined from around 100 mg/dL in control
cells by half in YAP1-overexpressing cells, whereas extracellular lactate levels (Figure 40 D)
were increased
≥
1.8-fold.
Tet- +Tet
SH-EP-TR-YAP1
S127A
LV
#10
#14
#17
6.0
6.5
7.0
7.5
8.0
pH value
- Tet
+ Tet
**
p=0.09
LV
#10
#14
#17
0
50
100
150
200
Glucose [mg/dl]
- Tet
+ Tet
p=0.08
p=0.09
LV
#10
#14
#17
0
50
100
150
200
Lactate [mg/dl]
- Tet
+ Tet
p=0.07
p=0.06
*
A
B
C
D
Figure 40 YAP1 activation in SH-EP-TR-YAP1S127A cells alters the extracellular glucose and lactate concentration.
Tetracycline-induced activation of YAP1S127A in SH-EP-YAP1S127A clones resulted in (A) an obvious color change of phenol
red-containing medium. Measurement of (B) pH-value of the medium in YAP1-activated cells (+Tet) and appropriate
controls (-Tet, LV) confirmed the acidification. (C) Extracellular glucose levels were decreased and (D) extracellular lactate
levels were increased upon YAP1 induction. Data of n=3. Statistical significance was assessed by using the unpaired
Student´s t-test; p* 0.01 < p ≤ 0.05.
LV
#10
#14
#17
0
50
100
150
200
Glucose [mg/dl]
- Tet
+ Tet
p=0.08
p=0.09
-Tet +Tet
Results
114
To further elucidate the glycolytic rate in YAP1-activated cells, a glycolytic rate assay with one
clone per cell line and associated empty vector controls was performed. Extracellular
acidification rates (ECAR), which is dominated by secreted lactate (Wu et al., 2007), and
oxygen consumption rates (OCR), which directly measures mitochondrial respiratory rate
(Mookerjee et al., 2015), were measured over time, while YAP1-activated and control cells
were treated with mitochondrial inhibitors Rotenone and Antimycin A (Rot/AA) as well as
glycolysis inhibiting glucose analog 2-deoxyglucose (2-DG), subsequently. The proton efflux
-2
0
2
4
6
8
10
0 10 20 30 40 50 60 70
ECAR(mpH/min)
Time(min)
SK-N-AS
Tet+
Tet-
ê
ê
Rot/AA
2-DG
-5
0
5
10
15
20
0 10 20 30 40 50 60 70
OCR(pmol/min)
Time(min)
SK-N-AS
ê 2-DG
ê
Rot/AA
-2
0
2
4
6
8
10
12
0 10 20 30 40 50 60 70
ECAR(mpH/min)
Time(min)
SH-EP
ê
ê
Rot/AA
2-DG
-2
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
OCR(pmol/min)
Time(min)
SH-EP
Tet+
Tet-
ê
ê
Rot/AA
2-DG
LV
LV
#17
#17
0
50
100
150
PER [pmol/min]
LV
LV
#17
#17
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
LV
LV
#4
#4
0
50
100
150
PER [pmol/min]
LV
LV
#4
#4
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
SH-EP-TR-YAP1S127A
LV
LV
#17
#17
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
LV
LV
#17
#17
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
-2
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
OCR(pmol/min)
Time(min)
SH-EP
Tet+
Tet-
ê
ê
Rot/AA
2-DG
-2
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
OCR(pmol/min)
Time(min)
SH-EP
Tet+
Tet-
ê
ê
Rot/AA
2-DG
SK-N-AS-TR-YAP1S127A
LV
LV
#17
#17
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
-2
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
OCR(pmol/min)
Time(min)
SH-EP
Tet+
Tet-
ê
ê
Rot/AA
2-DG
LV
LV
#4
#4
0
50
100
150
GlycoPER
[pmol/min]
Tet -
Tet +
-2
0
2
4
6
8
10
0 10 20 30 40 50 60 70
ECAR(mpH/min)
Time(min)
SK-N-AS Tet+
Tet-
ê
ê
Rot/AA
2-DG
A
B
C
D
Figure 41 YAP1S127A overexpression affects the glycolytic rate in neuroblastoma cell lines. The simultaneous measurement
of ECAR and OCR in (A) SH-EP-TR-YAP1S127A #17 and (B) SK-N-AS-TR-YAP1S127A #4 cells revealed increased extracellular
acidification and reduced oxygen consumption in YAP1-activated cells (+ Tet) compared to solvent control cells (-Tet). Plots
show one representative measurement of YAP1-inducible clones with mean ± SD of 6 technical triplicates (C, D)
Corresponding PER and herein included glycolytic PER values of both cell lines are represented as bar plots showing mean
± SD of two independent experiments, each performed in 6 technical triplicates. YAP1-activated cells (#, +Tet) demonstrated
higher overall and glycolytic PERs than attendant controls. ECAR = extracellular acidification rate, OCR = oxygen
consumption rate, Rot/AA = Rotenone/Actinomycin A, 2-DG = 2-deoxyglucose, PER = proton efflux rate, glycoPER =
glycolytic PER.
- Tet
+Tet
- Tet
+Tet
- Tet
+Tet
- Tet
+Tet
Results
115
rate (PER) and the glycolysis-derived PER (glycoPER) were calculated from these data in
consideration of the buffer factor and the CO2 contribution factor.
An induction of YAP1S127A in SH-EP-TR-YAP1S127A cells resulted in a 2-fold increase of the ECAR
(Figure 41 A). Application of mitochondrial inhibitors Rot/AA increased the extracellular
acidification in ethanol control cells 1.9-fold, but only 1.2-fold in YAP1-activated cells.
Simultaneously, the OCR decreased 3.5-fold in ethanol-treated control cells, but only about
half in YAP1-overexpressing SH-EP-TR-YAP1S127A. The subsequent glycolysis inhibition of the
cells with 2-DG inhibited the ECAR dramatically in both conditions, whereas the oxygen
consumption was not affected. Proton efflux rates of SH-EP-TR-YAP1S127A cells (Figure 41 C)
were calculated and revealed a 3-fold increase of proton release by YAP1-activated cells over
ethanol controls. Notably, tetracycline treatment alone elevated the PER and also the
glycoPER 1.5-fold.
The SK-N-AS-TR-YAP1S127A cells revealed a similar profile of glucose metabolism with effects
being less pronounced. Initially, the ECAR in YAP1-activated cells was 1.9-fold higher
compared to ethanol control cells and increased only minimally upon inhibition of the
mitochondrial oxidative phosphorylation (Figure 41 B). Upon application of 2-DG, the
acidification rate was reduced very fast in YAP1 high level cells and slowly in control cells.
Notably, the OCR was higher in control cells initially, but application of Rot/AA did not affect
the YAP1-induced and control cells differently. However, the glycolysis-driven PER was much
higher in YAP1 high level SK-N-AS-TR-YAP1S127A cells (81.3 pmol/min) than in all control cells
(averaged 33 pmol/min) (Figure 41 D).
Transcriptome data analysis of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells uncovered
significantly increased gene expression of transmembrane glucose transporters Glut2/3,
monocarboxylate transporters (MCT7/8/12) as well as glycolysis-catalyzing enzymes
hexokinase 2 and phosphofructokinase (Figure 42 A). These indicated genes regulated by YAP1
are correlated to the glucose metabolism including the import of glucose, its conversion into
lactate as well as the export of lactate from the cell (Figure 42 B). Genes of the citric acid cycle
and oxidative phosphorylation complexes were not found to be regulated in YAP1-activated
cells.
Results
116
Glut 2 / SLC2A2
Glut 3 / SLC2A3
HK2
PFKP
PFKFB4
MCT 4 / SLC16A3
MCT 7 / SLC16A6
MCT 8 / SLC16A2
MCT 12 / SLC16A12
20
25
210
215
220
Gene expression, normalized counts [log2]
#
#
#
#
°#
#
#
°#
Glucose
transporter
Glycolysis Lactate
transporter
Upreg. in SK-N-AS-TR-YAP1S127A
Upreg. in SH-EP-TR-YAP1S127A
Downreg. in SK-N-AS-TR-YAP1S127A
Downreg. in SH-EP-TR-YAP1S127A
1
0
1
2
3
4
5
Data 1
SK-N-AS-TR-YAP1S127A Tet -
SK-N-AS-TR-YAP1S127A Tet -
SK-N-AS-TR-YAP1S127A Tet +
SH-EP-TR-YAP1S127A Tet -
SH-EP-TR-YAP1S127A Tet +
SK-N-AS-TR-YAP1S127A Tet -
SK-N-AS-TR-YAP1S127A Tet +
SH-EP-TR-YAP1S127A Tet -
SH-EP-TR-YAP1S127A Tet +
SK-N-AS-TR-YAP1S127A -Tet
SK-N-AS-TR-YAP1S127A +Tet
SH-EP-TR-YAP1S127A -Tet
SH-EP-TR-YAP1S127A +Tet
SK-N-AS-TR-YAP1S127A -Tet
SK-N-AS-TR-YAP1S127A +Tet
SH-EP-TR-YAP1S127A -Tet
SH-EP-TR-YAP1S127A +Tet
Glucose
Import
Export
GLUT3GLUT2
HK2
Glucose
Glucose-6-P
Fructose-6-P
Fructose-1,6-BP
Glyceraldehyde-3-P
1,3-P-Glycerate
3-P-Glycerate
2-P-Glycerate
P-Enolpyruvate
Pyruvate
Lactate
Lactate
GPI
ALDO
GAPDH
PGK
PGAM
PK
LDHA
Fructose-2,6-BP
PFKFB4
PFKP
ENO3
MCT7 MCT8MCT4
Dihydroxy-
acetone-P
TPI
ALDO
Cytosol
Pentose
phosphate
pathway
Serine/Glycine
One-Carbon Units
TCA cycle
Amino
acids
Fatty
acids
Nucleotides
Figure 42 Glycolysis-associated genes are
upregulated in YAP1-activated
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAPS127A
cells. (A) Significantly upregulated (hash) or
downregulated (circle) genes (p<0.0001) of
YAP1-activated (+Tet) or ethanol control (-Tet)
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
are depicted as normalized counts. (B)
Schematic illustration of glycolysis and
branching pathways as well as biological
building blocks (nucleotides, amino acids, fatty
acids) with colors encoding the significantly
upregulated (black bordered box) and
downregulated (red bordered box) genes in
SH-EP-TR-YAP1S127A (blue boxes) and
SK-N-AS-TR-YAP1S127A (dark-yellow boxes).
Glut = glucose transporter, MCT = mono-
carboxylate transporter, HK = Hexokinase, GPI
= Glucose-6-phosphate isomerase, PFK =
Phosphofructokinase, PFKFB = PFK/Fructose-
2,6-Biphosphatase, ALDO = Aldolase, GAPDH =
Glyceraldehyde-3-phosphate dehydrogenase,
TPI = Triosephosphate isomerase, PGK =
Phosphoglycerate kinase, PGAM =
Phosphoglycerate mutase, ENO = Enolase,
PKM = Pyruvate kinase, LDHA = lactate
dehydrogenase A. Figure adapted from Kim
and Yeom, 2018
Results
117
Taken together, our results demonstrated a higher glucose uptake of YAP1-activated
neuroblastoma cells with additional upregulation of glucose transporter gene expression and
an impairment of oxygen consumption. Elevated extracellular lactate concentrations and
upregulated lactate transporter genes were accompanied by an increased ECAR, which proves
higher lactate production and secretion by YAP1-overexpressing cells.
5.18 Does YAP1 synergize with HIF-2
𝜶
?
