Mechanistic Study On Regulation Of Drug Resistance Of Pancreatic Cancer By Obg-like ATPase 1(OLA1)

Tumor is a typical multi-cause disease,which is comprehensively regulated by multiple intracellular genes and the tumor external environmental factors.Tumor multidrug resistance(MDR)is the main cause of tumor therapy failure,but the mechanisms of MDR are still unclear.Pancreatic cancer is highly malignant,and the 5-year survival rate is less than 8%.Chemotherapy is the main treatment for pancreatic cancer,while it is easy to develop drug resistance.Obg-like ATPase(OLA1)is a NTPase,belonging to the YchF subfamily of the Obglike GTPase family.OLA1 can hydrolyze both ATP and GTP.Similar to many drug resistance genes,OLA1 participates in cell energy metabolism.Abnormal energy metabolism is a significant feature of tumor,which is closely related to the drug resistance.Currently,no research about the influence of OLA1 on the drug resistance of pancreatic cancer has been reported.This study aims to investigate the effect and mechanism of OLA1 on the chemoresistance of pancreatic cancer,so as to explore new ways to treat pancreatic cancer.(1)Through immunohistochemical analysis,it was found that OLA1 was highly expressed in clinical pancreatic cancer.According to the patient's medication and drug response analysis,the clinical tumor tissues were divided into drug-resistant group and drug-sensitive group.Immunohistochemical analysis data indicated that OLA1 was highly expressed in chemotherapy-resistant cases.Through short-term drug treatment of drug-sensitive cell lines and the construction of long-term acquired drug resistance cell lines,it was found that OLA1 expression gradually increased with drug treatment time.By regulating the expression of OLA1 in vitro and in vivo,and observing the changes in the sensitivity of tumor cells to chemotherapeutic drugs,we found that OLA1 promoted pancreatic cancer drug resistance.(2)Through the transcriptomics study of OLA1 and DO enrichment analysis,it was found that OLA1 might affect the sternness of pancreatic tumor cells through the cell sternness regulator SOX2.Through the clone formation experiment,the microsphere formation experiment and the expression of cancer stemness marker(CD44+,CD133+),cell stemness regulating genes as well as proteins,it was found that OLA1 affected the regulation of pancreatic cancer stemness cell.Through the pathway analysis software IP A,it was found that OLA1 directly interacted with SOX2.Proteomic analysis of OLA1 immunocoprecipitation revealed that OLA1 and HHIP were co-immunoprecipated.The His pull down experiments of prokaryotic expression of OLA1 proved that OLA1 directly interacted with SHH.Fluorescence co-localization analysis could form an OLA1/SHH/HHIP complex in the nucleus,which leaded to an increase in the expression of HHIP in the nucleus and inhibited the negative feedback mechanism of the Hedgehog pathway.The experiment of subcutaneous xenograft tumor in NOD-SCID immunodeficiency mice verified that OLA1 affected the stemness and drug resistance of pancreatic cancer cells through the SOX2/GLI3/HHIP axis.Through TargetScan database,we found that miR-34a-3p was the upstream of OLA1,and confirmed that miR-34a3p targeted OLA1 3'UTR 492-499 sequence through dual luciferase reporter gene.The in vitro and in vivo experiments ulteriorly confirmed that miR-34a-3p inhibited the expression of OLA 1,thereby increasing the sensitivity of pancreatic tumor cells to chemotherapeutics.(3)Through the analysis of OLA1 transcriptomics,it was found that OLA 1 was significantly related to the adhesion characteristics of tumor cells.Through the analysis of cytological function,it was found that OLA1 promoted the wound healing ability,cell migration and invasion ability of pancreatic tumor cells.Through the analysis of molecular biology level,it was found that OLAl affected the EMT process through SNAIL/E-cadherin,which in turn affected the metastasis of pancreatic cancer cells.In order to explore whether OLA1 regulated drug resistance in other tumors,breast cancer and oral cancer were selected as the research objects.In breast cancer,the high expression of OLAl promoted the metastasis of cancer cells and improved chemotherapy resistance by activating the TGF?/SMAD3 axis.In oral cancer,OLAl inhibited OSCC cell metastasis through the TGF?/SMAD2/SMAD4 axis.Although OLA1 participated in the EMT process of breast and oral cancer through TGF?,its specific signal transduction mechanism has adopted two different strategies.This study reveals the molecular mechanism of OLA1 involved in pancreatic cancer drug resistance.It promotes pancreatic tumor cell stemness and inhibits Hedgehog negative feedback by regulating the SOX2/GLI3/HHIP axis,thus leading to pancreatic cancer drug resistance.Through the study of EMT process of different tumor cells by OLA1,we find that the mechanism of OLA1 mediating cancer cell metastasis is tissue-specific.This study indicates that OLA1 is a potential tumor drug resistance target,which is helpful to the development of molecular targeted therapy drugs for malignant tumors,and provides a new perspective for the research of new drug resistance targets.