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Coo dina ed down egula ion o Spinophilin and he ca aly ic
subuni s o PP1, PPP1CA/B/C, con ibu es o a wo se p ognosis
in lung cance
E a M. Ve dugo-Si ianes1,2, Lola Na as1,2, Sonia Molina-Pinelo1,2, I ene Fe e 2,3,
Al a o Quin anal-Villalonga3, Ja ie Peinado1,4, Jose M. Ga cia-He edia1,2,5, Blanca
Felipe-Ab io1,2, Sand a Muñoz-Gal an1,2, Juan J. Ma in1,2,6, Luis Mon uenga2,7, Luis
Paz-A es2,3 and Amancio Ca ne o1,2
1Ins i u o de Biomedicina de Se illa (IBIS), Hospi al Uni e si a io Vi gen del Rocío, Uni e sidad de Se illa, Consejo Supe io
de In es igaciones Cien í icas, Se illa, Spain
2CIBER de Cánce , Ins i u o de Salud Ca los III, Pabellón 11, Plan a 0, Mad id, Spain
3H120-CNIO Lung Cance Clinical Resea ch Uni , Ins i u o de In es igación Hospi al 12 de Oc ub e and CNIO, Mad id, Spain
4Radia ion Oncology Depa men , Hospi al Uni e si a io Vi gen del Rocío, Se illa, Spain
5Depa men o Vege al Biochemis y and Molecula Biology, Uni e si y o Se ille, Se ille, Spain
6Depa men o P edic i e Medicine and Public Heal h, Uni e sidad de Se illa, Se illa, Spain
7P og am in Solid Tumo s and Bioma ke s, Cen e o Applied Medical Resea ch (CIMA), Pamplona, Spain
Co espondence o: Amancio Ca ne o, email: [email p o ec ed]
Keywo ds: Spinophilin; PP1; bioma ke ; lung cance ; he apy
Recei ed: May 13, 2017 Accep ed: Sep embe 03, 2017 Published: Oc obe 26, 2017
Copy igh : Ve dugo-Si ianes e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License 3.0 (CC BY 3.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho
and sou ce a e c edi ed.
ABSTRACT
The sca old p o ein Spinophilin (Spinophilin, PPP1R9B) is one o he egula o y
subuni s o phospha ase-1 (PP1), di ec ing i o dis inc subcellula loca ions and
a ge s. The loss o Spinophilin educes PP1 a ge ing o pRb, he eby main aining
highe le els o phospho yla ed pRb. Spinophilin is absen o educed in app oxima ely
40% o human lung umo s, co ela ing wi h he malignan g ade. Howe e , li le is
known abou he ele ance o he coo dina ed ac i i y o p esence o Spinophilin and
i s epo ed ca aly ic pa ne s in he p ognosis o lung cance . In he p esen wo k,
we show ha he down egula ion o Spinophilin, ei he by p o ein o mRNA, is ela ed
o a wo se p ognosis in lung umo s. This e ec is mo e ele an in squamous cell
ca cinoma, SCC, han in adenoca cinoma. Down egula ion o Spinophilin is ela ed
o a dec ease in he le els o i s pa ne s PPP1CA/B/C, he ca aly ic subuni s o PP1.
A dec ease in hese subuni s is also ela ed o p ognosis in SCC and, in combina ion
wi h a dec ease in Spinophilin, a e ma ke s o a poo p ognosis in hese umo s.
The analysis o he genes ha co ela e o Spinophilin in lung umo s showed clea
en ichmen in ATP biosyn hesis and p o ein deg ada ion GO pa hways. The analysis
o he esponse o se e al common and pa hway- ela ed d ugs indica es a di ec
co ela ion be ween he Spinophilin/PPP1Cs a io and he esponse o oxalipla in and
bo ezomib. This inding indica es ha his a io may be a good p edic i e bioma ke
o he ac i i y o he d ugs in hese umo s wi h a poo p ognosis.
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Resea ch Pape
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INTRODUCTION
The Spinophilin (Spn, PPP1R9b) gene is loca ed
a 17q21.33, a cy ogene ic a ea equen ly associa ed
wi h mic osa elli e ins abili y and loss o he e ozygosi y
(LOH). LOH in ch omosome 17q21.3 has been obse ed
in di e en umo s, including b eas , o a ian, p os a e,
colo ec al, gas ic, enal and lung ca cinomas as well as in
sali a y gland ca cinosa coma, an ex emely agg essi e
neoplasm [1–6].