In order to identify pathways affected commonly in YAP1-activated SK-N-AS-TR-YAP1S127A and
SH-EP-TR-YAP1S127A cells, the differentially expressed genes were tested for significantly
enriched pathway annotations of the C2 collection (MSigDB) with an FDR-adjusted p-value of
p<0.05. Gene sets concerning DNA methylation, histone acetylation, EGF and HRAS signaling
as well as hypoxia were the six highest ranked ones (Tables A+B). Considering the observation
that YAP1 could induce aerobic glycolysis in neuroblastoma cells, similar to HIF proteins (see
section 1.2.7.4), a possible link between YAP1 and hypoxia was further investigated. Hypoxia
HIF1A
EPAS1
HIF3A
ARNT
VHL
EGLN1
EGLN2
EGLN3
2
0
2
2
2
4
2
6
2
8
2
10
2
12
2
14
2
16
Gene expression, normalized counts [log
2
]
#
#
SH-EP-TR-YAP1
S127A
Tet -
SH-EP-TR-YAP1
S127A
Tet +
SK-N-AS-TR-YAP1
S127A
Tet -
SK-N-AS-TR-YAP1
S127A
Tet +
Figure 43 EPAS1 is upregulated in YAP1-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
cells. Transcriptional levels of HIF proteins and HIF-regulators in YAP1-activated (+Tet) or ethanol
control (-Tet) cells of SH-EP-TR-YAP1S127A #10 and SK-N-AS-TR-YAP1S127A #2, presented as normalized
counts from RNA-sequencing data. EPAS1 is the only significantly upregulated (hash) gene
(p<0.0001). HIF = hypoxia-inducible factor, ARNT = aryl hydrocarbon receptor nuclear translocator,
EGLN =EGL-9 homolog.
SK-N-AS-TR-YAP1S127A -Tet
SK-N-AS-TR-YAP1S127A +Tet
SH-EP-TR-YAP1S127A -Tet
SH-EP-TR-YAP1S127A +Tet
SK-N-AS-TR-YAP1
S127A
-Tet
SK-N-AS-TR-YAP1
S127A
+Tet
SH-EP-TR-YAP1
S127A
-Tet
SH-EP-TR-YAP1
S127A
+Tet
Results
118
describes a limited supply of oxygen and thereby forces cells to adapt rapidly to oxygen-
deprived conditions including a shift to aerobic glycolysis, induction of angiogenesis and
vascularization among others (Greer et al., 2012). Two main drivers for transcriptional
regulation in hypoxic conditions are the hypoxia-inducible factors HIF-1α, encoded by HIF1A,
and HIF-2α, encoded by EPAS1 gene.
The analysis of RNA-sequencing data from YAP1-induced neuroblastoma cells revealed a
6.6-fold and 9.8-fold induction of EPAS1 expression in SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells, respectively (Figure 43). Thus, it was assumed, that a YAP1-
induced metabolic switch (see section 5.17) could be mediated via a YAP1-HIF-2α axis in
neuroblastoma cells. A possible protein stabilization of HIF-2α was examined via immunoblot
analysis of YAP1-activated cells as well as appropriate controls including solvent ethanol and
empty vector controls (Figure 44). Although gene transcription was induced in both cell lines,
SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A, a protein signal for HIF-2α could not be
detected.
In order to stabilize HIF-2α proteins and measure differences between YAP1 high-level
neuroblastoma cells and controls, the same experiment was performed under hypoxic
conditions. Tetracycline-treated cells and solvent controls of both YAP1-inducible cell lines
were exposed to normoxic standard tissue culture conditions (21% oxygen) or hypoxic
conditions (1% oxygen) for 72 hours.
In both cell lines, SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A, immunoblotted HIF-2α
protein was detectable and thus consequently stabilized under hypoxic compared to normoxic
LV #10 #17
Tet
- + - + - +
YAP1
HIF-2α
β-Ac7n
SH-EP-TR-YAP1
S127A
LV #4 #9
Tet
- + - + - +
YAP1
HIF-2α
β-Ac7n
SK-N-AS-TR-YAP1S127A
Figure 44 No HIF-2α stabilization in YAP1-activated neuroblastoma cells under normoxic conditions.
Representative immunoblots detecting protein levels of YAP1, HIF-2α and
𝜷
-Actin (loading control) in
YAP1-activated (+Tet) or ethanol control (-Tet) cells of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
clones are shown (n=2).
Results
119
conditions (Figure 45). Dependent on YAP1 levels, the signal of HIF-2α altered in strength: in
hypoxic ethanol control cells, lower HIF-2α levels were detected than in hypoxic YAP1-induced
cells. Higher YAP1 levels provoked increased HIF-2α signals in hypoxic cells. Please note, that
YAP1 undesirably was induced independently of tetracycline treatment in SH-EP-TR-YAP1S127A
#10 cells.
In addition to protein detection of HIF-2α, an analysis to identify an enrichment of HIF target
genes among the differentially expressed genes in the RNA-sequencing data was performed
comparing YAP1-activated to ethanol-treated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A
cells (Figure 46). After identification of all unique 1 kb promoter sites of annotated genes, the
presence of the common HIF DNA binding motif -RCGTG- (Wenger, Stiehl and Camenisch,
2005) was investigated in the promoter regions of significantly upregulated genes and found
in 3% of them. In comparison, motifs of the main YAP1-interacting transcription factors
TEAD1-4 (JASPAR 2018 database) were detected in 5% and 7% in SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A, respectively. Combinations of these putative TF binding sites were
found in 1-2% of all upregulated genes.
In summary, YAP1 activation induced EPAS1 gene transcription in neuroblastoma cells.
Ascertainable protein levels could only be found in hypoxic conditions, in which HIF proteins
are stabilized independently of YAP1. Nevertheless, the YAP1-mediated transcriptional
upregulation of EPAS1 could possess a very useful tool for neuroblastoma tumor progression
in challenging hypoxic conditions.
SK-N-AS-TR-YAP1
S127A
LV #9
HIF-2α
YAP1
β-Act
O
2Nox Hyp Nox Hyp
Tet
- + - + - + - +
SH-EP-TR-YAP1
S127A
LV #10
HIF-2α
YAP1
β-Act
O
2
Nox Hyp Nox Hyp
Tet
- + - + - + - +
Figure 45 HIF-2α protein levels in normoxic and hypoxic YAP1-activated neuroblastoma cells. Representative immunoblots
show protein levels of YAP1, HIF-2α and
𝜷
-Actin as loading control in hypoxic (Hyp) or normoxic (Nox) YAP1-activated (+Tet)
or solvent control (-Tet) cells of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A clones (n=2). HIF = hypoxia-inducible factor,
O2 = oxygen.
Results
120
3% 5%
2%
90%
HIF-mo.fs TEADmo.fs HIF+TEADmo.fs others
SH-EP-TR-YAP1
S127A
3%
7%
1%
89%
HIF-mo.fs TEADmo.fs HIF+TEADmo.fs others
SK-N-AS-TR-YAP1
S127A
Figure 46 HIF and TEAD binding site enrichment in YAP1-induced SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells.
Putative HIF and/or TEAD binding site are found significantly enriched amongst in the promotor regions of upregulated
genes in YAP1-activated SH-EP-TR-YAP1S127A (blue) and SK-N-AS-TR-YAP1S127A (yellow) cells.
Discussion
121
6. Discussion
Neuroblastoma is a heterogeneous tumor disease ranging from spontaneous regression to
ultra-high risk tumors with recurring neoplasms. Relapse of neuroblastoma happens in about
50 - 60% of all high-risk patients, who complete their therapy (Basta et al., 2016). In such cases,
the treatment options are very limited up to date, which leads to a mandatory search for
promising therapeutic targets and new therapeutic interventions. Previous results of our
working group revealed a gene expression pattern of active YAP1 in relapsed neuroblastomas
compared to their primary counterparts (Schramm et al., 2015). In various analyses we
investigated the role of YAP1 in this tumor entity and highlighted the oncogenic potential of
YAP1 in neuroblastoma progression. This section will outline the impact that YAP1 has on
neuroblastoma growth, migration potential and chemotherapy sensitivity. Furthermore, the
various effects that YAP1 has on cell metabolism, are put into a comprehensive context of
previous studies in both, neuroblastoma and other tumor entities.
6.1 YAP1 co-determines cell proliferation and viability of neuroblastoma cells
In this study, endogenous YAP1 levels were initially investigated in 19 neuroblastoma cell lines
with YAP1 mRNA levels correlating with its protein levels. Thus, YAP1 was found to be
heterogeneously expressed in neuroblastoma cell lines. This diversity reflects the variability
of YAP1 expression in primary neuroblastoma tumor tissues. With respect to different patients
features and cell backgrounds, a diverse neuroblastoma cell-line panel was screened for cell
growth upon YAP1 gene silencing, even if a direct connection between YAP1 levels and any
clinical parameter or neuroblastoma-specific recurrent genetic alteration could not be
established. The siRNA-mediated knockdown of YAP1 reduced the cell viability and
proliferation rates, albeit this effect was not observed in all neuroblastoma cell lines and cell-
growth inhibition was only moderate. The cell-context dependent growth limitation partially
resulted from G0/G1 cell cycle phase arrest and not from apoptosis, as it was demonstrated
in SK-N-SH cells. Aiming for a validation of cell-viability data achieved with siRNA-mediated
Discussion
122
YAP1 knockdown, shRNAs targeting YAP1 were lentivirally delivered to neuroblastoma cells
with a maximum transduction efficiency of 75-85%. The shRNA-mediated knockdown reduced
the YAP1 gene expression less efficiently and revealed no alteration of neuroblastoma cell
viability. Based on different sequences (junction-spanning exon 2/3 and 8/9 targeting via
siRNAs versus exon 2 and 9 targeting via shRNAs), RNA structures (double stranded siRNA
versus shRNA folded into a stem-loop structure), delivery (lipid transfection versus lentivirally
transduced shRNA-carrying vector), intracellular processing (RISC only versus additional
DICER-dependent processing) and effective RNA concentrations, siRNAs and shRNAs targeting
the same mRNA show variable silencing efficiency and off-target effects (Rao et al., 2009).
Especially the off-target effects were demonstrated to be more prominent in siRNA
approaches compared to shRNA-mediated gene silencing (Klinghoffer et al., 2010). The
disparity of cell viability data gained from neuroblastoma cell lines treated with either siRNAs
or shRNAs targeting YAP1 puts the target specificity into question. Since the reduced viability
of SH-EP-TR-YAP1S127A cells upon YAP1 knockdown could be reversed by the induction of
YAP1S127A , we considered the results obtained with siRNAs targeting YAP1 as YAP1-specific.
Our data were confirmed by Yang and co-workers, who also performed siRNA-mediated YAP1
knockdown in two neuroblastoma cell lines SH-SY5Y and SK-N-SH and found subsequent
impairment of cell proliferation (Yang et al., 2017). The cell line SH-SY5Y was not included in
our panel, but SK-N-SH cells were also significantly responsive to YAP1 perturbation. In
contrast, Shim and colleagues silenced YAP1 via shRNA-mediated knockdown in two
neuroblastoma cell lines, SK-N-AS and NLF, and could not find any significant cell growth
alteration in vitro, although a successful knockdown was evidenced with decreased expression
of YAP1 target genes CYR61 and CTGF. However, the in vivo growth of these cells was retarded
(Shim et al., 2020). Our data argue for a YAP1-mediated regulation of cell proliferation in
neuroblastoma cells. The diversity of cellular responses to a YAP1 knockdown suggests
additional factors that are necessary for a YAP1-mediated regulation of neuroblastoma cell
growth. With regard to YAP1-silenced SK-N-AS cells, that lack growth response in vitro but
show delayed tumor growth in vivo (Shim et al., 2020), the impact of the tumor
microenvironment and demanding conditions in physiological tumors additionally need to be
considered in the characterization of YAP1 in neuroblastoma pathogenesis. Therefore, further
Discussion
123
studies with appropriate modeling of human tumor microenvironment will shed light on the
role of YAP1 in neuroblastoma progression.
