Transcriptional Factor Gli2 Promotes Cisplatin Resistance By Regulating MDR1 In Human Epithelial Ovarian Cancer

Background: Ovarian cancer is the most fatal gynecological malignancy,and cytoreductive surgery and combination chemotherapy based on platinum and taxane have been the main treatment modalities for ovarian cancer.However,tumor chemotherapy efficacy is often limited due to multidrug resistance.Chemotherapy resistance is a key factor affecting the prognosis of ovarian cancer patients,especially platinum resistance.Among them,cisplatin(DDP)resistance remains a key challenge to improve clinical outcomes in ovarian cancer patients.Therefore,novel biomarkers for early prediction of chemoresistance properties of ovarian tumors and therapeutic strategies to reverse drug resistance are urgently needed.The Hedgehog(Hh)signaling pathway is a key regulator of embryonic development and homeostasis and plays an important role in chemoresistance in multiple tumors.Classical multidrug resistance is often associated with the expression of ATP-binding cassette transporters,and the gene encoding multidrug resistance protein 1(MDR1,also known as ABCB1)positively regulates chemotherapy resistance in many different types of cancer,including ovarian tumor.Our previous study found that Gli2 overexpression can lead to cisplatin resistance in ovarian cancer cells,but the specific regulatory mechanism is still unclear.Therefore,this study aimed to evaluate MDR1 as a potential downstream target of Gli2 and the role of the Gli2/MDR1 axis in promoting cisplatin resistance in ovarian cancer cells.Purpose: To explore the specific molecular mechanism of Hedgehog-Gli2 signaling pathway regulating ovarian cancer chemoresistance to cisplatin.Methods: To generate drug-resistant SKOV3/DDP cells,SKOV3 cells were grown for six months under continuous induction wherein the cisplatin concentration was steadily increased.Gli2 expression in ovarian cancer cells with varying cisplatin sensitivities was detected using western blot.Cell counting kit-8 assays were used to assess the cisplatin sensitivity of SKOV3,SKOV3/DDP,A2780,and A2780/DDP cells and reversal of cisplatin resistance in SKOV3/DDP and A2780/DDP cells.Cell proliferation was analyzed using 5-ethynyl-2′-deoxyuridine(Ed U)incorporation assays.The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays.Finally,xenograft ovarian cancer tumors were generated in nude mice,which were then treated with intraperitoneal cisplatin or phosphate-buffered saline(PBS)injections to investigate if Gli2 affected cisplatin resistance in ovarian cancer in vivo.Results: DDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive ovarian cancer cells.Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression,whereas it increased DNA damage,thereby sensitizing ovarian cancer cells to cisplatin.Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor(GANT61)in SKOV3/DDP and A2780/DDP cells.In cells stably overexpressing Gli2,treatment with gradient concentrations of verapamil,an MDR1 inhibitor,significantly inhibited MDR1 expression.Our findings indicate that downregulation of MDR1 expression may reverse ovarian cancer cell resistance to cisplatin.Moreover,dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2,with Gli2 positively regulating MDR1 expression.Finally,subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating ovarian cancer drug resistance.Conclusions: Gli2 is involved in the regulation of cisplatin chemotherapy resistance in ovarian cancer cells.Morever,we identified a mechanism by which Hedgehog-Gli signaling regulates ovarian cancer cisplatin chemoresistance by modulating MDR1 expression.