PMID：29313411.pdf

Full Terms&Conditions of access and use can be found athttp:/ by:University of PennsylvaniaDate:10 January 2018,At:02:39AutophagyISSN:1554-8627(Print)1554-8635(Online)Journal homepage:http:/ of WNT-CTNNB1 signaling upregulatesSQSTM1 and sensitizes glioblastoma cells toautophagy blockersMireia Nager,Marta Cresp Salln,Anna Visa,Charumathi Pushparaj,MariaSantacana,Anna Maci,Andre Yeramian,Carles Cant&Judit HerrerosTo cite this article:Mireia Nager,Marta Cresp Salln,Anna Visa,Charumathi Pushparaj,MariaSantacana,Anna Maci,Andre Yeramian,Carles Cant&Judit Herreros(2018):Inhibition ofWNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagyblockers,Autophagy,DOI:10.1080/15548627.2017.1423439To link to this article:https:/doi.org/10.1080/15548627.2017.1423439View supplementary material Accepted author version posted online:09Jan 2018.Submit your article to this journal View related articles View Crossmark data Publisher:Taylor&Francis Journal:Autophagy DOI:https:/doi.org/10.1080/15548627.2017.1423439 INHIBITION OF WNT-CTNNB1 SIGNALING UPREGULATES SQSTM1 AND SENSITIZES GLIOBLASTOMA CELLS TO AUTOPHAGY BLOCKERS Nager M,Salln MC$,Visa A$,Pushparaj C$,Santacana M#,Maci A$,Yeramian A,Cant C$&Herreros J*.IRBLleida.Departments of Basic Medical Sciences and Experimental Medicine$,University of Lleida.Immunohistochemistry Unit#,IRBLleida.Rovira Roure 80,25198 Lleida,Spain.*Corresponding author:j.herreroscmb.udl.cat Mireia Nager(PhD),Department of Basic Medical Sciences,University of Lleida,IRBLleida.Rovira Roure 80,25198 Lleida,Spain(mireia.nagercmb.udl.cat).Phone:00-34-973702214 Marta Cresp Salln(MSc),Department of Experimental Medicine,University of Lleida,IRBLleida,Rovira Roure 80,25198 Lleida,Spain(marta.crespimex.udl.cat).Phone:00-34-973702214 Anna Visa(MSc),Department of Experimental Medicine,University of Lleida,IRBLleida,Rovira Roure 80,25198 Lleida,Spain(annavisamex.udl.cat).Phone:00-34-973702214 Charumathi Pushparaj(PhD),Department of Experimental Medicine,University of Lleida,IRBLleida,Rovira Roure 80,25198 Lleida,Spain(charu_yamunayahoo.co.in).Phone:00-34-973702214 Downloaded by University of Pennsylvania at 02:39 10 January 2018 2 Maria Santacana(PhD),Immunohistochemistry Unit,IRBLLeida.Rovira Roure 80,25198 Lleida,Spain(msantacanairblleida.cat).Phone:00-34-973702937 Anna Maci(PhD),Department of Experimental Medicine,University of Lleida,IRBLleida,Rovira Roure 80,25198 Lleida,Spain(anna.maciamex.udl.cat).Phone:00-34-973702937 Andre Yeramian(PhD),Department of Basic Medical Sciences,University of Lleida,IRBLleida.Rovira Roure 80,25198 Lleida,Spain(andree.yeramiancmb.udl.cat).Phone:00-34-973702937 Carles Cant(PhD),Department of Experimental Medicine,University of Lleida,IRBLleida,Rovira Roure 80,25198 Lleida,Spain(c.cantimex.udl.cat).Phone:00-34-973702950 Judit Herreros(PhD),Department of Basic Medical Sciences,University of Lleida,IRBLleida.Rovira Roure 80,25198 Lleida,Spain(j.herreroscmb.udl.cat).Phone:00-34-973702950 Note:The authors declare no conflict of interests.ABSTRACT WNT-CTNN1B signaling promotes cancer cell proliferation and stemness.Furthermore,recent evidence indicates that macroautophagy/autophagy regulates WNT signaling.Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma(GBM),a devastating brain tumor.Inhibiting TCF,or silencing TCF4 or CTNNB1/-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and,in turn,autophagy.DKK1/Dickkopf1,a canonical WNT receptor antagonist,also induced autophagic flux.Importantly,TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation,and nuclear translocation of TFEB,a master regulator of lysosomal biogenesis and autophagy.TCF inhibition or silencing additionally affected GBM cell proliferation and migration.Autophagy induction followed by its blockade can promote cancer cell death.In agreement with this notion,halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent Downloaded by University of Pennsylvania at 02:39 10 January 2018 3 mechanism involving CASP8(caspase 8).In vivo experiments further underline the therapeutic potential of such dual targeting in GBM.Keywords:autophagy,CTNNB1,glioblastoma,SQSTM1,TCF,TFEB,WNT.Running title:Canonical WNT signaling inhibition triggers autophagy INTRODUCTION Gliomas are primary brain tumors classified into different malignancy grades(World Health Organization):diffuse astrocytoma(grade II),anaplastic astrocytoma(grade III)and glioblastoma multiforme(GBM;grade IV)1,2 GBMs are the most common and aggressive gliomas,presenting a poor prognosis and median survival below 14 months 3.GBM features are a high infiltrative/invasive capacity,apoptosis resistance,necrosis and angiogenesis 4.Despite treatment(extensive surgery,radiotherapy and chemotherapy with temozolomide),GBM recurrence is unfortunately very common.Therefore,there is an urgent need for novel therapies for this non-curable disease 5.Canonical WNT signaling plays important functions in development and cell proliferation,and its aberrant activation is linked to tumorigenesis 6,7.The main effector of the WNT canonical pathway is CTNNB1/-catenin 8,which acts together with transcription factors of the TCF/LEF(transcription factor/lymphoid enhancer binding factor)families to control expression of WNT target genes.In the absence of WNT ligands,CTNNB1 associates with the destruction complex that includes GSK3B(glycogen synthase kinase 3 beta),a Ser/Thr kinase that phosphorylates CTNNB1,leading to its proteasomal degradation.Upon WNT binding to FZD(frizzled class receptor)and LRP(LDL receptor related protein)receptors,the destruction complex destabilizes.Unphosphorylated CTNNB1 then escapes from degradation and translocates to the nucleus 8.WNT inhibitors,such as the extracellular antagonist of LRP5 and LRP6,DKK1/Dickkopf1,control WNT signaling.Unlike other cancers,CTNNB1 mutations are infrequent in GBM,and WNT signaling activation appears to occur by upregulation of WNT ligands 9 or epigenetic regulation of WNT inhibitors 2,10.Additional mechanisms include control by upstream protoncogenes 11,overexpression of the scaffold protein DVL2/Dishevelled2 12 and activation of CTNNB1 nuclear signaling 10,13.Macroautophagy(referred to as autophagy)is a conserved adaptive response that recycles cellular components to provide nutrients under metabolic cell stress.During autophagy,double-membrane vesicles,phagophores,engulf damaged proteins and organelles.Downloaded by University of Pennsylvania at 02:39 10 January 2018 4 After maturation into autophagosomes,these compartments then fuse with lysosomes,where the cargo is degraded,providing biosynthetic and energy precursors,thus contributing to the survival of tumor cells in unfavorable conditions 14.Autophagy in cancer plays a dual role.In early stages of tumorigenesis,it acts as a tumor suppressor preventing accumulation of damaged proteins and organelles.However,in advanced stages,autophagy provides tolerance against metabolic stress under conditions of nutrient deprivation and confers resistance to radio and chemotherapy 15.MTOR(mechanistic target of rapamycin)regulates cell growth,proliferation and anabolic processes,and its inhibition is central to autophagy induction 16.Interestingly,autophagy and WNT-CTNNB1 signaling appear inversely regulated in cancer.The WNT pathway component DVL2 maintains GBM cell proliferation through canonical and noncanonical WNT signaling 12.In