Screening And Functional Investigation Of Gemcitabine Resistance Related Molecules In Pancreatic Cancer

Pancreatic cancer is one of the most lethal malignancies with high morbidity and mortality.Pancreatic cancer is characterized by its late diagnosis,limited effective therapy,and extremely poor prognosis.The dismal prognosis is largely attributed to extreme chemoresistant phenotype of the tumor.Gemcitabine remains a standard chemotherapeutic agent for advanced pancreatic cancer and post-surgery adjuvant therapy.However,only few patients benefited from gemcitabine treatment.Intrinsic and acquired resistance to gemcitabine is the major obstacle to drug efficacy.Therefore,effective strategies are urgently needed to overcome drug resistance.The deregulation of the apoptotic machinery is one of the central events during development of gemcitabine resistance.Bcl-XL is well known as a major anti-apoptotic protein in pancreatic cancer,but the regulatory network of Bcl-XL is complicated and the detailed mechanism remains unclear.GATA binding protein 1(GATA1)is the founding member of the GATA transcription factor family.The role of GATA1 in pancreatic cancer remains unexplored.In this study,we found that GATA1 is highly expressed in pancreatic cancer tissues and patients with highly expressed GATA1 showed shorter overall survival(OS)and recurrence-free survival(RFS).Also,GATA1 status predicts gemcitabine response in PDAC patients.For the patients who received gemcitabine-based chemotherapy,those with highly expressed GATA1 had shorter OS and RFS,whereas for the patients who did not receive chemotherapy,GATA1 status did not predict their prognosis.We found GATA1 promotes cell proliferation in PDAC cells with cell proliferation assay and colony formation assay.Further investigation demonstrates GATA1 is involved in both intrinsic and acquired gemcitabine resistance in PDAC cells.Mechanistically,using dual luciferase assay and ChIP assay,we found that GATA1 upregulates Bcl-XL transcription by binding to its promoter.In vitro and in vivo experiments confirmed that GATA1 induces gemcitabine resistance through enhancing Bcl-XL mediated anti-apoptosis.GATA1 loses its ability to promote gemcitabine resistance in Bcl-XL knockdown PD AC cells,which implies that GATA1 regulates gemcitabine resistance in a Bcl-XL-dependent manner.Moreover,in PDAC patients,Bcl-XL expression is positively correlated with GATA1 level and predicts clinical outcomes and gemcitabine response.Cox regression analysis demonstrated that both GATA1 and Bcl-XL are independent prognostic factors for pancreatic cancer.Recently microRNAs were found to be involved in gemcitabine resistance in pancreatic cancer through different pathways.However,the specific mechanisms have not been fully elucidated.The efficacy of gemcitabine is largely dependent on drug uptake.Human equilibrative nucleoside transporter(hENT1)is the major membrane transporter for gemcitabine uptake.Current studies about hENT1 regulation are limited and miRNA in regulating hENT1 is still unclear.Through miRNA profiling,we found that miR-1197 expression was upregulated in gemcitabine-resistant cells.By cell viability assay and colony formation assay,our data showed that miR-1197 plays a critical role in intrinsic and acquired drug resistance in PDAC cells.Dual luciferase assay demonstrated that miR-1197 regulates hENT1 expression by binding to its 3’-UTR.Furthermore,we found that miR-1197 induces gemcitabine resistance in pancreatic cancer through hENT1 in vitro and in vivo.More importantly,miR-1197 was negatively correlated with hENT1 expression and miR-1197 predicts prognosis and gemcitabine response in PDAC patients.In conclusion,we identified two unreported molecules in gemcitabine resistance of pancreatic cancer,GATA1 and miR-1197.These molecules regulate gemcitabine sensitivity through anti-apoptosis and drug uptake pathway respectively.Moreover,GATA1 and miR-1197 both predicts prognosis and gemcitabine response in PDAC patients.Our study brings new insights in the mechanism of gemcitabine resistance and provides novel markers and potential targets for pancreatic cancer.