The investigation of YAP1 as a potential oncogene in neuroblastoma tumor progression is still
quite recent, but in many other tumor entities such as lung adenocarcinoma (Zhang et al.,
2015), malignant mesothelioma (Mizuno et al., 2012), ovarian cancer (Xia et al., 2014) and
sonic hedgehog (SHH)-mediated medulloblastoma (Fernandez-L et al., 2009) as well as normal
mammalian tissues (Schlegelmilch et al., 2011), regulation of cell growth and tissue
homeostasis by YAP1 is described extensively (reviewed in Piccolo, Dupont and Cordenonsi,
2014). Interestingly, the overexpression of constitutively active YAP1S127A in neuroblastoma
cell lines SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A revealed no additional advantage in
respect to cell cycling and the cell division rate in vitro. In other cancer entities such as ovarian
cancer (Xia et al., 2014), lung adenocarcinoma ((Zhang et al., 2015) or non-small cell lung
cancer (Yu et al., 2018), ectopic YAP1 expression has been shown to induce cell proliferation
and organ growth both, in vitro and in vivo. Even the transformation of normal cells such as
NIH-3T3 by YAP1 overexpression can increase proliferation rates and tumorigenic growth after
subcutaneous injection into mice (Nishimoto et al., 2019). The major disparity between
neuroblastoma and the other mentioned entities is the origin of cells, which is epithelial for
the latter and neural crest stem-like for the first. Concomitant with lineage specificities,
enormous differences in predominantly active transcription factors and regulatory networks
characterize a special cell identity. These distinctions might be one reason for various cellular
responses to activation of a transcriptional co-activator such as YAP1.
However, in contrast to our findings, Shen and colleagues recently postulated a YAP1-
mediated increase of cell proliferation. They overexpressed wildtype YAP1 in neuroblastoma
cells SH-SY5Y and found a larger number of YAP1 expressing cells being positive for the
proliferation marker Ki67 compared to vector control cells (Shen et al., 2020). Two reasons for
the contradiction to our results are considerable. Firstly, Shen et al. serum-starved the cells
for 24 hours prior to the analysis (Shen et al., 2020). Serum deprivation of epithelial cells leads
to a phosphorylation-mediated inactivation of YAP1 by LATS1/2 kinases entailing a reduction
of proliferation (Yu et al., 2012; Adler, Johnson et al., 2013; Plouffe et al., 2016). A YAP1
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phosphorylation upon serum-starvation was also prominent in an immunoblot of serum-
starved neuroblastoma cells by Shen et al., whereas the total YAP1 levels, displayed via
immunostaining of SH-SY5Y cells, were reduced in serum-free media and only visible in cell
nuclei, strongly indicating a lack of phosphorylation (Shen et al., 2020). The reduction of total
YAP1 levels was in line with our own finding of serum-deprivation leading to decreased YAP1
synthesis. An inactivating YAP1 phosphorylation in SH-EP and SK-N-AS wildtype cells upon
serum starvation could not be observed (supplementary Figure A). The discrepancy of
LATS1/2-mediated YAP1-phosphorylation in epithelial and neuroblastoma cells might be
reasoned by additional cell type-specific regulatory factors or varying speed of protein
degradation. The reduction of YAP1 abundance could either indicate a transcriptional
regulation of YAP1 in neuroblastoma cells upon limitation of mitogenic signals or it might be
a matter of extensive proteasomal degradation upon phosphorylation, which was not
measurable anymore after 72 hours of incubation. Nevertheless, a serum-starvation prior to
proliferation analysis resembles a YAP1 inhibition and limits the data comparability. Secondly,
pictures of Ki67-immunostained SH-SY5Y cells presented by Shen et al. revealed a relatively
low number of eGFP-tagged YAP1-positive cells in the whole cell population. Such a mixed cell
culture might lead to different proliferative capacities of cells overexpressing YAP1, as it has
been demonstrated for YAP1-overexpressing glioma cells, that revealed increased
proliferation rates when co-cultured with vector control cells (Liu et al., 2019).
As expected, cultivation of neuroblastoma cells in serum-reduced cell growth medium
reduced the cell viability. This viability reduction could partially be reverted by induction of
YAP1S127A in our Tet-ON YAP1-inducible cell line models. This finding implicates, that YAP1
mediates growth advantage to neuroblastoma cells with additional environmental stress or
nutrient limitations. But unlike the results from Shen et al., no alterations in proliferation rates
of serum-deprived cells were found upon induced YAP1S127A-overexpression. We speculate,
that active YAP1 induces a transcriptional program that releases tumor cells from growth-
factor dependent cell proliferation. In addition, induced YAP1 could enable neuroblastoma
cells to adapt their metabolism to overcome serum-starvation or other limitations to favor
tumor growth and cancer cell maintenance.
Taken together, our results reveal a cell context-dependent regulation of neuroblastoma cell
growth by YAP1. Additionally, YAP1 confers an advantage in regard to cell growth and
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maintenance for neuroblastoma cells, which have to withstand environmental stress such as
growth factor limitations.
6.2 YAP1 mediates a reduced sensitivity to cytotoxicity of chemotherapeutic
drugs to neuroblastoma cells
After a positive response to induction chemotherapy in most neuroblastoma cases, up to 60%
of high-risk neuroblastoma patients experience a progression or relapse, which is prevalently
metastatic and refractory to further therapy (Cohn et al., 2009; Basta et al., 2016; Berthold et
al., 2017). In 1998, the research group of P. Reynolds performed in vitro experiments with
isogenic neuroblastoma cell lines generated at diagnosis, after induction chemotherapy and
after treatment with myeloablative chemoradiotherapy. They presented dose-response data
proving the acquisition of therapy-correlated cytotoxic drug resistance without proposing any
mechanisms (Keshelava et al., 1998). Considering the reported YAP1-activated mRNA
expression pattern in neuroblastoma relapse tumors (Schramm et al., 2015), we hypothesized
that YAP1 promotes chemotherapy resistance in neuroblastoma.
In other cancer entities than neuroblastoma, the role of YAP1 in mediating drug resistance is
well described (Yoshikawa et al., 2015; Ciamporcero et al., 2016; Ma et al., 2016; Xiao et al.,
2016; Gujral and Kirschner, 2017; Lee et al., 2018; Zhou et al., 2019). In order to elucidate a
potential role of active YAP1 in mediating drug resistance to neuroblastomas, we tested the
efficacy of three chemotherapeutics from different ingredient groups in our YAP1-inducible
cell line models, the anthracycline doxorubicin, the topoisomerase II inhibitor etoposide and
the mitotic inhibitor and plant alkaloid vincristine. After drug treatment of YAP1S127A-activated
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells compared to ethanol-treated control cells,
an ATP-based cell viability assay was performed. It demonstrated higher readouts for YAP1-
activated cells in certain concentration ranges. The observation of up to 3-fold higher
luminescence values in YAP1-activated cells treated with low concentrations of
chemotherapeutic agents was unexpected. Since the assay detects ATP-levels, a possible
explanation could be a YAP1-mediated ATP-release of cells treated with low-dose
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chemotherapeutic agents. The theory of hormesis, which describes a stimulating effect of low-
dose toxic substances, could also be feasible in YAP1-activated neuroblastoma cells.
However, an application of low-dose chemotherapeutic agents to YAP1-activated
SH-EP-TR-YAP1S127A cells revealed a higher cell viability compared to control cells, which
indicates less sensitivity to the cytotoxicity of chemotherapeutic drugs. This beneficial effect
mediated by YAP1S127A was lost in high-dose ranges. In comparison, YAP1-activated
SK-N-AS-TR-YAP1S127A cells revealed a smaller effect, yet it was observed in broader
concentration ranges of the used agents. An overall fold resistance or metrics such as IC50
(half maximal inhibitory concentration) values could not be calculated because the dose-
response on cell viability occurred to be biphasic (e.g., SH-EP-TR-YAP1S127A #10, treated with
etoposide) instead of sigmoidal. Thus, our data only suggest less sensitivity of YAP1-activated
neuroblastoma cells to the cytotoxic effects of chemotherapeutic drugs of different
mechanisms of action.
To further elucidate the role of YAP1-mediated drug desensitization in a more elaborated
fashion, three-dimensional tumor growth of YAP1-activated as well as YAP1-silenced
neuroblastoma cells should be monitored during drug treatment.
However, confirming our finding, Yang et al. demonstrated a YAP1 dependency of tumorigenic
growth of cisplatin-resistant SH-SY5Y cells in vitro and in vivo, indicating a crucial role of YAP1
in promoting drug resistance in neuroblastoma cells (Yang et al., 2017). In line with our work,
Shim and colleagues recently silenced YAP1 via shRNA in SK-N-AS xenografts and found them
being more sensitive to cyclophosphamide treatment, highlighting the resistance-mediating
role of YAP1 in refractory neuroblastomas (Shim et al., 2020). YAP1 seems to confer resistance
not only to chemotherapeutical intervention, but it is also reported to minimize efficacy of
molecular therapy targeting MEK in RAS-activated neuroblastoma cells (Coggins et al., 2019).
Mechanistically, it remains unclear how YAP1 conveys the resilience against apoptosis
mechanisms in neuroblastoma. From studies in BRAF and RAS-mutant cancer cell lines it is
known, that YAP1 promotes resistance to RAF and MEK inhibitors via transcriptional
upregulation of anti-apoptotic Bcl-xL (BCL2L1) expression or via regulation of an EMT-like
genetic program in cooperation with FOS (Shao et al., 2014; Lin et al., 2015). Lee and
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collaborators found YAP1 upregulated in tyrosine-kinase inhibition (TKI) resistant lung cancer
cell lines, and Ghiso et al. linked YAP1-mediated resistance to the overexpression of AXL, a
survival-promoting protein transcriptionally regulated by YAP1 (Ghiso et al., 2017; Lee et al.,
2018). TKI-resistant neuroblastoma cells also depend on increased AXL levels (Debruyne et al.,
2016). In fact, mRNAs of BCL2L1, AXL, FOSL1 and FOSL2 were significantly upregulated in our
cell line models upon YAP1 activation (supplementary Table C) implying that respective
proteins might mediate YAP1-dependent resistance. However, our data do not allow any
statement to be made about this. The upregulation of EMT-promoting genes such as FOSL1
and FOSL2 induced by YAP1 will be discussed in more detail in section 6.4.
Another mechanism of cellular drug resistance is the expression of multi-drug resistance
genes encoding for ATP-binding cassette-transporter (ABC-transporter), which ferry the
chemotherapeutic drugs out of the cell (Fletcher et al., 2016). Although Murray et al. found
ABCC4 inhibition sensitizing neuroblastoma tumors to topoisomerase inhibitor Irinotecan,
other groups could not find a correlation of the main ABC transporters to a patient´s drug
response (Goto et al., 2000; De Cremoux et al., 2007; Murray et al., 2017). In our
transcriptome data from YAP1-activated cell lines, no evidence was found for regulation of
the most important ABC transporters such as MDR1 or ABCG2, indicating that YAP1 does not
directly regulate drug efflux in neuroblastoma to mediate resistance.
The interaction of tumor cells with their microenvironment comprises adhesion-dependent
and independent cell-cell-interactions with different cell types such as macrophages,
fibroblasts, immune cells, endothelial cells, pericytes and mesenchymal stroma cells
(reviewed by Balkwill, Capasso and Hagemann, 2012; Borriello et al., 2016). Beside secretory
signaling and cell-cell-junctions, the extracellular matrix (ECM), functioning as a scaffold
material and a natural barrier, plays a crucial role in mediating resistance (Shain and Dalton,
2001; Grantab, Sivananthan and Tannock, 2006; Balkwill, Capasso and Hagemann, 2012).