turn,autophagy negatively regulates WNT signaling by promoting DVL2 17 and CTNNB1 degradation 18,19.Furthermore,the TCF-CTNNB1 complex represses SQSTM1/p62 18,which is responsible for the binding to MAP1LC3/LC3(microtubule associated protein 1 light chain 3)that,once lipidated,associates with phagophores and is involved in cargo recognition 20.Finally,WNT signaling(involving GSK3B inhibition)activates MTOR and protein translation 21.Here we sought to elucidate the effects of inhibiting WNT-CTNNB1 signaling on autophagy in GBM cell lines and biopsy-derived cell cultures.To this end,we inhibited TCF-CTNNB1 signaling using the pharmacological inhibitor FH535,silencing TCF4 or CTNNB1 by shRNA/siRNA,or challenging the cells with the WNT receptor antagonist DKK1.We found that inhibition of WNT signaling in GBM upregulates SQSTM1 and,consequently,autophagic flux through MTOR inhibition and nuclear translocation of TFEB(transcription factor EB),a master regulator of autophagosome and lysosome biogenesis 22,23.Moreover,our data show that autophagy induction renders GBM cells more sensitive to cell death by autophagy blockers,which could be exploited in future therapies.RESULTS SQSTM1 levels increase in GBM and inversely correlate with CTNNB1 To test SQSTM1 protein levels in diffuse astrocytoma,anaplasic astrocytoma and GBM(grade IV)samples,we performed an immunohistochemical analysis in tissue microarrays(TMAs).SQSTM1 was detectable in grade II astrocytomas and its levels increased in GBMs(8.78 fold,GBM vs.astrocytomas;p=0.003)(Fig.1A and 1D).Further analysis using the GlioVis platform Downloaded by University of Pennsylvania at 02:39 10 January 2018 5 24 confirmed the increase of SQSTM1 mRNA levels in GBMs compared to non-tumor or astrocytoma samples(p0.001;Fig.1B).Previous studies in colorectal carcinoma cells 18 described an inverse relationship between SQSTM1 and CTNNB1.Data obtained from Gliovis supported this in GBM(Fig.1C;p=0.01),which was corroborated in our GBM biopsy-derived cell cultures and established cell lines(Fig.1D).A comparison of autophagic markers and the levels of CTNNB1 or CCND1(cyclin D1;a WNT target)indicated an inverse relationship in several GBM cases(Fig.1E),further suggesting a negative association between WNT-CTNNB1 signaling and autophagy or vice versa.WNT-CTNNB1 signaling regulates SQSTM1 through TCF-dependent transcriptional regulation To investigate how WNT-CTNNB1 signaling modulates SQSTM1,GBM cell lines were treated with WNT3A to activate WNT-CTNNB1 signaling or with FH535(a TCF inhibitor that affects CTNNB1 recruitment)25 to inhibit it.WNT3A decreased SQSTM1 mRNA in the A172 cell line(Fig.2A and see below).On the contrary,SQSTM1 expression(at both the mRNA and protein levels)increased after treatment with FH535 in U251-MG and U87-MG cell lines and in GBM primary cultures(Figs.2A and 2B).We confirmed that WNT3A and FH535 activate or inhibit WNT signaling,respectively,by the TOP-Flash assay and expression of the target gene CCND1(Fig.S1).A172 cells displayed higher SQSTM1 protein levels than U251-MG or U87-MG cells(Fig.S2),which may explain why SQSTM1 could not be further increased by FH535,but significantly decreased upon treatment with WNT3A.To validate the results obtained using a pharmacological inhibitor of TCF,we transduced cells with lentiviruses carrying a shRNA targeting TCF4.Cells expressing the shRNA against TCF4 showed a 60-80%decrease in TCF4 mRNA,compared to cells expressing a scrambled shRNA(Fig.2C).Parallel to this,cells expressing the shRNA of TCF4 increased SQSTM1 mRNA(1.8-2.3 fold vs.scrambled;Fig.2C)and SQSTM1 protein levels(Fig.2D),consistent with the FH535 results.Thus,TCF inhibition or silencing raise