Low le els o Spinophilin exp ession ha e been
ound in lung adenoca cinoma [7], head and neck
cance [8], hepa ocellula ca cinoma [9], human gas ic,
small in es ine and colo ec al adenoca cinoma [10, 11],
glioblas oma [12] and b eas cance [13, 14]. In all cases,
down egula ion o Spinophilin co ela ed wi h a highe
malignan g ade, mo e agg essi e biological beha io and
esis ance o he apies, leading o as e elapse and poo e
pa ien su i al. Fu he mo e, he loss o Spinophilin also
co ela ed wi h p53 mu a ions.
Analysis o human b eas umo s showed ha
Spinophilin down egula ion inc eased he s emness
p ope ies and he exp ession o s em- ela ed genes
(Sox2, KLF4, Nanog and OCT4) [14]. B eas umo
s em cells appea ed o ha e low le els o Spinophilin
mRNA, and Spinophilin loss co ela ed wi h inc eased
s em-like cell appea ance in b eas umo s, as indica ed
by an inc ease in CD44+/CD24- cells. A educ ion o he
le els o PPP1CA mimicked he cance s em-like cell
pheno ype o Spinophilin down egula ion, sugges ing
ha he mechanism o Spinophilin in ol es PP1a [14].
Spinophilin is a sca olding p o ein in e ac ing wi h mo e
han 30 pa ne p o eins, including p o ein phospha ase 1
(PP1) and F-ac in [15–17]. Howe e , he physiological
ele ance o some o hese in e ac ions emains o be
de e mined. Spinophilin pe o ms impo an unc ions in
he ne ous sys em whe e i is implica ed in egula ing
spine mo phology and densi y, synap ic plas ici y and
neu onal mig a ion [15]. Spinophilin also egula es se en-
ansmemb ane ecep o s and may link hese ecep o s
o in acellula mi ogenic signaling e en s ha a e
dependen on p70S6 kinase and he small G p o ein-GEF
Rac. Spinophilin also in e ac s wi h doubleco in, an ac in-
binding p o ein wi h an es ablished ole in he subcellula
a ge ing o PP1. Spinophilin enhances PP1-media ed
dephospho yla ion o doubleco in [18] concomi an wi h
PP1 localiza ion in he cy osol [19, 20]. Thus, localiza ion
o he doubleco in–Spinophilin–PP1 complex in he
cy osol inhibi s PP1 phospha ase ac i i y, leading o
glioma cell dea h e ec s ia a mi o ic spindle ca as ophe.
A he unc ional le el, Spinophilin egula es
PP1 ac i i y, he eby main aining highe le els o
phospho yla ed pRb [21]. This e ec con ibu es o an
inc ease in p53 ac i i y h ough he inc ease in ARF
p o ein. Howe e , in he absence o p53, educed le els
o Spinophilin inc ease he umo igenic p ope ies o
cells. Spinophilin knockou mice ha e a educed li espan,
an inc eased numbe o umo s and inc eased cellula
p oli e a ion in some issues, such as he mamma y duc s.
In addi ion, he combined loss o Spinophilin and p53
ac i i y in mouse models leads o an inc ease in mamma y
ca cinomas, con i ming he unc ional ela ionship
be ween p53 and Spinophilin [22].
The da a sugges ha he egula ion o PP1 ac i i y
is he mechanism by which Spinophilin ac s o p oduce
i s umo supp esso ac i i y [23]. Howe e , li le is
known abou he ele ance o he ca aly ic subuni s o
PP1 in cance p ognosis and speci ically i s coo dina ed
egula ion wi h Spinophilin.
PP1 se ine/ h eonine phospha ases a e mul ime ic
enzymes assembled om a small numbe o ca aly ic
subuni s wi h one o hund eds o egula o y subuni s.
The egula o y subuni p o ides p ecision and speci ici y
o he a ge [24]. These phospha ase egula o s do no
sha e ex ensi e sequence conse a ion. Ins ead, hey a e
iden i ied by hei physical in e ac ion and unc ion [24].
