ETS Family Transcription Factor GABPA Inhibits The Malignant Phenotype Of Bladder Cancer By Regulating Luminal-basal Differentiation

Telomerase reverse transcriptase(TERT)is the core component of telomerase.TERT can activate telomerase and thus can transform normal cells to tumor cells which have an immortal feature,the activity of TERT in differentiated human cells is strictly controlled,however a variety of malignant tumors,such as bladder cancer and glioma carry TERT promoter mutations which leads to an abnormal TERT activation,The ETS family transcription factor GAB PA and its partner GABPB1 coordinately activate mutated TERT promoters in these tumor cells,resulting in the increased telomerase activity.GABPA depletion or the disruption of the GABPA/GABPB1 complex by knocking down GABPB1 was shown to inhibit telomerase,thereby eliminating the tumorigenic potential of glioblastoma cells.GABPA/B1 is proved to be a cancer therapeutic target.However,the role of GABPA in BC is unclear.Here,we unexpectedly observed that GABPA ablation inhibited TERT expression,but robustly promoted proliferation,stemness,and invasive phenotypes and cisplatin resistance in BC cells,while its overexpression exhibited the opposite effects.Overexpression of GABPA also inhibited in vivo metastasis in a xenograft transplant model.Mechanistically,GABPA directly activates the transcription of FoxAl and GATA3 which are,key transcription factors driving luminal differentiation of urothelial cells.Consistently,TCGA/GEO dataset analyses show that GABPA expression is correlated positively with luminal phenotypes while negatively with basal signatures.Luminal tumors express higher GABPA than the basal tumors.Lower GABPA expression is associated with the GABPA gene methylation or deletion(especially in basal subtype of BC tumors),and predicted significantly shorter patient survival based on the analyses of TCGA and our BC patient cohort.Taken together,GABPA dictates luminal identity of BC cells and inhibits aggressive diseases in BC by promoting cellular differentiation despite of its stimulatory effect on telomerase/TERT activation.Given these biological functions and its frequent methylation and/or deletion,GABPA serves as a tumor suppressor rather than oncogenic factor in BC.The effect of GABPA effect on oncogenesis is context-dependent and its targeting for telomerase inhibition in BC may promote disease metastasizing.Aimswe manipulate the expression of GABPA in the bladder cancer to research how it regulates the expression of TERT mRNA.observe GAPBPA directly stimulate the transcription of FoxA1 and GATA3 which can promote the luminal differentiation.Further more,we are able to observe effects of GABPA on proliferation,invasion,stemness and Cisplatin resistance in bladder cancer cell.It is meaningful to explore the function of GABPA in the initiation and development processes of bladder cancer on molecular biology level.Our findings may provide a new target for the diagnosis and treatment of BC.Methods:(1)The TCGA and GEO databases were used to collect and compare the pathological classifications of bladder cancer,including the correlation between the expression of GABPA and TERT in the luminal and basal subtypes,and the mRNA expression of GABPA and FoxA1/GATA3.(2)A total of 112 BC patients who underwent surgery in Second Hospital of Shandong Uni versity from 2006 to 2016 were included in the study.Immunohistochemistry was used to detect differences in GABPA and FoxAl expression in paraffin sections of collected bladder tumor pathological samples(3)By culturing three types of bladder cancer cells,including J82,SW1710,and HT1197,we explored the underlying mechanisms.We used GABPA siRNA transfection to knock down GABPA expression and up-regulated the expression of GABPA by introducing ectopic expression of GABPA in these cells,We then extracted the RNA in the cells after 48h and the total protein after 72h,and used real-time quantitative PCR and western-blot technologies to detect the effect of knockdown or high expression.The next step is to verify that whether GABPA has an activating effect on FoxA1 and GATA3 transcription by using ChIP assay,BrdU,cell flow cytometry,Trans-well,cisplatin resistance and other experiments are used to detect the changes in tumor cell proliferation,invasion and drug resistance after knockdown and overexpression of GABPA.