SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer.

SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer Imanol Zubiete-Franco a,1 , Juan L. García-Rodríguez a,1 , Fernando Lopitz-Otsoa a,1 , Marina Serrano-Macia a , Jorge Simon a , Pablo Fernández-Tussy a , Lucía Barbier-Torres a , David Fernández-Ramos a , Virginia Gutiérrez-de-Juan a , Sergio López de Davalillo a ,OnintzaCarlevaris b , Adolfo Beguiristain Gómez c , Erica Villa d , Diego Calvisi e , César Martín f ,EdurneBerra b , Patricia Aspichueta g,k , Naiara Beraza a , Marta Varela-Rey a ,MatiasÁvila h , Manuel S. Rodríguez i , José M. Mato a , Irene Díaz-Moreno j , Antonio Díaz-Quintana j ,TeresaC.Delgado a, ⁎, María L. Martínez-Chantar a, ⁎ a Liver Disease and Liver Metabolism Lab, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160 Derio, Bizkaia, Spain b Physiopathology of the Hypoxia-Signalling Pathway Lab, CIC bioGUNE, 48160 Derio, Bizkaia, Spain c University Hospital of Donostia, 20014 Donostia, Gipuzkoa, Spain d Department of Gastroenterology, Azienda Ospedaliero-Universitaria & University of Modena and Reggio Emilia, 41124 Modena, Italy e Institute of Pathology, University Klinic of Regensburg, 93053 Regensburg, Germany f Instituto Biofisika (CSIC, UPV/EHU) and Departamento de Bioquímica y Biología Molecular, UPV/EHU, 48940 Leioa, Spain g Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain h Hepatology Department, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, 31008 Pamplona, Spain i UbiCARE, Advanced Technology Institute in Life Sciences (ITAV)-CNRS-IPBS, 31106 Toulouse, France j Instituto de Investigaciones Químicas (IIQ) –Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla –Consejo Superior de Investigaciones Científicas (CSIC), 41092 Sevilla, Spain k Biocruces Health Research Institute, 48093 Barakaldo, Bizkaia, Spain abstractarticle info Article history: Received 13 June 2018 Received in revised form 13 December 2018 Accepted 14 December 2018 Available online 26 December 2018 Background: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC). Methods: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involvedin regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity,wasperformed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC. Findings: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using sitedirected mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmicshuttlingandfuelinghepatomacellgrowth.Molecular modellingofSUMO modifiedLKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors. Interpretation: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. Fund: This work was supported by grants from NIH (US Department of Health and Human services)- R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.- C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017–87301-R and SAF2014–52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013–2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015–71017/BMC Keywords: LKB1 SUMO HCC SIRT1 STRADα EBioMedicine 40 (2019) 406–421 ⁎Corresponding authors at: CIC bioGUNE, Ed. 801A Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain. E-mail addresses: [email protected] (T.C. Delgado), [email protected] (M.L. Martínez-Chantar). 1 Joint first authors. https://doi.org/10.1016/j.ebiom.2018.12.031 2352-3964/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientificay Técnica y Innovacion 2013–2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007–2009 and 2011–2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971–16 (P.A.), MINECO:SAF2015–64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16–251 (M.S.R.), MINECO - BFU2016–76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sourceshad no involvement instudy design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Liver Kinase B1 (LKB1) is a 50 kDa serine/threonine kinase ubiquitously expressed in adult and fetal tissues, particularly in pancreas, liver, testes and skeletal muscle [1]. LKB1 is an upstream activator of 14 kinases from the ARK (AMP-activated protein kinase-related kinase) family including AMP-activated protein kinase (AMPK). In fact, the defective activation of AMPK in LKB1-null cells can be rescued by reexpression of LKB1 [2,3]. AMPK functions as a cellular energy sensor to provide metabolic adaptations under ATP-deprived conditions such as starvation and oxygen deprivation (hypoxia) [4]. Under these circumstances, AMPK activation results in stimulation of bioenergetic