Recently, the research group around L. Chesler published evidence of relapse and widespread
metastasis in a cyclophosphamide-treated chemo-resistant MYCN-driven mouse model
Th-MYCNCPM32. Profound changes in ECM composition with a greater share of collagens and
fibronectin-1 as well as an enrichment in mesenchymal subtype of neuroblastoma cells in the
refractory recurrences were described (Yogev et al., 2019). Therefore, the structure of the
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surrounding ECM has decisive influence on the neuroblastoma tumor cell features, and vice
versa, the cells shape their own ECM. In a transcriptome analysis approach, we found that
YAP1S127A overexpression in neuroblastoma cells also increased the expression of ECM
molecules such as integrins, collagens and fibronectin-1 in vitro, what might enable the cells
to tolerate higher dosages of chemotherapeutics in vivo. Two main downstream pro-survival
signaling pathways, the AKT signaling and the ERK1/2 cascade (Edwards et al., 2005; Deville
and Cordes, 2019), were transcriptionally activated in our YAP1-induced cell lines. Both are
known big players in conveyance of chemotherapeutic drug resistance to cancer cells
(reviewed in Salaroglio et al., 2019; Liu et al., 2020).
In summary, we found YAP1-activated neuroblastoma cells being less sensitive to cytotoxic
drugs of different classifications than their control counterparts. Further effort is needed to
enlighten and intervene in the mechanisms by which YAP1 promotes drug tolerance to
neuroblastoma cells. Our data additionally provide first evidence of potential mechanisms of
how YAP1 mediates chemotherapy tolerance by reorganization of the neuroblastoma ECM
composition and regulating anti-apoptotic and EMT-related gene expression as well as pro-
survival signaling pathways.
6.3 YAP1 promotes motility of neuroblastoma cells
The movement of cells is a physiologic process already necessary for the earliest embryonic
development. In week three of embryogenesis, neural crest cells (NCC) migrate through the
whole embryo to build structures such as adrenal medulla, ganglia, smooth muscles and
others (Simões-Costa and Bronner, 2013). Neuroblastomas originate from NCC that already
committed to sympathoadrenal lineage, but it is not known, if the malign transformation
starts very early and cells migrate afterwards to their target areas or if the oncogenic genetic
lesions occur at a later time point after migration already occurred (Cheung and Dyer, 2013).
However, Hindley and colleagues found YAP1 being expressed in a CD15-/CD44+/CD49d+
neural crest precursor-similar neural subset of neural cell cultures and is also synthesized in
NCCs of chick embryos (Hindley et al., 2016). Additionally, they postulated that YAP1 together
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with retinoic-acid signaling determines the migration potential of these cells (Hindley et al.,
2016). In 2019, another research group showed YAP1 promoting NCC migration in avian
embryos with a requisite crosstalk between YAP1 and Wnt as well as BMP signaling (Kumar,
Nitzan and Kalcheim, 2019). These data associate YAP1 with a highly motile cell phenotype
and also assign YAP1 a determinant role in migration processes. In NCC-derived
neuroblastoma tumors, activation of YAP1 was initially found in tumor relapses (Schramm et
al., 2015), which are frequently metastatic (Basta et al., 2016) and thus, encompass migrating
tumor cells. With respect to the fact, that stage 3/4/4s neuroblastoma more often relapse
than stage 1/2 (Simon et al., 2017), EFS probability was displayed as a function of YAP1
expression of neuroblastoma patients, where event-free is defined as the time period until
first recurrence, tumor progression, secondary malignancy or patient´s death (Monclair et al.,
2009). The analysis revealed, that the EFS probability of stage 3 and 4s neuroblastoma patients
was lower when high YAP1 expression levels were detected. Such tumors are characterized
by an infiltration across the body midline (vertebral column) or disseminations into lymph
nodes, liver, skin and bone marrow (Simon et al., 2017). This increased probability for disease
progression or recurrence of disseminating and infiltrating neuroblastomas suggests a
correlation between the high YAP1 levels and a high cell migration capability. The presence of
distant metastasis is also true for stage 4 neuroblastoma, however, most of these tumors are
already driven by potent oncoproteins such as MYCN, TERT, ATRX, ALK or Ras/MAPK, which in
turn regulate cell migration themselves (Hasan et al., 2013; Pugh et al., 2013; Brady et al.,
2020; Koneru et al., 2020). Interestingly, Yang et al. found YAP1 protein abundance correlated
to disease stages through immunohistochemical staining of primary neuroblastoma tumors
(Yang et al., 2017), which we could not verify in expression data from neuroblastoma data sets
comprising all INSS stages. Supporting the idea of an INSS-stage independent YAP1 expression
pattern, most established neuroblastoma cell lines comprise heterogeneous YAP1 expression
levels, although they were established from tumors of high-risk patients with stage 3 or stage
4 disease, only (Thiele, 1998).
YAP1-driven cell migration has been shown for NCC (Hindley et al., 2016), but also for
neuroblastoma (Seong et al., 2017; Yang et al., 2017; Cai et al., 2020) and other tumor entities
such as glioblastoma (Shah et al., 2019), NSCLC (Yu et al., 2018), ovarian carcinoma (Xia et al.,
2014), mammary carcinoma and melanoma (Lamar et al., 2012). We observed the inhibition
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of cell migration in YAP1 silenced cells and increased cell motility in YAP1S127A-overexpressing
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells. A YAP1 knockdown resulted in decreased
“wound” closure in SK-N-SH and SK-N-BE cells, but not in IMR-5 and SK-N-FI. Here, congruent
results to data obtained by Yang et al. and Cai et al. were achieved, who transiently silenced
YAP1 in SH-SY5Y, SK-N-SH, SK-N-AS and SK-N-BE(2) cells and found reduced invasion and
migration abilities (Yang et al., 2017; Cai et al., 2020). The lack of a significant response in two
of four analyzed neuroblastoma cell lines might be attributed to the short observation period
of 48 hours. Furthermore, the YAP1-mediated effect on the cell migration could be
superimposed by the cell proliferation, that is also modified by YAP1 silencing. The increased
cell migration of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells overexpressing
constitutively active YAP1S127A was determined by using the transwell migration assay. Herein,
the effect of cell proliferation blurring the migratory phenotype of cells is diminished upon the
active process of cell translocation through a small pore membrane, which requires
cytoskeletal rearrangement processes and a turnover of cell-cell as well as cell-matrix
junctions. Together with the observed upregulation of migration-associated genes in YAP1-
induced cells, our data evidence the regulation of neuroblastoma cell motility by YAP1.
RNA-sequencing analysis of SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
overexpressing constitutively active YAP1S127A revealed significantly upregulated migration-
associated genes that were related to ECM reconstructions, cytoskeletal rearrangements and
cell-cell as well as cell-ECM interactions, showing that YAP1 co-determines the ECM structure
and thus, can intervene in migration processes. The assembly and disassembly of substrate
anchors of the cell, so called focal adhesion (FA), is crucial for cell motility and is described to
be regulated by YAP1 (Nardone et al., 2017; Mason et al., 2019). Our data revealed a
significant upregulation of integrins, main component of FAs, as well as some additional FA-
associated transcripts (e.g., genes for vinculin, myosin, tensin and paxillin) in YAP1-induced
SK-N-AS-TR-YAP1S127A cells. Qiao and colleagues uncovered a link between dynamic
cytoskeleton rearrangements necessary for migration and metastasis and our protein of
interest: YAP1 positively regulates the RhoA inactivator ARHGAP29, thereby inhibits the ROCK-
LIMK-cofilin axis and reduces the cellular stiffness, which in turn endorses a metastatic
phenotype (Qiao et al., 2017). Both YAP1S127A-inducible neuroblastoma cell lines exhibited an
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upregulation of ARHGAP29 (supplementary Table C) representing a possible YAP1-mediated
mechanism of neuroblastoma cell migration.
Seong and coworkers established a metastatic neuroblastoma mouse model by intracardial
injection of neuroblastoma SK-N-AS cells that subsequently gave rise to metastasis in bones
and the CNS. These were reinjected into mice and the in turn resulting metastasis were found
to be YAP1-activated and more metastatic than parental cells. Knockdown experiments
confirmed the YAP1 dependence of metastatic subpopulation growth (Seong et al., 2017). The
question of whether active YAP1 is a driver or a consequence of metastasis development,
remains unanswered. Another strong indicator for YAP1 as a key player in neuroblastoma
metastasis development is the high proportion of YAP1 positive cells in lymph node metastasis
compared to primary neuroblastoma sites, as recently reported by Cai et al. (Cai et al., 2020).
In another pediatric tumor, YAP1 also acts as a mediator of cell motility and metastasis
development. Only recently, an Ewing sarcoma subtype exhibiting low levels of oncoprotein
EWS-FLI1 (also known as EWS-ETS) has been shown to exhibit enhanced YAP1/TAZ/TEAD
signaling. The treatment of EWS-FLI1low Ewing sarcoma cells with YAP1-TEAD inhibitor
Verteporfin inhibited cell migration and metastasis development in vitro and in vivo, without
impairing the cell proliferation (Bierbaumer et al., 2021). Our in vitro data, that prove a YAP1-
mediated regulation of neuroblastoma cell migration, are considered as being supported by
the in vivo data that were obtained by other researchers.
Taken together, our data present evidence for a YAP1-mediated regulation of neuroblastoma
cell migration and related ECM reconstructions, potentially linking the activation of YAP1 in
recurrences and metastasis to previous migration events. For the sake of completeness it
should be mentioned that in turn ECM bidirectionally orchestrates YAP1 activity via
mechanical forces (Dupont et al., 2011) tight and adherens junctions (Kim and Gumbiner,
2015; Park et al., 2019), Wnt signaling (Park et al., 2015), RTK and GPCR signaling (reviewed in
Piccolo, Dupont and Cordenonsi, 2014; Meng, Moroishi and Guan, 2016; Rausch and Hansen,
2020). Although we did not make mechanisms of YAP1-induced neuroblastoma-ECM
interactions the subject of our investigation, this might be very interesting to follow up due to
the fact, that dissemination of tumor re-initiating cells and subsequent relapse development
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forces tumor cells to migrate through and interact with many different tissues and
corresponding ECM. Moreover, the development of relapses is still one factor of worse
outcome for neuroblastoma patients and it needs further efforts to understand the molecular
biology and define druggable targets. The data collected in this study show, that YAP1 might
be such a target molecule by promoting drug resistance and migration in neuroblastoma cells.
6.4 Does YAP1 induce EMT in neuroblastoma?
The transition of epithelial cells to a mesenchymal phenotype in order to allow migration and
invasion is a phenomenon occurring in both, normal physiology and tissue cancer biology
(Yang et al., 2020). Neural crest cells undergo EMT to reach the state of migratory NCCs via
induction of signaling pathways (e.g., TGF
β
/BMP, Wnt, RTK), transcription factors (e.g.,
SNAI1/2, Twist1, Sox8/9/10), cadherin switch/shedding and matrix remodeling (Theveneau
and Mayor, 2012). Cancer cells are also able to change their phenotype stepwise, which is
preliminary for detachment from tumor bulk, subsequent enhancement of motility for the
migration through surrounding tissues as well as ECM, and invasiveness into and out of the
circulation in order to outgrow metastasis at distant locations. The malign transition thereby
proceeds similar to the developmental EMT via external and internal signaling (Theveneau and
Mayor, 2012). Thus, evidence of EMT cannot only be provided by the transcriptional activity
of transcription factors, but also needs proof of changes in cellular properties (Yang et al.,
2020).
We have shown that an activation of the protein YAP1 in neuroblastoma cell lines enhanced
the cell migration potential and increased the cellular tolerance to chemotherapeutic drugs.