SQSTM1 expression in GBM cells.Because CTNNB1 acts together with TCF,we next investigated the role of CTNNB1 in SQSTM1 regulation.First,we transfected GBM cells with siRNAs against CTNNB1 or GAPDH(used as a control).We observed decreased CTNNB1 and increased SQSTM1 levels in U251-MG and U87-MG cells upon transfection of the CTNNB1 siRNA(Fig.2E).By contrast,cells expressing a stabilized mutant form of CTNNB1(S37Y)that is not degraded,presented lower levels of SQSTM1 compared to cells transfected with wild-type(WT)CTNNB1(Fig.2F).Downloaded by University of Pennsylvania at 02:39 10 January 2018 6 Together,these results confirm that CTNNB1-TCF signaling transcriptionally represses SQSTM1 in GBM.Inhibition of WNT-CTNNB1 signaling increases the autophagic flux in GBM Next,we aimed to understand the functional implications of the SQSTM1 regulation by WNT-CTNNB1 signaling.As SQSTM1 acts as a cargo receptor of phagophores,we studied the autophagic flux upon inhibition of WNT signaling.First,we reduced the activity of TCF with FH535.Analysis of autophagy markers showed an increase of MAP1LC3A/B-II in treated cells,indicative of increased autophagosome numbers.These results were paralleled by lower levels of polyubiquitinated proteins in GBM cells treated with FH535(Fig.3A),suggesting an enhancement of autophagy.To confirm whether the deregulation observed corresponds to an induction of autophagic flux,we used bafilomycin A1(Baf),which prevents autophagosome-lysosome fusion and blocks autophagic flux.Increased autophagic flux would elevate MAP1LC3A/B-II upon addition of Baf.If the increase in MAP1LC3A/B-II in treatment plus Baf is higher than that of Baf alone,results should be interpreted as autophagic flux induction.Instead,MAP1LC3A/B-II levels unaltered by Baf should be interpreted as autophagy blockade and accumulation of autophagosomes 26.We treated GBM cells with or without FH535 for 24 h together with Baf(last 2 h).Treatment with FH535 and Baf showed higher levels of MAP1LC3A/B-II compared to Baf alone(Fig.3B),indicating autophagy induction by FH535.Importantly,similar results were obtained in GBM primary cultures(Fig.3B).These results showed that autophagic flux is augmented by inhibition of TCF in GBM.To assess the involvement of WNT signaling in autophagy regulation at an upstream level of the WNT-CTNNB1 pathway,we treated GBM cell lines with DKK1(a WNT antagonist acting at the receptor level).We treated cells with DKK1 alone or together with Baf.Results showed increased MAP1LC3A/B-II levels in cells cotreated with DKK1 and Baf compared to cells treated with Baf alone(Fig.3C),showing that DKK1 induced autophagy.The regulation of autophagy by inhibition of WNT signaling was additionally addressed by transfecting with a tandem-fluorescent tagged MAP1LC3B plasmid(ptfLC3/mRFP-EGFP-LC3)27.Autophagosomes formed from phagophores that incorporated ptfLC3 appear yellow(as a result of the colocalization of mRFP and EGFP fluorescence),which distinguished them from autolysosomes(presenting a red fluorescence due to quenching of EGFP in the lysosomes).Rapamycin(an MTOR inhibitor)increased the number of red puncta(Fig.3D).In contrast,treatment with the autophagy blocker chloroquine(CQ)elevated the number of yellow puncta Downloaded by University of Pennsylvania at 02:39 10 January 2018 7 (Fig.3D).FH535 or DKK1 produced a red puncta pattern,similar to that obtained with rapamacyin,demonstrating increased numbers of autolysosomes and autophagy induction(Fig.3D).A comparison of the pattern obtained after FH535 plus CQ vs.FH535 alone confirmed an intact autophagic flux(data not shown).Finally,we used immunocytochemistry against SQSTM1 and MAP1LC3A proteins to