pathways and inhibition of ATPand NADPH-consuming processes such as biosynthesis and proliferation. It is particularly relevant in this respect that AMPK inhibits the mammalian target of rapamycin complex 1 (mTORC1) pathway [5] with concomitant downregulation of the glycolytic pathway [6,7]. Indeed, silencing LKB1 in mouse embryonic fibroblasts promotes a metabolic switch to aerobic glycolysis, also called the Warburg effect, a hallmark of highly proliferative tumor cells, supported by mTOR activity and driven by hypoxia inducible factor (HIF)-1α[8]. Moreover, AMPK can regulate energy expenditure by modulating mitochondrial biogenesis and controlling the expression of oxidative enzymes [9–11]. Alternatively, LKB1-induced activation of AMPK signaling can offer a protective effect by allowing the cell time to attempt to reverse the aberrantly high ratio of AMP/ATP, that otherwise can cause cell death [12]. Germlinemutations or deletions in the LKB1 geneare responsible for thePeutz-Jeghers Syndrome (PTS),a cancer-proneautosomal dominant inherited disorder [1,13]. Likewise, somatic mutations of LKB1 gene are involved in the development of sporadic cancers, such as cervical, prostate and lung cancers, amongothers [14–16]. Although genetic evidence supports the tumor-suppressive role for LKB1, other evidence revealed that LKB1 may also exhibit pro-oncogenic functions. In the context of liver disease, a controlled balance in hepatic LKB1 levels has been described as a gatekeeper of hepatocyte proliferation during regeneration [17,18]. Furthermore, LKB1 expression or activity have been previously shown to be augmented in Hepatocellular Carcinoma (HCC), the most common type of liver cancer, especially related to bad prognosis and late stage HCC [19,20]. The mechanisms underlying the oncogenic role of LKB1 in HCC remain rather unexplored. LKB1 localization within the cell is critical for theregulation of its activity. LKB1 has a N-terminal regulatory domain in the most N-terminal regionafter the kinase domain and a C-terminal regulatory domain [21]. LKB1 has also two specific recognition sequences called nuclear localization sequences (NLS) in its N-terminal region [22], that allow the shuttling between cytoplasm and nucleus of LKB1 in mammalian cells. LKB1 nucleocytoplasmic shuttling is mediated by cofactors, such as the STe20-Related ADaptor (STRADα) and mouse protein 25 (MO25) [2]. Briefly, STRADαinduces relocalization of LKB1 from the nucleus to the cytoplasm whereas MO25 stabilizes the STRADα-LKB1 interaction [23,24]. On the other hand, STRADαinhibits nuclear import of LKB1 by competing with karyopherin importin-αfor binding to LKB1 [25]. To date, several regulating mechanisms have been proposed to explain LKB1 cellular localization: i) aberrant expression of STRADαwas associated with nuclear LKB1 during corticogenesis [26]; ii) the orphan nuclear receptor Nur77 can bind and sequester LKB1 in the nucleus [27]; and finally iii) reversible post-translational modifications of LKB1 have been shown to regulate its stability and activity [19,28,29]. Post-translational modification is a criticaleventin thedynamicregulation of protein stability, location, structure, function, activity and Research in context Evidence before this study LKB1 is a serine threonine kinase protein with an ambiguous role in cancer progression. LKB1 expression or activity is augmented in bad prognosis and late stage liver cancer. LKB1 cellular localization is relevant for its activity. LKB1 has been shown to be a target of SUMO post-translational modifications. SUMO-mediated modifications are known to play an important role in protein target subcellular localization during cancer progression. Added value of this study In this study, we show that LKB1 overexpression in human hepatoma cells during hypoxic stress promotes tumor cell growth and survival. Moreover, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC as well as in liver biopsies of HCC patients. Finally, our data suggest that LKB1 SUMOylation is aberrant in liver cancer being essential for the regulation of its subcellular localization and oncogenic role. Implications of all the available evidence Our results support a potential role for LKB1 SUMOylation as a novel oncogenic mechanism in HCC and thereby the putative therapeutic potential of protein-based, peptidyl and small molecule inhibitors of various SUMO specific proteases isoforms. 