Additionally, the SK-N-AS-TR-YAP1S127A cells, but not the SH-EP-TR-YAP1S127A, revealed an
upregulation of EMT transcription factors SNAI2, RUNX1/2 and TWIST2 upon YAP1 activation,
indicating an EMT-like switch in these cells. In neuroblastoma, which is a more mesenchymal
tumor compared to epithelial carcinomas (Tan et al., 2014), cells have been shown to
transition from adrenergic to a mesenchymal or NCC-like phenotype and vice versa (Boeva et
al., 2017; Van Groningen et al., 2017). Van Groningen and colleagues established gene
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133
signatures to which neuroblastoma cells can be assigned either to the MES type expressing
VIM and FN1 or to the DBH, PHOX2A/B and GATA2/3 expressing ADRN subtype (Van
Groningen et al., 2017). Interestingly, all mesenchymal cells highly express YAP1 (Van
Groningen et al., 2017). Boeva et al. in parallel drafted these two distinct cell populations on
the basis of transcriptional core regulatory circuitries (CRC) including the TFs PHOX2B, HAND2
and GATA3 driving the sympathetic noradrenergic cells and AP-1 family TFs determining the
NCC-like cells (Boeva et al., 2017). Both research groups could correlate the mesenchymal cell
phenotype to reinforced drug resistance. Whether the frequently refractory relapsed
neuroblastomas contain more MES cells remained unclear, as the results were different in
both studies (Boeva et al., 2017; Van Groningen et al., 2017). In fact, a very recent publication
confirmed the finding of a mesenchymal super enhancer signature. It has been found
predominantly in neuroblastoma relapses and metastasis, including the upregulation of
mesenchymal CRC transcription factor target genes compared to corresponding primary
tumors (Gartlgruber et al., 2020). The comparison of our transcriptome data of YAP1-induced
SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A cells with the MES and ADR signatures defined
by van Groningen et al. and Boeva et al. revealed a high proportion of the MES gene sets
upregulated and ADR genes downregulated in SK-N-AS-TR-YAP1S127A, but not in SH-EP-TR-
YAP1S127A (Boeva et al., 2017; Van Groningen et al., 2017).
In line with our data, Shim et al. perturbed YAP1 in neuroblastoma cell lines NLF and IMR-5
and found YAP1 positively regulating mesenchymal genes such as OCT4, SOX2, IL6ST and JAK1
(Shim et al., 2020). We hypothesized that an activation of YAP1 in neuroblastoma cells could
initiate a transition into the mesenchymal phenotype and thereby favor the progression of
relapse and metastasis. One of the remaining questions is, why only SK-N-AS and not SH-EP
cells are driven into a mesenchymal phenotype. We think, this transition is a cell type specific
process, possibly additionally influenced by the microenvironment. SK-N-AS cells are
categorized as mesenchymal by Gartlgruber et al. and Boeva et al. ranked them as
intermediate cell type, whereas SH-EP cells are irrevocably indicated as mesenchymal by all of
them (Boeva et al., 2017; Van Groningen et al., 2017; Gartlgruber et al., 2020). Activation of
the mesenchymal protein YAP1 in already mesenchymal cells possibly drives different
mechanisms compared to a more adrenergic cell, that still achieves an advantage by transition
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into a motile, resistant and thereby more aggressive mesenchymal phenotype. The different
cellular phenotypes and backgrounds of SK-N-AS and SH-EP cells might also be the reason for
the finding of only a minimal number of genes commonly up- and downregulated in both,
when YAP1 was induced.
Pursuing the idea of the two cellular phenotypes within neuroblastoma tumors, with SH-EP
being the more mesenchymal and YAP1 high expressing cell line, a higher chemotherapeutic
drug tolerance would be expected in SH-EP-TR-YAP1S127A compared to SK-N-AS-TR-YAP1S127A
cells. In fact, SK-N-AS-TR-YAP1S127A cells tolerated higher concentrations of etoposide,
doxorubicin and vincristine indicating additional mechanisms to provoke drug resistance in
neuroblastoma cells.
However, in other cell systems and tumor entities, YAP1 has been shown to drive EMT-like
switches such as in the neuroblastoma cell line SK-N-AS (Overholtzer et al., 2006; Zhao et al.,
2008; Lamar et al., 2012; Diepenbruck et al., 2014; Shao et al., 2014; Yu et al., 2018; Park et
al., 2019; Kandasamy et al., 2020). Park et al. found YAP1 driving EMT in a RAC-1 and Wilms
Tumor-1 dependent manner (Park et al., 2019). The research group of W. Hahn found an
oncogenic-KRAS specific transcriptional regulation of EMT genes by binding of YAP1 to the
mesenchymal transcription factor FOS in human colon cancer cells (Shao et al., 2014). Also, in
NSCLC tumors YAP1 drives an EMT-like program via TEAD-dependent induction of the EMT
transcription factor SLUG (Yu et al., 2018). YAP1 is a transcriptional co-activator, that cannot
directly bind DNA, but interacts with a wide range of transcription factors such as TEAD
(Vassilev et al., 2001; Li et al., 2010), AP-1 (Zanconato et al., 2015; Liu et al., 2016; Park et al.,
2020), SMAD (Ferrigno et al., 2002; Grannas et al., 2015), RUNX (Zaidi et al., 2004; Qiao et al.,
2016), ERBB4 (Komuro et al., 2003; Haskins, Nguyen and Stern, 2014), TBX5 (Rosenbluh et al.,
2012), tp73 (Strano et al., 2001) and Brd4 (Zanconato et al., 2018) to mediate regulation of
various cellular processes. This allows for a flexible and very “customized” response, which
may be why different cell systems yield such variable downstream signaling of YAP1.
According to the current stage of knowledge, cancer cells undergo a partial EMT (pEMT),
marked by coexpression of epithelial and mesenchymal markers, rather than a complete
transition to epithelial or mesenchymal phenotype (Nieto et al., 2016; Yang et al., 2020).
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Panchy et al. recently published an analysis of transcriptomic data from various carcinomas
calculating summary scores for epithelial and mesenchymal genes from single cancer-cell
samples and found heterogeneous distribution of the tumors to five different clusters
representing epithelial, mesenchymal and three intermediate subtypes. Interestingly,
migration-associated genes increased dramatically in an intermediate state and not in the final
mesenchymal state (Panchy et al., 2020). Two years earlier it has already been shown on
single-cell level, that cells from one single tumor differ in their pEMT state with distinct, state-
dependent migration potentials and also different chromatin region accessibilities
(Pastushenko et al., 2018). YAP1 induced in one of two neuroblastoma cell lines a switch into
mesenchymal direction, and it is still unclear if this might be a full transition. Only the EMT
transcription factors, SNAI2, RUNX1/2 and TWIST2, were upregulated in induced
SK-N-AS-TR-YAP1S127A cells suggesting, if at all, only a partial EMT. Time- and state-dependent
measurement of EMT transcription factor transcriptome levels in E to M transitioning
MCF-10A cells revealed a well-orchestrated differential gene expression over time and state
(Panchy et al., 2020). Also, in neuroblastoma cells, a single-cell analysis revealed
transitional-state characteristic expression cluster of phenotype-specific marker genes (Yuan
et al., 2020). This demonstrates that not all factors and proteins need to be expressed at the
same time via EMT, but there are clusters of molecules that characterize a transition stage.
Cellular EMT-related traits such as motility and drug resistance were affected by YAP1
perturbation. Genes regulating ECM reconstructions, enabling not only cellular movement but
also creating space and premises for cell migration, were upregulated as well. The timing of
cellular transition processes should also be considered; three days after YAP1 activation, a
partial transition at most can be expected (Panchy et al., 2020), as EMT is known to take about
some days both, in vitro and in embryonic development (Schünke, Schulte and Schumacher,
2006; Tran et al., 2011; Karacosta et al., 2019). Moreover, the obligatory epigenetic
regulations such as DNA methylation, histone modifications and miRNA and long-non coding
RNA deregulations, that are necessary for transition of cells during EMT, have not been
studied in our system (Sun and Fang, 2016; Wang, Dong and Zhou, 2020). Thus, due to the
short activation time we are not allowed to judge about a possible complete EMT driven by
YAP1.
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The data provided in this study demonstrate that activated YAP1 can, dependent on the cell-
context, initiate a pEMT-like state in neuroblastoma cell lines, although we cannot make a
final statement about the nature of the YAP1-induced switch from adrenergic-to-
mesenchymal neuroblastic cells. In line with our hypothesis, all neuroblastoma cells already
assigned to or transitioning into mesenchymal phenotype are shown to express high levels of
YAP1 (Boeva et al., 2017; Van Groningen et al., 2017; van Groningen et al., 2019; Shim et al.,
2020).
6.5 YAP1 intervenes in the metabolism of neuroblastoma cells
Referring to the initial finding of YAP1-activated mRNA signature in neuroblastoma relapses
and a subsequent acknowledgement of YAP1 activation in aggressive, migratory and often
metastatic neuroblastoma cells by our own and other research groups, we sought for
YAP1-driven metabolic adaption that favors metastatic tumor growth such as described for
melanoma (Lee et al., 2019) or colorectal cancer (Kuo et al., 2019). As nicely reviewed by Koo
& Guan and Yamaguchi & Taouk, YAP1 is linked to glucose metabolism, lipid metabolism,
amino acid metabolism and also G-protein coupled receptors (GPCR) sensing metabolites (Koo
and Guan, 2018; Yamaguchi and Taouk, 2020). Effects of active YAP1S127A on neuroblastoma
cell metabolism were examined on transcriptome level and in functional assays.
The finding of YAP1 shaping the central carbon metabolism of human cells is described for
many cell types including normal tissues (Cox et al., 2018) and tumor cells (Cosset et al., 2017;
Lin and Xu, 2017; Song et al., 2017; White et al., 2019). In addition, glycolysis is reported to
vice versa dictate YAP1 signaling through various mechanisms (Mo et al., 2015; Wang et al.,
2015; Peng et al., 2017; Bhattacharya, Azambuja and Simoes-Costa, 2020). In this work, only
YAP1-driven downstream effects were subjected. Our data revealed no alteration of
extracellular glucose and lactate concentrations in YAP1-silenced neuroblastoma cells.
Concomitantly, after the observation of rapid medium acidification in YAP1S127A-activated
SH-EP-TR-YAP1S127A cell cultures, the measurement of extracellular glucose and lactate
concentrations revealed decreased extracellular glucose levels and increased extracellular
Discussion
137
lactate levels. In addition, the ECAR and OCR of YAP1-activated SH-EP-TR-YAP1S127A and
SK-N-AS-TR-YAP1S127A cells was detected simultaneously with a subsequent inhibition of
mitochondrial electron transport chain and glycolysis. The ECAR, which is mainly determined
by cellular lactate secretion, was increased in YAP1-activated cells and showed a reinforced
response to 2-DG application inhibiting the glycolysis pathway. The OCR, predominantly
driven by mitochondrial respiration, was impaired to a less extend in YAP1-activated SH-EP-
TR-YAP1S127A cells upon mitochondrial inhibition. The transcriptome analysis of YAP1-
activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells revealed an upregulation of
some glycolysis genes and genes encoding for lactate and glucose transporters.
Analysis of glycolytic flux and lactate secretion of kidney cells individually overexpressing one
of 24 glycolysis genes revealed only four key nods regulating glycolysis: glucose import, lactate
export and fructose-1,6-bisphosphate production. Additional analysis of oncogene-driven
glycolysis as well as glycolytic genes upregulated across 21 solid tumor entities revealed an
additional fourth step being decisive for glycolysis perpetuation: the phosphorylation of
glucose by HK2 (Tanner et al., 2018). In our experimental setup, YAP1S127A induced different
glycolytic genes such as Glut2/3, HK2, PFKP and MCT4/7/8 in both cell line models, all being
part of the key steps described by Tanner et al. (Tanner et al., 2018). Thus, the YAP1S127A-
activated cells gained the ability to enhance the utilization of extracellular glucose and the
release of lactate, although only a few glycolysis genes were differentially expressed.
Our results indicate that YAP1-activated cells increase their glucose consumption, metabolize
that glucose into lactate and release that metabolite into the extracellular space.
Furthermore, mitochondrial respiration does not seem to be essential for maintenance of
YAP1-induced neuroblastoma cells. The metabolic phenomenon of glucose degradation to
lactate in the presence of oxygen has been described around 100 years ago by Otto Warburg
and is nowadays referred to as Warburg effect (Warburg, Wind and Negelein, 1927). C. and G.