The e a e 4 di e en ca aly ic PP1 iso o ms, PPP1CA/B/C,
de i ed om 3 di e en genes, plus an al e na e splicing
o PPP1CC [25–27]. Due o he b oad spec um o ac i i y
o each ca aly ic subuni , no much in o ma ion ega ding
hei ole in umo s o hei pa hological alue has been
published. In glioblas oma, PP1A p o ein exp ession
showed no co ela ion wi h p ognosis in all cases o
on s a i ica ion based on IDH1 o ATRX exp ession.
Howe e , nuclea PP1A exp ession is a s ong independen
p edic o o poo o e all su i al in p53-posi i e GBMs
only [28]. PPP1CA u ina y con en was also associa ed
wi h ecu ence in bladde umo s [29]. Finally, analysis
o human umo s sugges s ha one o he PPP1CA alleles
migh be los in a high pe cen age o kidney and colo ec al
ca cinomas [30].
Because he speci ici y and p ecision o each PPP1C
iso o m is gi en by he egula o y subuni and we de ec ed
down egula ed Spinophilin in a subg oup o lung umo s,
we in es iga ed he e ec o Spinophilin egula ion o he
PP1 ca aly ic subuni s in human lung umo s.
RESULTS
Loss o Spinophilin in lung umo s
Spinophilin down egula ion in human lung umo s
is a causal e en ha igge s an inc ease in umo igenic
p ope ies, con ibu ing o hei malignan s a us [7].
We ha e p e iously desc ibed [7, 10] ha he cu o o
down egula ed o lowe le els o SPINOPHILIN p o ein
is equal o below 50% o he p o ein le els ound in
non- umo al cells o he same issue. Down egula ion
o SPINOPHILIN can be obse ed by immunos aining
(Figu e 1A) comp ising a ound 30% samples. On he o he
hand, we quan i ied he le els o Spinophilin by mRNA
exp ession le els and de ec ed app oxima ely 30% o
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umo s wi h le els o Spinophilin lowe han a e age (=
0.0113) (Figu e 1B). This pe cen age b oadly co ela ed
wi h he pe cen age o samples showing down egula ion
o Spinophilin by immunohis ochemis y [7]. A mo e
in-dep h analysis using public ansc ip omic da abases
shows a simila pe cen age o umo samples wi h
down egula ed Spinophilin (Supplemen a y Figu e 1).
Fu he mo e, his down egula ion o Spinophilin is mo e
p onounced in squamous umo s han in adenoca cinoma
(Figu e 1C-1E).
A simila pe cen age o cases showed educed
Spinophilin le els independen o whe he he analysis
was pe o med on mRNA o p o ein, sugges ing ha
Spinophilin down egula ion occu s ei he by he loss o
he 17p21 locus o in mos cases h ough he egula ion
o mRNA le els. To u he explo e his esul , we
analyzed Spinophilin p omo e me hyla ion in ma ched
lung umo samples s. non- umo al samples om he
same pa ien [31] (Supplemen a y Table 2). We ound
ha his gene showed inc eased me hyla ion in umo al
samples s. non- umo samples (Table 1). The inc eased
me hyla ion, howe e , occu s ega dless o whe he
he samples a e om adenoca cinoma o squamous
ca cinoma.
Figu e 1: Loss o Spinophilin in human umo s. (A and B) Rep esen a i e pho o o di e en lung umo s wi h di e en Spinophilin
le els. (C) The le els o Spinophilin mRNA in a coho o 72 human lung umo s desc ibed in e e ence [7] we e analyzed acco ding o he
p ocedu e desc ibed in M&M. The mean alue o mRNA le els o Spinophilin in all samples calcula ed and umo s dis ibu ed acco ding o
his mean alue. High Spinophilin > mean alue; Low Spinophilin > mean alue. (D) Analysis o Spinophilin mRNA le els in samples om
he coho o [64]. (E) Analysis o Spinophilin mRNA le els in samples om he coho o [65]. (E) Analysis o Spinophilin mRNA le els in
samples om he coho o [66]. In all h ee cases (D, E and F) he di e ences o he Spinophilin mRNA le els be ween ADC and SCC we e
s a is ically signi ican (p<0.05) ADC: Lung Adenoca cinoma; LCLC: La ge Cell Lung Ca cinoma; SCC: Squamous Cell Lung Ca cinoma.