407I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 interaction with other proteins. Recently, Ritho and colleagues have described for the first time LKB1 Small Ubiquitin-related MOdifier (SUMO)-mediated modifications and its implication under metabolic stress circumstances where SUMO-mediated modification of LKB1 is essential in promoting its interaction with its downstream target AMPK via a SUMO-interacting motif (SIM) essential for AMPK activation [29]. In addition, the SUMO protein modification has an extensive and critical role in the adaptive cellular response to hypoxia [30–34]. Chronic hypoxia is an important micro-environmental factor for establishing theaggressiveness of HCC by promoting tumor invasion and metastasis [35,36]. Thus, SUMOylation appears to be upregulated in many types of cancer, including HCC [37–39]. Previously, expression of both the SUMO E2 conjugating enzyme (Ubc9) and the E3 SUMO-protein ligase CBX4 were found to be upregulated and related to poor prognosis in HCC [38,39]. In this study, we aimed to explore the functional role of LKB1 in hepatocarcinogenesis and progression with special focus on the role playedby SUMOylated LKB1in themodulation of its cellular localization in hepatoma cells during hypoxic stress and in liver cancer. 2. Materials and methods 2.1. Cell lines Huh-7, Hep G2, and PLC/PRF/5, human hepatoma cell lines; and MLP-29, mouse liver progenitor cells were used. All cells were grown at 37 °C in a humidified atmosphere of 5% CO 2 –95% air and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Spain), unless otherwise stated. 2.2. Cell transfection Cell lines were transiently transfected using Lipofectamine 2000 (Invitrogen, USA), according to the manufacturer's instructions. Briefly, Lipofectamine 2000 (2.5 μl/1 μg DNA) was diluted in OPTI-MEM (Gibco) medium for transfections and incubated for 5 min. After incubation, the mixture was added to OPTI-MEM containing plasmid DNA (1 to 6 μgdepending on the experiment), and incubated for at least 20 min at room temperature (RT) to allow for the formation of the DNA-Lipofectamine complexes. DNA-lipofectamine complexes previously formed were added to the culture plates containing the corresponding culture medium with 10% FBS but without antibiotics and with the cells in suspension. Plasmid DNA used are listed in Supplemental Table 1. Putative SUMO sequence binding sites on LKB1 were analyzed using the SUMOplot Analysis Program (http://www.abgent.com/SUMOplot, Abgent, USA). The 4 highest ranking sites predicted by SUMOplot were used to create the mutants (lysines 96, 97, 178 and 235). Lysines were mutated to arginines (R). An additional acetylation LKB1 mutant (K48R) was also created. The mutant LKB1 plasmid constructs were created using the QuickChange kit for directed mutagenesis (Stratagene, USA), according to the manufacturer's instructions, with two complementary oligonucleotides and with the pcDNA3-FLAG LKB1 plasmid as a template. The products were sequenced (STABvida, Portugal). 2.3. Cell treatments The selective inhibitor of sirtuin 1 (SIRT1), Ex-527 (Sigma-Aldrich) in DMSO, was given to cells for 24 h at 30 μM. Cycloheximide, a eukaryote protein synthesis inhibitor was added to cells at 50 μg/ml in H 2 O. 2.4. Hypoxia in vitro For hypoxia experiments, cells were incubated in an Invivo 2 400 hypoxia Workstation (Baker Ruskinn, USA) at 1% O 2 for until 72 h and lysed/fixed in the hypoxia chamber. 2.5. Serum deprivation in vitro Cells were grown without FBSfor a 24-hour period and compared to cells maintained on 10%FBS. 2.6. Mouse models All animal experiments were performed according to the ARRIVE guidelines and carried out in accordance with the National Institutes of Health guide for thecare and use of Laboratory animals (NIH Publications N0.8023, revised 1978) and the guidelines of European Research Council for animal care and use. Adult male C57BL/6 mice and mouse models that develop HCC at 8-months old, such as the glycine Nmethyltransferase (Gnmt) deficient (Gnmt −/− )and wild type (Gnmt +/+ )mice, were bred and housed in the animal unit of CIC bioGUNE, which is an AAALAC-accredited facility. Animals were housed under controlled temperature (22 °C) and humidity conditions in a 12 h light/dark cycle with ad libitum access to food and water. 