Cori. and O. Warburg independently found tumor traversing blood exhibiting high lactate
levels and low glucose levels compared to blood in control veins, which they explained by
„aerobic glycolysis“ of cancer cells (Cori and Cori, 1925).
Discussion
138
Similar to our data collected in neuroblastoma, Cosset and co-workers found glucose
transporter Glut3 positively regulated by YAP1 in an integrin
𝑎vβ3
-expressing subpopulation
of glioblastoma (Cosset et al., 2017). Congruent to genes we found affected by YAP1
overexpression in neuroblastoma, the downregulation of glycolytic genes Glut3, HK2 and PFKP
with subsequent alteration of ECAR was reported by White et al. in YAP1 knockdown NF2-
deficient tumor cells (White et al., 2019). In our hands, a siRNA-mediated YAP1 silencing had
no effect on extracellular glucose or lactate levels in neuroblastoma cell lines. One could argue
that other regulatory mechanisms compensate for YAP1 loss. We rather think that the
metabolism of neuroblastoma cells growing in standard culture conditions, that are explicitly
composed as a glucose-rich, cell growth promoting and well-being microenvironment, does
not rely on YAP1-driven glycolysis. In such conditions, endogenous YAP1 mainly co-determines
the growth of neuroblastoma cells, as already shown in knockdown experiments. In turn, the
overexpression of YAP1 causes changes in cell identity and metabolism of neuroblastoma cells
rather than proliferative capacity. Only in challenging conditions such as nutrient and growth
factor limitations, YAP1 activation provides a cell viability benefit. Assuming such challenging
conditions in vivo, data from White et al. backed our data: without additional stress sources
such as glucose and glutamine limitation, YAP/TAZ depleted cells in vitro lack the dramatic
drop in tumor growth observed in vivo in YAP1-depleted orthotopically-transplanted renal
kidney tumor cells (White et al., 2019). Since in vitro YAP1 silencing in breast cancer cells
reduced glycolysis gene expression and lactate levels (Lin and Xu, 2017) such as in the
NF2-deficient cells, the lack of glycolysis regulation upon YAP1 knockdown in our study seems
to be neuroblastoma-specific.
However, the overexpression of YAP1S127 drives the transcriptional upregulation of target
genes encoding glycolysis proteins as well as transporters for glucose and lactate, thereby
conveying a metabolic switch into aerobic glycolysis. The pro-tumorigenic features of
enhanced glycolysis resulting in i) fueling the cellular metabolism, ii) adaption to hypoxic
conditions and iii) producing lactate as precursor for gluconeogenesis, regulator of
microenvironment and mediator of immune escape have been reviewed extensively (Gatenby
and Gillies, 2004; Hirschhaeuser, Sattler and Mueller-Klieser, 2011; Liberti and Locasale, 2016;
Parks, Mueller-Klieser and Pouysségur, 2020). We showed that YAP1 activation in
Discussion
139
neuroblastoma cells resulted in increased glycolysis and subsequent enhanced lactate
production. In regard to unaltered or even decreased gene expression of gluconeogenesis
enzymes (GPI, PEPCK, ENO3) lactate might predominantly serve as a molecule modulating
microenvironment and immune response for YAP1-activated neuroblastoma cells, which still
needs to be clarified. The YAP1-mediated induction of genes regulating de novo serine and
glycine synthesis as well as mitochondrial folate cycle indicates the re-routing of glucose-
derived carbon units into additional anabolic pathways beside glycolysis, which could be
further exploited in time-resolved metabolome analysis. The association between YAP1 and
cell adaption to hypoxia will be discussed later.
Recently, a close relationship between glycolytic flux and EMT has been highlighted, which is
valid in cancer cells as well as in developmental biology. Bhattacharya et al. reported
enhanced glycolytic activity as an obligate prerequisite for EMT and cell migration in neural
crest cells, the progenitors of neuroblastoma cells, in vivo (Bhattacharya, Azambuja and
Simoes-Costa, 2020). In addition, YAP1 was found to drive these EMT-related genes via tissue-
specific enhancers, although data only evidenced glycolysis-regulated YAP1 expression and
not vice versa (Bhattacharya, Azambuja and Simoes-Costa, 2020). Our data prove, that YAP1
overexpression in turn enhances aerobic glycolysis of neuroblastoma cells and that YAP1
initiates an EMT-like phenotype switch in SK-N-AS-TR-YAP1S127A. Other groups reported YAP1-
induced mesenchymal or undifferentiated cellular phenotypes in neuroblastoma (Shim et al.,
2020) and embryonal rhabdomyosarcoma (Slemmons et al., 2015), and also enhanced
glycolysis determined by YAP1 expression has been shown for glioblastoma (Cosset et al.,
2017) and breast cancer (Lin and Xu, 2017), but no triangle-correlations between aerobic
glycolysis, EMT and YAP1 have been reported by now in a single tumor entity. Thus, an open
question remains if YAP1-induced glycolysis directly promotes pEMT-like phenotype changes
in neuroblastoma or if both occur in parallel.
In summary, active YAP1 is able to induce the Warburg effect and, cell-context dependently,
it causes a mesenchymal phenotype switch in neuroblastoma cells. Such YAP1-activated cells
likely possess a high potential to foster tumor development by fueling anabolic pathways for
Discussion
140
cell growth, remodeling the tumor microenvironment and facilitating immune escape by
subsequent lactate production.
Amino acids and one carbon metabolism provide a large amount of the building blocks needed
for the generation of biological macromolecules such as proteins, lipids and nucleic acids. In
addition, regulation of a redox balance, genetic and epigenetic cell status is maintained by
those (DeBerardinis, 2011; Ducker and Rabinowitz, 2017). In SH-EP-TR-YAP1S127A cells, one of
two YAP1-inducible neuroblastoma cell line models, YAP1 enhanced expression levels of
enzymes catalyzing the metabolic network of serine and glycine biosynthesis, cysteine-
generating transsulfuration pathway and mitochondrial part of folate cycle. The regulation of
serine metabolism by YAP1 is, up to date, only hypothesized based on a mRNA correlation
analysis in LKB1-deficient breast cancer cells by Wu and colleagues (Wu et al., 2017). In
neuroblastoma, high expression level of PHGDH, the first and rate-limiting enzyme of de novo
serine synthesis pathway (SSP), is predominantly present in MYCN-amplified cell lines and
tumors and sensitized them to PHGDH inhibition (Xia et al., 2019; Arlt et al., 2021). In other
entities, tumor metastasis express high levels of PHGDH and lead to a routing of glucose-
derived carbon units into serine and glycine metabolism as well as one carbon metabolism
(Samanta et al., 2016; Ngo et al., 2020), but also primary tissues show high protein abundance
of PHGDH (The Human Protein Atlas: PHGDH). PHGDH and SSP are known to induce drug
resistance to cancer cells. Dependent on ATF4, PHGDH causes an accumulation of
𝑎
-ketoglutarate (
𝑎
KG) and NAPDH, an important antioxidant functioning as electron donor for
glutathione (GSH) and thioredoxin synthesis (Harris and DeNicola, 2020), and thereby
mediates TKI resistance (Wei et al., 2019). Ross and colleagues identified the serine and folate
metabolism as essential contributors to MAPK-cascade-mediated BRAFV600E inhibitor
resistance in cancer cells (Ross et al., 2017). PHGDH as part of serine biosynthesis is a strong
mediator of drug resistance comprising all classifications of chemotherapeutic drugs as well
as targeted therapies such as TKI (Jing et al., 2015; Zhang and Bai, 2016; Yoshino et al., 2017;
Zaal et al., 2017; Dong et al., 2018). Possibly, YAP1-driven drug tolerance towards
chemotherapeutic agents in part also relies on activation of SSP, which needs further to be
investigated. Additionally, main serine pathway regulator ATF4 was induced in YAP1-active
SH-EP-TR-YAP1S127A cells (supplementary Table C), which also strengthens the context-
Discussion
141
dependent importance of serine metabolism in YAP1 pathogenesis. The activation of ATF4 by
YAP1 has also been shown by Wu and collaborators, who postulated the regulation of YAP1
via PERK-eIF2
𝑎
-ATF4 axis after endoplasmic reticulum stress induction, but also showed an
ATF4 induction in YAP1 overexpressing liver cells (Wu et al., 2015).
In the neuroblastoma cell line model SH-EP-TR-YAP1S127A, YAP1S127A activation also enhanced
expression of genes regulating the mitochondrial folate metabolism. This part of one-carbon
metabolism is crucial in cancer cells to generate serine-derived one-carbon units
(Labuschagne et al., 2014), which in turn are transferred to formate and subsequently
supplied into nucleotide synthesis (Ducker et al., 2016). Knockout of SHMT1/2, which encode
serine-converting enzymes, impaired tumor growth of colorectal carcinoma cells HTC-116 in
vivo (Ducker et al., 2017). A SHMT2 knockdown in xenografted neuroblastoma cells also
prolonged the tumor growth significantly, emphasizing the importance of these enzymes for
tumor development (Ye et al., 2014). In a reactive oxygen species (ROS)-promoting condition
like hypoxia, SHMT2 is part of the redox-balancing machinery in neuroblastoma cells (Ye et al.,
2014). The folate cycle provides substantial amounts of antioxidant NADH and NADPH to
maintain redox homeostasis (Fan et al., 2014). HEK293T cells fed with [2,3,3-2H]-serine were
shown to reduce the incorporation of NADH into malate after SHMT2 or MTHFD2 silencing,
proving that most NADH is generated in MTHFD2-dependent mitochondrial serine-
metabolism (Locasale, 2013; Ducker et al., 2016). Also GSH, one output of transsulfuration
pathway which we also found to be upregulated in YAP1S127A-induced SH-EP-TR-YAP1S127A
cells, scavenges ROS and thereby regulates redox balance (Locasale, 2013). Considering the
reduced oxygen consumption rate of YAP1-activated neuroblastoma cells, our data suggest a
YAP1-mediated suppression of oxidative phosphorylation in SH-EP-TR-YAP1S127A, which in turn
fosters the reduction in intracellular ROS levels. White and co-workers have also shown, that
YAP1 maintains the redox balance by upregulation of glycolysis genes such as HK2 and PFKFB4
and reducing oxidative phosphorylation and thus, ROS production (White et al., 2019).
ALDHL2, which catalyzes conversion of 10formyl-THF into THF and CO2 under NADPH
production, has been shown to promote metastasis by raising the antioxidant defense
(Piskounova et al., 2015). YAP1 also induced ALDHL2 gene expression in our YAP1-inducible
Discussion
142
neuroblastoma cell line, which additionally exhibited a migratory and drug tolerant
phenotype.
In conclusion, we could show that YAP1 can transcriptionally activate gene expression of SSP,
mitochondrial folate cycle and transsulfuration pathway in neuroblastoma cells. The
evidenced increase of glucose consumption and upregulation of key glycolysis genes ensures
the multistep conversion of glucose into 3-phosphoglycerate, which is the substrate for the
SSP enzyme PHGDH and potential precursor molecule for the folate cycle and transsulfuration
pathway. Thus, active YAP1 might enable neuroblastoma cells to reroute glucose-derived
carbon units into these pathways to possibly maintain the redox homeostasis, which requires
further investigation.