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Dec eased Spinophilin le els p edic ed poo
ou come in lung cance pa ien s
To e alua e whe he SPINOPHILIN le els
we e associa ed wi h clinical ou come, we co ela ed
SPINOPHILIN immunohis ochemical s aining wi h
pa ien disease- ee in e al (DFI) and o e all su i al
(OS). Dec eased SPINOPHILIN le els we e associa ed
wi h a poo e OS (p=0,022) and DFI (P=0.020) in pa ien s
wi h lung cance (Figu e 2A and 2B). Mul i a ia e analyses
con i med ECOG and s age as independen p edic i e
ac o s o PFS. Rega ding o e all su i al, only s age
Table 1: Me hyla ion o Spinophilin p omo e in lung umo s
Spinophilin me hyla ion NON- umo Tumo S uden ’s T- es P alue
Adenoca cinoma 0.44 (n=13) 0.52 (n=13) <0.0001
SCC 0.44 (n=10) 0.51 (n=10) 0.009
The da a show he a e age mean o me hyla ion (see Me hods) in he analyzed samples (n=num o samples analyzed).
Figu e 2: Su i al p obabili y o pa ien s wi h lung cance acco ding o Spinophilin le els. (A) O e all su i al (OS) and
(B) Disease-F ee In e al (DFI). (C) O e all su i al (OS) and (D) Disease-F ee In e al (DFI) in pa ien s wi h low Spinophilin acco ding
o p53 le els.
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e ained independen p ognos ic signi icance in he Cox
mul iple eg ession model (Supplemen a y Table 3). The
analysis o concu en molecula al e a ions showed a
co ela ion wi h nuclea p53 s aining [7]. The e o e, we
measu ed he p edic i e alue o SPINOPHILIN in human
lung umo s wi h nuclea accumula ion o p53 (Figu e 2C
and 2D). We ound ha low SPINOPHILIN co ela ed
wi h high le els o nuclea p53 (>10%), as an indica o
o mu an p53, and his pa e n (low SPINOPHILIN and
mu an p53) co ela ed wi h a wo se p ognosis in bo h OS
and DFI (Figu e 2C and 2D).
To u he assess his hypo hesis, we e alua ed
he associa ion o Spinophilin mRNA le els wi h umo
esponse o pa ien su i al in independen coho s o
publicly a ailable da abases (Supplemen a y Figu e
1). Low le els o Spinophilin mRNA in lung umo
issue samples o di e en da abases we e always
p edic i e o wo se su i al p obabili y, co ela ing
wi h he indings o immunohis ochemis y on he
SPINOPHILIN p o ein.
Consequen ly, low le els o Spinophilin mRNA
we e also associa ed wi h a sho e disease- ee in e al
(DFI) and OS in hese se ies o pa ien s wi h lung umo s,
highligh ing he ele ance o Spinophilin as a p edic i e
ac o (Figu e 2 and Supplemen a y Figu e 1).
Dec eased le els o PPP1Cs a e ela ed wi h high
isk and p edic ed poo ou come in lung SCC
cance pa ien s
SPINOPHILIN is a egula o y subuni o PP1 and
binds one o he ca aly ic subuni s, PPP1CA, B o C,
o ming a he e odime wi h PP1 phospha ase ac i i y.
The e o e, we measu ed whe he he le els o hese
ca aly ic subuni s a e ela ed o su i al p obabili y in
umo s o he lung. To his end, we selec ed he TCGA
da abases o adenoca cinoma o SCC speci ic umo s.
We obse ed ha o adenoca cinoma, he pa ien s wi h
low le els o PPP1CA o B ha e a signi ican ly highe
isk o dec eased su i al han pa ien s wi h high le els
o exp ession o hese genes (Figu e 3A). The opposi e
e ec is obse ed o PPP1CC. Howe e , he su i al
p obabili y o he pa ien s does no change signi ican ly
when all h ee PPPC ca aly ic subuni s a e conside ed
(Figu e 3B).
When we s udied SCC umo s, we obse ed a mo e
homogeneous beha io and he pa ien s wi h low le els
o PPP1CA, B o C ha e a signi ican ly highe isk han
pa ien s wi h high le els o hese genes (Figu e 3C).
Fu he mo e, pa ien s wi h low le els o hese genes ha e
a signi ican ly poo e su i al p obabili y (Figu e 3D).