2.7. In vivo hypoxia treatment Three-month-old C57BL/6 mice males were exposed to systemic hypoxia in a sealed workstation (Baker-Ruskinn InvivO 2 400, Cultek). The final 10% O 2 environment was reached after a 2 h30 period in which oxygen levels were gradually reduced. Feeding and light cycles were kept uniform in the hypoxia workstation. Control mice were also exposed to the same chamber but under a 21% O 2 environment. Mice were euthanized by cervical dislocation inside the chamber, and the dissected organs were directly fixed. 2.8. Human HCC samples The work described has been carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans after obtaining informed consent. Surgically resected liver specimens of 22 patients with HCC (10 Hepatitis C, 10 ASH and 2 NASH) were examined. The Basque Biobank (http://www.biobancovasco.org) provided the data and type of biospecimen. In addition, we have used frozen paired HCC tumor and surrounding tissue liver biopsies obtained during tumor resection (n = 6) as well as HCC liver biopsies from a set of samples previously classified as fast growing (n = 3) or slow growing (n = 3). The latter samples were included in a previously published larger study, which included two cohorts of patients with cirrhosis of any etiology on ultrasound surveillance (training set = 78 and validation set = 54). At first identification of HCC, they underwent two CT scans 6 weeks apart with no treatment in-between. Fast growing patients has a median tumor doubling time of 42 days while for the slow growing patient's median tumor doubling time was 97 days [40]. US-guided liver biopsies were initially obtained and used to generate a microarray (Agilent Whole Human Genome Oligo) according to the MIAME guidelines. Gene expression data is available at the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo) under the accession number: GSE54236. 2.9. Protein extraction and western blotting analysis Protein extraction and Western blotting analysis was performed as previously described [41]. Primary antibodies and their optimal incubation conditions are detailed in Supplemental Table 2. 2.10. Crystal violet viability assay Cell viability was assessed using crystal violet staining (SigmaAldrich). 408 I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 2.11. Scratching wound-healing assay Cells were seeded to confluence over 12 mm coverslip after an overnight transfection. A pipette tip (200 μl) was used to scratch a straight line through all the wells. Media was changed twice to remove dead and unattached cells. The wells were then placed in hypoxia during 72 h. Pictures of the scratch were taken using an Eclipse TS100 microscope (Nikon, Japan). 2.12. Subcellular proteome extraction kit ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) from Merck (Spain) was used. 2.13. Immunocytofluorescence Cells seeded on 12 mm coverslips were fixed in PBS 4% paraformaldehyde (Santa Cruz Biotechnology, USA). Coverslips were then blocked and permeabilized with PBS containing 0.1% BSA, 10% goat serum and 0·2% Triton X-100 for 30 min at RT. After blocking, the coverslips were washed in PBS and incubated overnight in a humid chamber with the primary antibody (FLAG 1:100) in PBS. Coverslips were washed in PBS and incubated during 1 h at RT in blocking solution with DAPI but no Triton X-100 with secondary antibody (dilution 1:200, Cy3 conjugated anti-mouse or FITC-conjugated anti-rabbit, Jackson ImmunoResearch laboratories, USA). Coverslips were mounted in Dako fluorescence mounting medium (Dako, Denmark). Five Images from each experimental condition were taken using an Axioimager D1 (Zeiss). Blind quantification of LKB1 positive nuclear cells versus total number of stained cells was performed manually by an immunohistochemistry technician. 2.14. Nickel-Histidine affinity purification using nickel-nitriolotriacetic acid (Ni 2+ -NTA) beads Cells were transfected with the respective constructs and His 6 - SUMO as described. After treatments, His 6 -SUMOylated proteins were purifiedaspreviouslydescribedby usinglow density Ni 2+ -NTA-agarose beads (ABT, Spain) [19,42]. 2.15. Protein immunoprecipitation assays Total protein extracts were immunoprecipitated with 5 μg of IgG1 (BD Pharmigen), anti-LKB1 or anti-STRADαantibodies by using A/G PLUS-Agarose Beads (Santa Cruz). 