(4)J82 cells expressing ectopic GABPA(J82/GABPA)and empty vector control cells(J82/control)were injected into 6-week-old male BALB/c nude mice through the tail vein to evaluate the effect of GABPA on metastasis in vivo.After 10 weeks,the mice were sacrificed.Livers,lungs and other organs were collected,and treated with BOUIN’S fixative.HE staining and immunohistochemistry were performed to detect the expression of ki67 in the sections to assess tumor invasion and metastasis in mice(5)Student’s t test,Mann-Whitney U-test,and Chi2-test or Fisher’s exact test were used for univariate analysis.Spearman’s Rank-Order Correlation was applied to determine correlation coefficient p and P value.P values<0.05 were considered as statistically significant.Survival analyses were performed with log-rank test.Levels of GABPA transcripts were classified into low and high groups using median separation.All the tests were two-tailed and P values<0.05 were considered as statistically significant.Result:(1)By analyzing the TCGA dataset,however,we unexpectedly found that GABPA mRNA was highly abundant in luminal tumors.TERT expression was analyzed simultaneously,and there was no significant difference between these two BC subtypes(2)Using IHC to analyze the expression of GABPA and FoxAl in the bladder cancer tissue as well as adjacent normal bladder tissues derived from patients with bladder cancers,we can clearly see that in BCs GABPA correlates positively with FoxA1(3)In bladder cancers cell lines,we can conclude that FoxAl and GATA3 which are two key factors of luminal differentiation are the downstream targets of GABPA by manipulating GABPA expression in BC-derived J82,SW1710 and HT1197 cells and then analyzing changes in FoxAl and GATA3 mRNA and protein levels.At the same time,it is surprisingly to observe that the CDKN1A and CDKN1B which are the downstream targets of FoxAl and GATA3 are also correlate positively with the expression of GABPA.(4)In BrdU and cell flow cytometry experiments,it can be observed that the down-regulation of GABPA in tumor cells obviously leads to higher proliferation ability.On the contrary,when the GABPA overexpression plasmid is transferred into the cell,the proliferation of bladder cancer cells are obviously inhibited.Compared with the control group,the bladder cancer cells transfected with GABPA siRNA in the Trans-well experiment had significantly enhanced invasive properties.At the same time,after transferring into GABPA overexpression plasmid,the invasiveness of tumor cells was significantly reduced compared with the control group.In the cisplatin resistance experiment,we knocked down the expression of GABPA in bladder cancer cells and found that compared with the control group,its resistance to cisplatin was significantly enhanced(5)In vivo metastasis of BC cells in a mouse model,J82 cells with ectopic GABPA expression(J82/GABPA)and their control counterparts(J82/Control)were injected into two groups of nude mice(6/group)via the tail vein,respectively.Mice were killed 10 weeks later,and their lungs and livers were examined for tumor cell seeding,by making into paraffin sections,After immunohistochemical detection of ki67,HE staining and BOUIN’S fixative treatment,we found that the overexpression of GABPA in tumor cells inhibited the metastasis of bladder cancer cells in xenograft mouse models.Conclusion:1、Confirming the expression of GABPA in bladder cancer patients is of great significance to the survival and prognosis of patients.The survival rate and prognosis of the high expression group are better than those of the low expression group。2、GABPA promotes the luminal differentiation of bladder cancer by regulating FoxA1 and GATA3,the key transcription factors for the differentiation of luminal cells.GABPA is likely to impose positively influence to promote the bladder cancer to form luminal phenotype,and prevent cancer from infiltrating into the muscle layer to form more malignant basal subtype.3、At the cellular level,the overexpression of GABPA inhibited the proliferation and invasion of bladder cancer cells,and enhanced the anti-tumor effect of cisplatin which is a chemotherapy drug to treat bladder cancer cells.4、In vivo experiments,compared with the control group,GABPA’s overexpression group has an inhibitory effect on the metastasis of bladder cancer cells in nude mice.