Transcriptome data of YAP1-induced neuroblastoma cells revealed enhanced gene expression
for amino acid transporters in both cell lines, SK-N-AS-TR-YAP1S127A and SH-EP-TR-YAP1S127A
cells, although the effect was stronger in the latter. In addition, expression of cytosolic and
mitochondrial glutaminolysis pathway genes GOT1, PSAT1 and GTP2, were induced in
SH-EP-TR-YAP1S127A cells, yet glutaminase genes GLS1 and GLS2 were not affected. Amino acid
transporters and subsequent mTOR signaling have been shown to be regulated by YAP1 via
direct YAP1-TEAD1 transcriptional regulation in hepatocellular carcinoma (Park et al., 2016)
Also in other tissues and tumor entities, the YAP1-controlled transcriptional activation of
SLC1A5, SLC7A5, SLC38A1, GLS, GLUL, GOT1 and PSAT1 was described extensively (Hansen et
al., 2015; Bertero et al., 2016; Edwards et al., 2017; Liu et al., 2017; Yang et al., 2018). In cancer
cells, amino acids are primarily used for protein synthesis to cover the biosynthetic demand
in fast proliferating cells (Vettore, Westbrook and Tennant, 2020). We have shown that YAP1
activation enhances amino acid transporter and synthesis genes in already transformed and
fast proliferating neuroblastoma cells with lack of further increase of cell growth. This raises
the question of what the additional amino acids are used for.
It is still vague, how YAP1 is activated in neuroblastoma cells and when it gets activated. Some
reports highlight the activation of YAP1 in neuroblastoma relapse and metastasis (Schramm
et al., 2015; Seong et al., 2017; Cai et al., 2020), others found a correlation between YAP1
Discussion
143
mRNA and tumor stages (Yang et al., 2017). We think, YAP1 might be a “survival protein”
supporting cells, that are exposed to challenging conditions such as nutrient limitation, drug
treatment or hypoxia. In this case, the upregulation of amino acid transporters might not have
a great value in artificial cell culture conditions but could cover the need for energy and
biomolecules in such physiological conditions.
Yue and colleagues described a transcriptional activation of essential amino acid transporters
by MYC proteins, which initiated MYC translation in a positive feedback loop to maintain the
oncogenic metabolism including glycolysis, SSP and glutaminolysis of neuroblastoma and
lymphoma cells (Yue et al., 2017). It is entirely possible, that an accumulation of amino acids
also maintains high YAP1 expression levels. A mTOR-mediated post-translational upregulation
of YAP1 has been demonstrated in perivascular epithelioid cell (PEC)-omas in vitro and in vivo
(Liang et al., 2014). Since it has already been shown that intracellular high amino acid levels
lead to an activating translocation of mTORC1 (Bar-Peled and Sabatini, 2014), YAP1-induced
upregulation of amino acid transporters could close such a positive feedback loop to sustain
its activity. However, based on the herein presented data no statement can be made about
the actual protein synthesis or the transport of amino acids in YAP1-activated neuroblastoma
cells. Thus, follow up experiments are needed to verify or disprove this hypothesis.
The direct YAP1-mediated upregulation of glutaminolysis genes GOT1, PSAT1 and GTP2 that
was found in YAP1-activated SH-EP-TR-YAP1S127A cells, has already been shown in breast
cancer cells (Yang et al., 2018). After glutamine uptake is secured by upregulation of glutamine
transporter SLC38A1, GLS enzymes catalyzing the first step of glutamine conversion are
interestingly not affected by YAP1S127A expression in both, breast cancer and neuroblastoma
cells (Yang et al., 2018). In addition to a transcriptional regulation of GLS1 and GLS2 (Gao et
al., 2009), the activity of GLS enzymes is modulated via posttranslational modifications (Han
et al., 2018). Thus, the conversion of glutamine into glutamate could be secured in YAP1-
activated neuroblastoma cells without upregulation of the GLS1/2 expression.
Molecules emerging from the glutamine conversion are amino acids such as aspartate, alanine
and serine, epigenetically active
𝑎
KG and antioxidative GSH (Yoo et al., 2020). All of that are
Discussion
144
able to support and partially mediate the drug resistant and aggressive phenotype of YAP1-
expressing neuroblastoma cells.
Taken together, our data reveal an upregulation of genes encoding enzymes of the amino acid
metabolism in YAP1-activated cells. Follow-up experiments should include metabolomics of
YAP1-activated neuroblastoma cells, to evaluate amino acid levels and their presumable
contribution to resistance mechanisms as well as the antioxidative shielding of the cells.
The reduction or abolishment of oxygen supply frequently leads to hypoxic microregions
within neuroblastoma (Pietras et al., 2007; Chen et al., 2015) as well as in other solid tumors
(Höckel and Vaupel, 2001). Cells are able to tailor oxygen-limited conditions down to a O2
partial pressure of 8-10mmHg, whereas individual O2-dependent cellular functions vary in
their critical thresholds (Höckel and Vaupel, 2001). Neuroblastoma cell trait changes
accompanying the hypoxic adaptation include induction of the Warburg effect (Qing et al.,
2010), development of drug resistances (Hussein et al., 2006) and increase of cell motility
(Chen et al., 2015). All of them have been highlighted in this work for YAP1-activated
neuroblastoma cells. The search for gene expression alterations of hypoxia-related enzymes
resulted in YAP1-mediated upregulation of EPAS1, encoding for hypoxia-inducible factor
(HIF)-2
𝑎
, in both cell line models SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A. The research
group around S. Påhlman evidenced a HIF-2
𝑎
-driven initiation of an aggressive and immature
NCC-like phenotype of neuroblastoma cells in vitro and in vivo (Nilsson et al., 2005; Pietras et
al., 2007). Thus, we initially hypothesized that YAP1 binds to and thus stabilizes HIF-2
𝑎
in
normoxic conditions to initiate metabolic changes upon YAP1 activation in neuroblastoma
cells. The protein detection via western blot technique revealed, that only in hypoxic (1%
oxygen) and not normoxic conditions higher HIF-2
𝑎
protein signals could be detected in YAP1-
activated cells compared to control cells. Thus, alterations of glycolytic rates in YAP1-
overexpressing neuroblastoma cells are likely not HIF-2α-mediated.
HIF-2
𝑎
was reported to be stabilized in long-term hypoxic neuroblastoma cells as well as in
cells incubated in normoxic conditions in both, in vitro and in vivo (Nilsson et al., 2005;
Holmquist-Mengelbier et al., 2006; Pietras et al., 2007, 2009). Herein presented results could
Discussion
145
not verify HIF-2
𝑎
abundance in SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cell line models,
that were cultured under normoxic conditions. Unfortunately, different neuroblastoma cell
lines were used in both in vitro studies, diminishing the data comparability. Furthermore,
Nilsson et al. separated and probed 60-80 µg protein lysates, whereas for our study only 10
µg protein were applied, which could explain result contradictions due to detection limitations
of western blot technique. In addition, equal protein loading into SDS-gels was not evidenced
by Nilsson and colleagues, reducing their data validity (Nilsson et al., 2005).
Well characterized target genes of HIF-2
𝑎
such as VEGFA and BHLHE40 (Persson et al., 2020)
were only upregulated in one YAP1-activated cell line each (supplementary Table C), which
does not clearly answer the question of commonly YAP1-HIF-2
𝑎
regulated gene expression.
The analysis of TF binding-motif enrichment among the significantly upregulated genes in
YAP1S127A-activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells revealed HIF DNA
binding motifs in 4-5% of the transcripts. Although this seems a very minimal proportion, the
DNA binding motif of well-described and main interaction partner TEAD was only present in
7-8% of all upregulated genes. Putting both in proportion, HIF proteins might actually co-
determine the YAP1-induced phenotype. Follow-up experiments including proximity ligation
assay and chromatin-immunoprecipitation with DNA sequencing (ChIP-Seq) of YAP1-
overexpressing neuroblastoma cells will shed light on a potential interaction of HIF-2
𝑎
and
YAP1 and their target sequences.
To our knowledge, the regulation of HIF-2
𝑎
via YAP1 is not known yet. Ma et al. demonstrated
the regulation of YAP1 by HIF-2
𝑎
in colon cancer, but without identification of any positive
feedback loop pointing to a vice versa regulation (Ma et al., 2017). Further, the GPRC5A-
mediated induction of YAP1 by HIF-1
𝑎
is requisite for cancer cell adaption to hypoxic
conditions (Greenhough et al., 2018). In hepatocellular carcinoma, a direct protein interaction
between HIF-isoform HIF-1
𝑎
and YAP1 has been demonstrated to induce aerobic glycolysis.
Since this glycolysis induction could be interrupted by verteporfin treatment, an appropriate
complex formation and downstream signaling is dependent on YAP1-interacting TEAD (Chen
et al., 2018). The two HIF-isoforms HIF-1
𝑎
and HIF-2
𝑎
share structural commonalities, and
thus, one could imagine an interaction of YAP1, TEAD and HIF-2
𝑎
as well, but this has further
Discussion
146
to be investigated. In regard to challenging therapeutic interventions of HIF-signaling, it is
valuable to identify tumor-specific HIF-interaction partners as surrogating target molecules.
Taken together, we could demonstrate an upregulation of EPAS1 in YAP1-activated
neuroblastoma cell lines. Consequently, the HIF-2
𝑎
abundance was higher in YAP1-activated
cells, when hypoxic conditions resulted in a consolidation of HIF-2
𝑎
. Contrary to our
assumption, HIF-2
𝑎
has not been stabilized by YAP1 in standard culture conditions. However,
the YAP1-induced EPAS1 upregulation might be an additional mechanism of HIF-2
𝑎
regulation
in hypoxic neuroblastoma cells.
6.6 Conclusions and Outlook
Cancer cells are highly proliferating and thus in a permanent need of biological building blocks.
Additionally, tumor cells frequently face challenging situations including space and nutrient
limitation, hypoxia, immune attacks as well as therapeutic interventions. In order to overcome
growth limitations and promote tumor development against the odds, cellular adaption
systems orchestrating the cell metabolism and escape strategies are requisite. YAP1 is one
conductor of such contextual adjustments in neuroblastoma cells, as we have demonstrated
in this study. Cell growth and viability was impaired in many YAP1 silenced neuroblastoma cell
lines. An important question remains about determining factors such as genetic aberrations
or active transcription factor networks that dictate YAP1 dependence of neuroblastoma cell
growth. The herein evidenced tolerance to chemotherapeutic drug disposition and increased
cell migration potential of YAP1-activated cells likely favors metastasis and relapse formation,
which still remains to be proven in appropriate biological systems such as in vitro three-
dimensional models or animal models. A cellular mesenchymal phenotype switch and
metabolic rewiring provoked by YAP1 paves the way for tumor cell migration and
re-settlement at distant sites. In addition, antioxidative potential of activated pathways as well
as transcriptional activation of HIF-2
𝑎
shields neuroblastoma cells against ROS-mediated
apoptosis. We suggest targeted and shot-gun proteomic with simultaneous mass
spectrometry-based metabolomic approach to identify proteins and metabolites altered by
Discussion
147
YAP1 expression in different neuroblastoma cell lines of various phenotype. Considering the
EPAS1 upregulation as well as physiological conditions, experimental setups in oxygen-limited
conditions would additionally be of great value.
Altogether, our data highlight YAP1 as a protein that promotes neuroblastoma tumor
progression. In particular, the presence of YAP1 in relapse neuroblastoma and metastasis
suggests a crucial role of YAP1 in conveying a survival benefit to cells in challenging conditions
and therefore offers an attractive molecule for targeted therapy. By mediating cell plasticity
to neuroblastoma cells, YAP1 is able to determine important hallmarks of cancer (e.g.,
sustained proliferation signaling, resisting cell death, activating invasion and metastasis,
deregulating cellular energetics (Hanahan and Weinberg, 2011)). In addition, a strong CD274
(PD-L1) and IL6R upregulation was observed, yet not further investigated, in both YAP1-
inducible cell line models (supplementary Table C). Thus, the influence of YAP1 activation on
immune escape and tumor-promoting inflammation absolutely needs to be clarified.