2.16. SUMO binding entities (SUBEs) SUBEs, earlier described and validated [43], were used as a capturing system for endogenous SUMOylated LKB1. Liver tissue (75 mg) from Gnmt −/− and Gnmt +/+ as well as fromHCC patients were used. Briefly, livers were lysed in lysis buffer [50 mM Tris pH 8.5; 150 mMNaCl, 5 mM EDTA, 1% Igepal, supplemented with 1× protease inhibitor cocktail (Roche) and 50 μM of PR-619 (ubiquitin and ubiquitin-like isopeptidases inhibitor, LifeSensors)]. Lysates were centrifuged at 14000 ×gand the supernatant was incubated with 50 μlofGSTagarose beads containing 50 μg of SUBEs or GST and 1 mM DTT (Dithiothreitol) for 2 h, at 4 °C. Beads were then pulled down by centrifugation, 1000 ×gfor 5 min, and 1/10 of theunbound fractionwas saved for western blot analysis (flow through-FT). Washes were carried out using 30 column volumes of wash buffer (50 mM Tris pH 8.5; 50 mM NaCl, 5 mMEDTA and 1%Igepal). Elutionswereperformed in one column volume of 2 Laemmli Buffer. For Western blot analysis, samples were separated in NUPAGE 4–12% BT Gels, 1.5 mm, 15Well. 2.17. Modelling Computational analysis of the various LKB1 species used the X-ray diffraction coordinates of the LKB1-STRADα-MO25αcomplex by Zequiraj et al. (2009; pdb 2WTK). Highly mobile regions of LKB1 were absent and, therefore, needed modelling. For this purpose, 250 structures were generated using Modeller 9v7 [44] and classified according to their Discrete Optimized Protein Energy (zDOPE) scores to select the model with its lowest value. Molecular Dynamics trajectories were computed with the AMBER 16 package [45], using the 14SB force field [46]. For the acetylated lysine residue, Papamokos' parameterizations [47] were used. Simulations run under periodic boundary conditions in orthorhombic boxes. Initially, the minimum distance between protein and cell faces was 10 Å. PME electrostatics were set with the Ewald summation cut-off at 9 Å. Sodium counter-ions neutralized the charges of the system. The structures were solvated with SPC water molecules [48]. Protein side-chains were energy-minimized (100 steepest descent and 1400 conjugate gradient steps)down to a RMS energy gradient of 0·01 kJ mol-1 Å-1. Afterwards, solvent wassubjected to 1000 steps of steepest descent minimization followed by 500 ps NPTMD computations using isotropic molecule position scaling and a pressure relaxation time of 2 ps at 298 K. Temperature was regulated with Berendsen's heat bath algorithm [49], with a coupling time constant equal to 0·5 ps. The density of the system reached a plateau after ca. 150 ps simulation. Then, for each protein, the whole system was energy minimized and submitted to NVT-MD at 298 K, using 2·0 fs integration time steps. Snapshots were saved every 100 ps. SHAKE algorithm (Ryckaert et al., 1977) was used to constrain bonds involving hydrogen atoms. Coordinate files were processed using CPPTRAJ [50]. Further processing was made in Origin 16 (Originlab) and graphic displays were built in UCSF Chimera [51]. 2.18. Immunohistochemistry Paraffin-embedded sections (5 μmthick)offormalin-fixed liver samples were initially deparafinized in xylene or xylene-substitute and rehydrated through graded alcohol solutions. Specific antibodies and experimental conditions used in immunohistochemistry can be found in supplemental Table 3. For the analysis, images were taken with an upright light microscope (Zeiss, Germany). The average sum of intensities and stainedarea percentage of each sample was calculated using FRIDA software (http://bui3.win.ad.jhu.edu/frida/,JohnHopkins University). 2.19. Statistical analysis Data is expressed as mean ± SEM (standard error of the mean). Statistical significance was estimated using the Mann-Whitney Utest. A p value of b0·05 was considered significant. 3. Results 3.1. LKB1 offers survival and invasiveness advantage to human hepatoma cells during hypoxic stress LKB1 is endogenously expressed in human hepatoma cells (Suppl. Fig. 1a). These results are in agreement with earlier evidence showing augmented expression of LKB1 in rodent hepatoma cells [52–54]. In order to further explore the functional role of LKB1 in HCC progression, we transiently overexpressed LKB1 in Huh-7 human hepatoma cells both after serum deprivation (0% FBS) or under hypoxic (1% O 2 ) conditions in comparison with cells under normoxia (21% O 2 ) and cultured with 10% FBS. LKB1 ectopic transient overexpression in Huh-7 hepatoma cells results in comparable levels of LKB1 after 24 h of hypoxic, serum deprivation or normoxic stimuli (Fig. 1a, Suppl. Fig. 1b). 