Figu e 3: Highe isk and lowe su i al p obabili y o pa ien s wi h lung cance acco ding o lowe mRNA le els o he
ca aly ic subuni s o PP1. (A and C) Risk o wo se su i al p obabili y acco ding o mRNA le els in pa ien s wi h adenoca cinoma (le ,
A) o squamous cell ca cinoma ( igh , C). (B and D) Su i al p obabili y (log ank) o pa ien s wi h lung cance acco ding o he mRNA le els
o he join ca aly ic subuni s o PP1. Values we e aken abo e o below he a e age o each subuni e alua ed. High o low isks we e aken
acco ding o he alues o igu e (A) and (C). (B) Lung adenoca cinoma; (D) Squamous cell ca cinoma. The TCGA coho was used.

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As a egula o y subuni , we s udied he co ela ion
o Spinophilin mRNA exp ession wi h hose o he
di e en ca aly ic subuni s. We analyzed he le els o
PPP1Cs mRNA in samples om Supplemen a y Table
1. We de ec ed a di ec ela ionship be ween he le els o
Spinophilin and hose o each ca aly ic subuni (Figu e 4A,
Figu e 4: Su i al p obabili y o pa ien s wi h lung cance acco ding o he join mRNA le els o he indi idual
ca aly ic subuni s o PP1 and Spinophilin. (A) Co ela ion be ween he mRNA le els o Spinophilin and he ca aly ic subuni s o
PP1 (S uden ’s T- es ; ***=p<0.001). We analyzed he le els in a coho o 70 samples om ou coho om Supplemen a y Table 1, o
which we had mRNA (see Ma e ials and Me hods). We quan i a ed by Q-RT-PCR he le els o PPP1CA, PPP1CB and PPP1CC and plo ed
acco ding o he low o high le els o Spinophilin mRNA (g aph). The g aph shows he dis ibu ion o he PP1 ca aly ic subuni acco ding
o he ca ego iza ion o samples in high o low Spinophilin acco ding o i s a e age. Fu he mo e we plo ed one o one co ela ion o
PPP1CA/B/C mRNA le els o Sphinophilin mRNA le els in each sample. The index o co ela ion ( ) and s a is ical signi icance (p) o
he Pea son co ela ion is included in he inse in each g aph. (B) Su i al p obabili y (log ank) o pa ien s wi h lung cance acco ding o
he mRNA le els o he join indi idual ca aly ic subuni s o PP1 and Spinophilin. Values we e aken abo e o below he a e age o each
subuni e alua ed. High o low isks we e aken acco ding o he alues o Figu e 3A. The TCGA coho was used.
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box g aph was analyzed by S uden ’s T- es ; inse also
shows Pea son ). The e o e, we nex measu ed whe he
he combina ion o egula o y Spinophilin and ei he o he
ca aly ic subuni s migh ha e some p edic i e capabili y.
We ound ha in adenoca cinoma umo s, he combina ion
had no clea e ec on su i al p ognosis (Figu e 4B).
Howe e , he p ognosis capabili y o low Spinophilin
combined wi h low le els o PPP1CA/B o C is clea e in
pa ien s wi h SCC umo s o he lung (Figu e 4B).
Finally, we combined low Spinophilin and low
PPP1CA/B and C le els and analyzed he p edic i e
capabili y o su i al in pa ien s wi h adenoca cinoma
o SCC umo s. We obse ed a clea and signi ican poo
p ognosis in pa ien s wi h umo s wi h low le els o
combined Spinophilin/PPP1CA, B and C only in pa ien s
wi h SCC umo s (Figu e 5).
While he analysis o he me hyla ion o Spinophilin
showed inc eased me hyla ion, he analysis o PPPCs
subuni s me hyla ion showed a dec eased me hyla ion
mean, in umo s s. non- umo samples, in PPP1CA and
PPP1CB, and inc eased me hyla ion mean in PPP1CC
(See Supplemen a y Table 4). The e o e, he mechanism
o egula ion mus be di e en o all h ee iso o ms. I
may include ansc ip ional egula ion o cell adap a ion
h oughou he g ow h o he umo .
Analysis o he GO e ms co ela ing o
Spinophilin
Pa ien s wi h lung umo s wi h low Spinophilin
le els showed clea and signi ican poo p ognosis.
The e o e, new he apeu ic al e na i es o hese pa ien s
a e needed. To explo e his poin , we looked o genes
ha co ela ed posi i ely and nega i ely o Spinophilin
(PPP1R9B) in he TCGA da abase. We selec ed genes
wi h a co ela ion >0.350 o <-0.350, o iden i y genes
ha co ela e posi i ely o nega i ely o Spinophilin
le els in umo s (Supplemen a y Table 5). Nex , we
iden i ied he GO e ms ela ed o hese genes using he
En ich web po al (Supplemen a y Table 6A and 6B).