To understand regulatory networks leading to the broad variation of YAP1-mediated
modifications, we propose the broad-based quest of neuroblastoma-specific well-established
and de novo transcriptional interaction partners of YAP1 using protein-protein-interaction
approaches with subsequent DNA sequencing. However, the major challenge will be the
exploration of YAP1-activating mechanisms and expression patterns in primary, metastatic
and relapsed neuroblastoma: transcriptional levels are only moderately altered in bulk RNA-
sequencings of neuroblastoma tumors. Physiological tracking and transcriptional profiling of
single cells might enlighten us about YAP1 expression levels in individual tumor cells. Such
approaches will clarify, if clonal selection of highly YAP1-expressing cells give rise to refractory
metastatic and/or recurrent outgrowth of neuroblastoma cells or if metastasized cells exhibit
mechanisms to short-term activate YAP1, possibly only on protein level. Furthermore, single-
cell proteomics of primary and relapse tumors is suggested, since the protein activity is
predominantly conducted by protein phosphorylation cascades and it possibly might be a
proteomic rather than transcriptional regulation leading to YAP1-activated mRNA-signatures
in relapse tumors.
148
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149
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8. Appendix
8.1 Supplemental Figures
YAP1
pYAP1(S127)
β-Ac/n
SH-EP SK-N-AS
105210
FCScontent[%]
105210
FCScontent[%]
Supplementary Figure A YAP1 protein synthesis is reduced in serum-starved
SH-EP and SK-N-AS wildtype cells. SH-EP and SK-N-AS wildtype cells were cultured
in standard cell culture media containing different volumes of FCS (0%, 1%, 2%, 5%,
10%) for 72 hours. Protein detection via immunoblots revealed a decrease of YAP1
and pYAP1(S127) protein abundance upon reduced FCS concentrations in SH-EP
cells. SK-N-AS cells displayed low, albeit also decreasing YAP1 protein amounts and
no positive signals for pYAP1(S127). Gel load was detected by β-Actin probing.
Representative pictures of three independent experiments are shown.
Appendix
178
Supplementary Figure B Plasmid maps for shRNA-mediated YAP1 knockdown.
Appendix
179
Supplementary Figure C Plasmid maps for tetracycline-inducible YAP1S127A overexpression.
Appendix
180
8.2 Supplemental data of gene analysis
Table A Top ten list of the C2 collection of genes (MSigDB) enriched for the commonly, significantly upregulated genes in
SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
Designation C2 collection gene set
Counts
Adj. p-value
Gene symbols
WANG_METHYLATED_IN_BREAST_CANCER
9
0.0000003583
THBS1, SLC7A5, DUSP1, F3,
SLC3A2, UPP1, FOSL1, EPHA2,
GADD45A
SENESE_HDAC1_AND_HDAC2_TARGETS_UP
16
0.0000007266
TGM2, IL6R, NAV3, ST3GAL1,
RAB3B, UBASH3B, PTX3, OSMR,
FOSL1, BCL2L1, WWC1, CD55, FN1,
IL1RAPL1, IL7R, EGFR
ZWANG_CLASS_3_TRANSIENTLY_INDUCED_BY_EGF
16
0.0000007518
ADM, FAM84B, DLC1, DUSP1,
ETS1, NCEH1, EMP1, LMO7,
DUSP14, LATS2, WWC1,
PPP1R15A, EPHA2, CD55, PLK2,
EZR
KIM_WT1_TARGETS_UP
16
0.0000020892
THBS1, ARHGAP29, ST3GAL1, F3,
ETS1, SLC7A1, ATP13A3, CYR61,
PTX3, FGF1, WT1, FLNC, FOXD1,
FOSL1, EPHA2, ACTN1
BILD_HRAS_ONCOGENIC_SIGNATURE
15
0.0000159729
COL12A1, NAV3, MBOAT2, DUSP1,
CD274, SH2D5, PTX3, UPP1, FOSL1,
UAP1, PPP1R15A, PVR, EPHA2,
CD55, FN1
GROSS_HYPOXIA_VIA_ELK3_DN
12
0.0000159729
ADM, NEDD4L, ETS1, CYR61, UPP1,
CTGF, PPP1R15A, PLK2, GADD45A,
ANKRD1, VLDLR, SLC7A11
PLASARI_TGFB1_TARGETS_10HR_UP
12
0.0000624303
PAPSS2, NGF, NPPB, NUAK1,
DUSP14, CTGF, FOSL1, MGLL,
PPP1R15A, PVR, CRY1, ANKRD1
HALMOS_CEBPA_TARGETS_DN
7
0.0000624303
STC2, ARHGAP29, CYR61, CTGF,
GADD45A, TFAP2C, ERBB3
WANG_SMARCE1_TARGETS_UP
14
0.0000624303
COL12A1, EPAS1, MFAP5, JDP2,
SERPINB7, IRS1, PTX3, TAGLN,
WT1, CD55, FN1, VLDLR, PRR5L,
WARS
MCBRYAN_PUBERTAL_BREAST_4_5WK_UP
12
0.0001379103
THBS1, TGM2, STC2, F3, PTX3,
FIGN, WWC1, FN1, PLK2, EZR,
TFAP2C, ERBB3
Appendix
181
Table B List of the C2 collection of genes (MSigDB) enriched for the commonly, significantly downregulated genes in YAP1-
activated SH-EP-TR-YAP1S127A and SK-N-AS-TR-YAP1S127A cells
Designation C2 collection gene set
Counts
Adj. p-value
Gene symbols
MIKKELSEN_MCV6_HCP_WITH_H3K27ME3
6
0.0310475457
STMN3, ALDH1A3, LMO1,
NPTXR, RASSF4, ACVRL1
RIGGI_EWING_SARCOMA_PROGENITOR_DN
6
0.0310475457
ALDH1A3, GNG2, NRP2, PDE4B,
PTN, DPYSL3
VART_KSHV_INFECTION_ANGIOGENIC_MARKERS_UP
5
0.0362776770
CXCR4, NRP2, ANGPTL2, PTN,
TGFB3
Table C List of selected genes significantly upregulated in either YAP1-activated SK-N-AS-TR-YAP1S127A or
SH-EP-TR-YAP1S127A cells or both
Gene
symbol
Gene ID
SK-N-AS-TR-YAP1S127A
SH-EP-TR-YAP1S127A
Mean
Log2 FC
Adj. p-value
Mean
Log2 FC
Adj. p-value
ARHGAP29
ENSG00000137962
12313.03
2.1362
0.0000000
2559.44
1.8381
0.0000000
ATF4
ENSG00000128272
-
-
-
9738.45
1.0717
0.0000000
AXL
ENSG00000167601
6439.22
5.0711
0.0000000
37836.60
0.8296
0.0000989
BCL2L1
ENSG00000171552
1621.29
1.1489
0.0000003
4615.44
1.4227
0.0000000
BHLHE40
ENSG00000134107
454.10
1.0017
0.0188589
4516.47
-0.8563
0.0049071
CD274
ENSG00000120217
370.85
2.4982
0.0000000
7412.04
1.8439
0.0000000
FOSL1
ENSG00000175592
1983.75
2.1643
0.0000003
7304.09
1.5179
0.0489252
FOSL2
ENSG00000075426
2052.95
1.8010
0.0000000
-
-
-
IL6R
ENSG00000160712
1062.31
3.0532
0.0000000
560.93
0.9176
0.0203914
VEGFA
ENSG00000112715
-
-
-
3302.53
1.0237
0.0000116
182
Appendix
183
8.3 Acknowledgement / Danksagung
An dieser Stelle möchte ich mich bei den zahlreichen Menschen bedanken, die zum Entstehen
dieser Arbeit beigetragen und diese überhaupt erst möglich gemacht haben.
Mein besonderer Dank gilt Prof. Johannes H. Schulte für die großartige Möglichkeit der Arbeit
in seinem neuen Team. Seine fachliche Anleitung und Unterstützung sowie die gleichzeitig
gewährte Forschungsfreiheit ermöglichten es mir mich in den letzten sechs Jahren sowohl
beruflich als auch persönlich weiterzuentwickeln!
Weiterhin danke ich Prof. Roland Lauster sowie Prof. Angelika Eggert für den
wissenschaftlichen und darüber hinausgehenden Diskurs und zudem Prof. Hyun-Dong Chang
für die Begutachtung meiner Dissertation.
Großen Dank bringe ich Dr. Falk Hertwig und Dr. Jörn Tödling für ihre unermüdliche
Diskussionsbereitschaft und ihre unerschöpflichen Ideen entgegen. Dank ihrer exzellenten
Expertise, gepaart mit Akribie und einer guten Portion Humor, konnte ich sehr viel lernen und,
auch in entmutigenden Situationen, immer wieder neue Kraft und Motivation schöpfen, um
dieses Projekt erfolgreich zu beenden. Dass du, Falk, tapfer meine kreativitätsfördernde
Jazzmusik aus einer Musikbox von fragwürdiger Qualität ertragen hast, rechne ich dir bis heute
hoch an.
Ganz herzlich danke ich Melanie für ihre unermüdliche experimentelle Unterstützung,
insbesondere bei der Pflege meiner unzähligen Einzelklone, und Annika für ihre raffinierten
Klonierungszaubereien und ihre wirklich immer fortwährende gute Laune. Aleix danke ich für
ihre Bereitschaft, die Rätsel der Mausgenetik mit mir gemeinsam zu entschlüsseln. Dr. Filippos
Klironomos gebührt mein Dank für seine bioinformatische Analysen. Bei Dr. Nicole Hübener
bedanke ich mich für ihre Klarheit und Furchtlosigkeit im Bürokratiedschungel der Charité.
Ganz besonderer Dank gilt meinen Promotionsgefährt:innen Mareike, Birte, Annabell, Laura,
Lena, Sabine und Heathcliff für die vielen gesprächigen Stunden in der „Zelle“, die illustren
Appendix
184
Kaffeepausen, die geteilte Freude der kleinen und großen Erfolge, die aufmunternden Worte
nach Lehrstunden in Demut und die nicht versiegenden Quellen an Schokolade in unseren
Büros.
Allen Kolleg:innen meiner Arbeitsgruppe und der AG Eggert bin ich zutiefst dankbar für eine
wunderbare Arbeitsatmosphäre und für die legendären „Beer hours“, Karnevalsfeiern,
Retreats mit Schnitzeljagd und eine Geburtstagsparty in San Fran, die alle Zeugnis des team
spirit sind, den ich mit euch zusammen erleben durfte.
All meinen Freund:innen, insbesondere Cam Loan, Cori, Rike, Marleen, Anne und Sebastian,
danke ich für fruchtbare Diskussionen und ihre emotionale Unterstützung während der
Studien- und Doktorandinnenzeit! Ihr habt mich gestärkt, motiviert, ermutigt, getröstet, mit
Kaffee und gutem Essen versorgt, zum Lachen gebracht und abgelenkt, wenn es nötig war. Ihr
seid, jeder auf seine ganz eigene Weise, ein Vorbild für mich, und dafür danke ich euch von
Herzen!
Meiner Familie gilt mein größter Dank! Meinen Eltern Heidi und Volker danke ich von Herzen
für die vielen Jahre der Unterstützung und für das Vertrauen in mich und meine Fähigkeiten.
Ihr habt mir stets ermöglicht meinen eigenen Weg zu gehen, wofür ich euch wirklich dankbar
bin! Meiner Schwester Tini danke ich unbeschreiblich für ihre Wertschätzung, ihren
unerschütterlichen Glauben an mich, für das mitreißendste Lachen dieser Welt und das Bild
der Krake in meinem Kopf. Dank unserer Tochter Tilda, die meine Laborzeit im April 2019
spontan für beendet erklärt hat, gewinne ich eine neue Perspektive auf viele Dinge und bin
dankbar für jeden Augenblick, den sie mich so glücklich macht. Meinem Mann Henning danke
ich für die vielfältige Unterstützung und Motivation in all den Jahren. Ohne deine Hilfe wäre
diese Arbeit, ganz besonders in der Kita-losen Pandemiezeit, nicht möglich gewesen. Du (und
mittlerweile auch Tilda) zeigst mir jeden Tag mit deiner Liebe, was im Leben - neben der
Wissenschaft - wirklich zählt. Danke!