409I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 Fig. 1. Liver Kinase B1(LKB1) offers survival and invasiveness advantage to humanhepatoma cells duringhypoxic stress. LKB1 overexpression was induced in human hepatoma Huh-7 cell line by using the pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1) and compared to control overnight transfection with the pcDNA™3.3-TOPO® plasmid (Ctrl), followed by 24 h treatment under control conditions of normoxia and complete media (21%oxygen, 10% serum), serum deprivation (SD) and hypoxia (1% oxygen, 10% serum). a.RepresentativeWestern blot ofLKB1, its downstream target AMP-activated protein (AMPK) and phosphorylated AMPK at Thr172 and hypoxia inducible factor (HIF1α), a hypoxic marker, are shown. [β-actin was used as loading control]. Quantifications are shown in Suppl. Fig. 1b; b. Cell viability as detected by staining of attached cells with crystal violet dye; c. Time-course of cell viability and cell migration using a wound-healing scratch assay after LKB1 overexpression under hypoxia; d. Representative immunofluorescence staining for LKB1 (FLAG) in Huh-7 hepatoma cells and quantification of the percentage of LKB1 nuclear positive staining cells. Scale bar corresponds to 50 μm; and e. Western blot of LKB1 levels in cytoplasmic and nuclear fractions [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control for cytoplasmic fractions and Histone H3 for nuclear fractions]. Quantifications are shown in Suppl. Fig. 1b. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p b0·05 and **p b0·01 are indicated (Mann-Whitney Utest). 410 I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 Fig. 2. Liver KinaseB1 (LKB1) isSUMOylated by SUMO-2in human hepatoma cells. a. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO1, 2, or 3, with the pcDNA3-FLAG-LKB1 Wild type plasmid (LKB1 WT) in the presence and absence of ubiquitin conjugating enzyme 9 (UBC9).b. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 with the LKB1 WT plasmid in the presence of the different SUMO E3 ligases PIAS 1, 2α,2β,and4.c.Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and with the LKB1 WT plasmid in the presence of the different SUMO-specific proteases, SENPs 1–7. d. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and with the LKB1 WT plasmid or the pcDNA3-FLAG-LKB1 Kinase Dead K78I plasmid. e. Immunoprecipitation assay between LKB1 and the STe20-Related ADaptor (STRADα) cofactor after STRADαoverexpression in Huh-7 human hepatoma cells. Normalized quantifications relative to inputs are shown below the panel. 411I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 LKB1 is an upstream activator of AMPK promoting the phosphorylation of AMPK Thr-172 in the activation loop of its αsubunit in response to metabolic stress in order to inhibit biosynthesis and proliferation [2,3]. Herein, LKB1 overexpression increased phosphorylation of AMPK at Thr-172 except during hypoxic stress, a condition characterized by high HIF-1αlevels (Fig. 1b, Suppl. Fig. 1b). Interestingly, after 24 h of hypoxia, LKB1 upregulation was associated with increased cell viability in comparison with control and serum deprived cells, both in Huh-7 (Fig. 1b) and another cell line of mouse liver progenitor cells, the MLP-29 cells (Suppl. Fig. 2a). A time course indicates that whereas hepatoma cells growth is hampered during hypoxia, LKB1 overexpression is able to induce cell growth under these conditions (Fig. 1c). Furthermore, hypoxia is known to unleash the invasive potential of tumor cells. Scratch wound-healing assay revealed that LKB1 overexpression also provides and invasiveness advantage to tumor cells under hypoxia (Fig. 1c). LKB1 cellular localization plays an important role its activity. During serum deprivation and normoxia conditions LKB1 actively shuttles between the nucleus and cytoplasm whereas under hypoxia, the LKB1 nucleocytoplasmic shuttling is hampered and LKB1 is more present in the nucleus (Fig. 1d, e, Suppl. Fig. 1c). Overall, LKB1 overexpression provides growth survival and invasiveness advantage to hepatoma cells during hypoxic stress which agrees with previous evidence from our laboratory and others showing that LKB1 expression is induced in HCC tumors [19,20], tumors characterized by a highly hypoxic environment. 3.2. Increased LKB1 SUMOylation in human hepatoma cells As previously mentioned, SUMOylation post-translational modifications are critical during hypoxia and thereby relevant in HCC [38,39]. In mammals, there are five SUMO paralogues, being SUMO-1, -2 and -3 Fig. 3. Liver Kinase B1 (LKB1) is SUMOylated by SUMO-2 at Lys178 in human hepatoma cells. a. Schematic representation of LKB1 showing the Nuclear (NLS) localization and the Kinase domain (KDN). SUMOylation LKB1 mutants used are also shown and described. b. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with His-SUMO-2 and the LKB1 SUMO mutants, LKB1 K96R, LKB1 K97R, LKB1 K178R and LKB1 K235R. Normalized quantifications relative to inputs are shown below each panel. 412 I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 more studied, compared to SUMO-4 and -5 [55–57]. Hepatic SUMO-1 protein levels are low, on the contrary to other tissues such as lung, uterus and prostate, whereas SUMO-2/3 levels in the liver are high [58]. On this regard, we have found that mice exposed to hypoxia show increased liver LKB1 nuclear expression and SUMO-2/3 levels (Suppl. Fig. 3a). Furthermore, we show that endogenous LKB1 SUMOylation by SUMO-2/3 is induced in Huh-7 hepatoma cells after 24 h of hypoxia in comparison with cells grown under normoxic conditions (Suppl. Fig. 3b). To further explore the role of SUMO-mediated modifications of LKB1 in hepatoma cells, Ni 2+ -NTA agarose beadpulldowns were performed in Huh-7 human hepatoma cells after cotransfection of pcDNA3-FLAG-LKB1 wild type (WT) and pcDNA3-His 6 - SUMO1, pcDNA3-His 6 -SUMO2 or pcDNA3-His 6 -SUMO3 plasmids. Our results show that LKB1 is mostly modified by SUMO-2, in a SUMOconjugating enzyme UBC9 dependent process, both in Huh-7 human hepatoma cells (Fig. 2a) and in MLP-29 cells (Suppl. Fig. 4a). Cotransfection with the E3 SUMO-protein ligases PIAS, especially PIAS 1, further increased LKB1 SUMOylation by SUMO-2 in Hu7–7 cells whereas co-transfection with SUMO-specific proteases, particularly SENP2, and SENP-1 and -3, decreased LKB1 SUMOylation (Fig. 2b,c). In Fig. 4. Liver Kinase B1 (LKB1) SUMOylation at Lys178 by SUMO-2 regulates human hepatoma cell survival by hampering LKB1 nucleocytoplasmic shuttling. LKB1 overexpression was induced in Huh-7 cells with pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1 WT) or the pcDNA3-FLAG-LKB1 K178R plasmid (LKB1 K178R) in the presence of His-SUMO-2. a. Western blot analysis of LKB1, [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control]. Quantifications are shown in Suppl. Fig. 4a; b and c. Cell viability as detected by staining of attached cells with crystal violet dye and number of cells; d. Immunoprecipitation assay between LKB1 WT and LKB1 K178R and the STe20-Related ADaptor (STRADα) cofactor after STRADαoverexpression in Huh-7 human hepatoma cells. Normalized quantifications relative to inputs are shown below the panel; e. Representative immunofluorescence staining for LKB1 (FLAG) in Huh-7 cells and quantification of the percentage of LKB1 nuclear positive staining cells. Scale bar corresponds to 50 μm. f. LKB1 levels in cytoplasmic (Cyto) and nuclear fractions (Nuc) [Glyceraldehyde 3-phosphate (GAPDH) was used as loading control for cytoplasmic fractions and Histone H3 for nuclear fractions]. Quantifications are shown in Suppl. Fig. 4b. At least triplicates were used per experimental condition. Data is shown as mean ± SEM. *p b0·05 is indicated (Mann-Whitney Utest). 413I. Zubiete-Franco et al. / EBioMedicine 40 (2019) 406–421 agreement, SENP2 has been previously reported to play a critical role in the control of HCC cell growth [59]. The SUMOylation of LKB1 does not depend on LKB1 kinase activity domain as a kinase dead (KD) mutant, pcDNA3-FLAG-LKB1 K78I, was equally modified by SUMO-2 (Fig. 2de ). Finally, SUMO-2-mediated modification of LKB1 reduced the interaction between LKB1 and STRADα, a co-factor involved in LKB1 nuclear export, as shown by immunoprecipitation assay, during STRADαoverexpression (Fig. 2e). Fig. 5. Liver Kinase B1 (LKB1) is modified by SUMO-2 in Lys178 after its acetylation at Lys48 in human hepatoma cells. a. Schematic representation of LKB1 showing the Nuclear (NLS) localization, acetylation domain (AD) and the Kinase domain (KDN). Acetylation LKB1 mutant used is shown. b. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection with the pcDNA3-FLAG-LKB1 Wild type plasmid (LKB1 WT), His-SUMO-2 and treatment with sirtuin 1 (SIRT1). c. Ni 2+ -NTA agarose bead pulldown in Huh-7 human hepatoma cells after transfection the LKB1 WT, LKB1 acetylation mutant, LKB1 K48R or the LKB1 SUMOylation mutant, LKB1 K178R, with His-SUMO-2, in the presence and absence of the Ex-527, the SIRT1 inhibitor. Normalized quantifications relative to inputs are shown below each panel; d. 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