The GO e ms ha co ela ed posi i ely o Spinophilin
we e en iched in se e al biological p ocesses, such as
ch oma in modi ica ion, ATP biosyn he ic p ocesses,
emb yo de elopmen and he egula ion o GTPase
ac i i y (Figu e 6). Al e na i ely, GO e ms ha co ela ed
nega i ely o Spinophilin, and he e o e may be en iched
in umo s wi h low Spinophilin, we e he egula ion o
p o ein deg ada ion, ATP biosyn he ic p ocesses and he
egula ion o he cell cycle (Figu e 6).
These GO e m en ichmen assays sugges ha
se e al pa hways in e e e wi h he aim o inding
al e na i e he apies. P ocesses such as he egula ion o
p o ein deg ada ion, ATP biosyn he ic p ocesses and he
egula ion o he cell cycle being nega i ely co ela ed
seemed mo e sui able because hey a e al e ed in he
absence o Spinophilin. The e o e, we es ed he e ec o
me o min, a egula o o he ATP biosyn he ic p ocess,
and bo ezomib, an inhibi o o p o ein deg ada ion, in
a panel o lung cance cell lines and hei co ela ion o
Spinophilin.
Analysis o he co ela ion be ween Spinophilin
and PPP1Cs in a panel o lung cance cell lines
and hei ela ionship wi h he d ug esponse
Fi s , we analyzed he le els o exp ession o
Spinophilin in a panel o 17 lung cance cell lines
(Supplemen a y Table 7), and we obse ed a di e en
pa e n o exp ession (Figu e 7A). The e o e, we classi ied
he panel as high Spinophilin cell lines (H1437, H1781,
Figu e 5: Su i al p obabili y o pa ien s wi h lung cance acco ding o he join mRNA le els o he ca aly ic subuni s
o PP1 and Spinophilin. Su i al p obabili y (log ank Cox) o pa ien s wi h lung cance acco ding o he mRNA le els o he join
ca aly ic subuni s o PP1 and Spinophilin. Values we e aken abo e o below he a e age o each subuni e alua ed. High o low isks we e
assessed acco ding o he alues o Figu e 3A. The TCGA coho was used.
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H2009, H358, Calu3 and Nuli1) and low Spinophilin cell
lines (H1650, H1975, H2228, H226, H3122, H460, H520,
HCC827, Calu1, A549 and NL20).
Then, we s udied he co ela ion o Spinophilin
exp ession wi h hose o he di e en ca aly ic subuni s
in he panel o 17 di e en lung cance cell lines
(Supplemen a y Table 8). As in he case o umo s,
we de ec ed a di ec ela ionship be ween he le els
o Spinophilin and he le els o each ca aly ic subuni
(Figu e 7B, inse shows also Pea son ; Supplemen a y
Table 8). As in umo s, cell lines wi h low Spinophilin also
con ained low le els o each ca aly ic subuni .
Wi h he aim o inding a d ug ha may be ac i e in
lung cell lines wi h low Spinophilin le els, we subjec ed
his panel o 17 cell lines o di e en ea men s o ob ain
he IC50 o he esponse in each cell line (Supplemen a y
Table 9). We speci ically es ed cispla in and e oposide as
common ea men s o lung umo s as well as oxalipla in
as a pla inum-de i ed compound wi h a di e en
spec um o ac i i y. Finally, we also es ed me o min
as a modula o o ATP biosyn hesis and bo ezomib as an
inhibi o o p o easomal deg ada ion [32, 33] because hese
wo mechanisms seemed o be highly ela ed o genes ha
co ela ed o Spinophilin le els. Then, we co ela ed he
IC50 o each d ug wi h he le els o Spinophilin, PPP1Cs
o he a io among hem and calcula ed he co ela ion
index (Pea son ). We ound ha none o he ac i i ies
o hese d ugs co ela ed wi h he le els o exp ession o
any o he es ed genes indi idually (Table 2). Howe e ,
we ound a clea co ela ion be ween he a io be ween
Spinophilin and PPP1CA o PPP1CB and he ac i i y o
oxalipla in o bo ezomib (Table 2, Supplemen a y Figu e
2). Al e na i ely, we did no obse e a co ela ion be ween
me o min ac i i y and Spinophilin o PP1 alues.
These da a sugges ha he lowe a io be ween he
le els o exp ession o hese genes may be a good ma ke
o he esponse o oxalipla in o bo ezomib. The e o e,
umo s wi h lowe le els o Spinophilin migh espond
be e o oxalipla in and/o bo ezomib depending on he
le els o PPP1CA/B. Howe e , his unc ional hypo hesis
needs mo e esea ch and o be alida ed in animal models.
DISCUSSION
Down egula ion o Spinophilin, ei he in p o ein o
mRNA, is ela ed o wo se p ognosis in lung umo s. This
e ec is mo e ele an in squamous cell ca cinoma han in
adenoca cinoma. Down egula ion o Spinophilin is ela ed
o a dec ease in he le els o PPP1CA/B/C, he ca aly ic
subuni s o PP1 and pa ne s o Spinophilin. A dec ease
in hese subuni s is also ela ed o a poo p ognosis in
SCC and is obse ed mo e clea ly in combina ion wi h
Figu e 6: GO e m en ichmen by genes ha co ela e posi i ely o nega i ely o Spinophilin le els. The analysis by he
En ich po al was pe o med on he genes om Supplemen a y Table 5. The genes we e ob ained by R2 analysis in da a om he TCGA
da abase.
Onco a ge 105204
www.impac jou nals.com/onco a ge
a dec ease in Spinophilin. PP1 has been iden i ied as he
majo enzyme ha dephospho yla es pRb du ing mi osis
[34, 35] and plays an impo an ole in he G1/S ansi ion
[36]. Al hough he e is li e a u e suppo ing he ole o
PP1 egula ion o pRb in i o [37–39], ou wo k is he
i s o suppo he down egula ion o he componen s o
he PP1 he e odime as a di ec con ibu ion o lung cance
and as a p edic o o a wo se p ognosis o hese pa ien s.
Figu e 7: Co ela ion be ween he mRNA le els o Spinophilin and he ca aly ic subuni s o PP1 in a panel o umo
cell lines. We analyzed a coho o 17 cell lines desc ibed in Supplemen a y Table 7. (A) We analyzed he exp ession le els o Spinophilin
in he coho o 17 cell lines desc ibed in Supplemen a y Table 3. We di ided he panel in o high and low Spinophilin, conside ing high
Spinophilin hose cell lines wi h exp ession > 0.01: H1437, H1781, H2009, H358, Calu3 and Nuli1 cell lines; and low Spinophilin he ones
wi h exp ession < 0.01: H1650, H1975, H2228, H226, H3122, H460, H520, HCC827, Calu1, A549 and NL20 cell lines. (B) We de ec ed
a di ec ela ionship be ween he le els o Spinophilin and he le els o each ca aly ic subuni . The g aph shows he dis ibu ion o he PP1
ca aly ic subuni acco ding o he ca ego iza ion o samples wi h high o low Spinophilin acco ding o i s a e age. We conside ed high
Spinophilin: H1431, H1781, H2009, H358, Calu3 and Nuli1 cell lines, and low Spinophilin: H1650, H1975, H2228, H226, H3122, H460,
H520, H827, Calu1, A549 and NL20 cell lines. Fu he mo e, he inse shows Pea son’s and i s s a is ical signi icance.
Table 2: Pea son co ela ion ( ) be ween he IC50 o he di e en ea men s and he le els o he indica ed genes
Spinophilin
PPP1CA PPP1CB PPP1CC Spinophilin/
PPP1CA*
Spinophilin/
PPP1CB*
Spinophilin/
PPP1CC*
me o min -0.2567736 -0.0305505 0.0471795 0.0072612 -0.1737827 -0.2997706 -0.3934922
oxalipla in 0.1098795 -0.0758366 -0.2116024 0.2138328 0.5907283 0.6886702 0.0679746
cispla in 0.0367228
0.06338384
0.0771211 -0.1303417 -0.1979426 -0.1891476 -0.0018704
e oposide 0.0134608 -0.0350167 -0.0008127 -0.0196913 -0.0707570 -0.0349190 -0.0275979
bo ezomib -0.0652384 -0.3027230 -0.0553075 0.2275598 0.7996544 0.4329986 0.0972166
* indica es he a io be ween he le els o Spinophilin and he ca aly ic subuni indica ed.