The Function And Regulation Mechanism Of LAT2 In The Chemoresistance Of Pancreatic Cancer

BackgroundPancreatic cancer is one of extremely malignant tumors with a poor prognosis.Chemoresistance is an important factor resulting in poor prognosis of patients with pancreatic cancer.Therefore,exploring the molecular mechanism regulating chemoresistance is of great significance for improving the prognosis of patients with pancreatic cancer.With the development of research on energy metabolism and tumor microenvironment,reprogrammed energy metabolism has become an emerging hallmark of cancer in recent years.Transporters have been reported to be amino acid sensors involved in controlling mTOR recruitment and activation,which is crucial for the growth of both normal and tumor cells.L-type amino acid transporter 2(LAT2),encoded by the SLC7A8 gene,is a Na+-independent neutral amino acid transporter and is responsible for transporting neutral amino acids,including glutamine,which can activate mTOR.Our research group constructed the lncRNA-miRNA-mRNA regulatory network related to gemcitabine chemoresistance through high-throughput sequencing technology and bioinformatics analysis,in which LAT2,as downstream of the regulatory network,is involved in the regulation of chemoresistance of pancreatic cancer.ObjectiveTo detect the expression of LAT2 in pancreatic cancer tissues and evaluate its clinical significance.To clarify the function of LAT2 on biological behaviors of pancreatic cancer including cell proliferation,apoptosis and chemosensitivity,as well as the regulatory effects and potential mechanisms of glutamine metabolism and glycolysis.To explore whether LAT2 can induce chemoresistance of pancreatic cancer by regulating glutamine metabolism and glycolysis.To verify whether the downstream pathway of LAT2 can be an effective therapeutic target for pancreatic cancer.MethodsThe LAT2 and Lactic dehydrogenase B(LDHB)expression levels in pancreatic cancer tissues were detected by immunohistochemical technique and their prognostic value was assessed.Changes in metabolic phenotype,glycolysis,chemosensitivity,proliferation and apoptosis were detected by XF energy metabolism analyzer,flow cytometry and CCK8 assay in pancreatic cancer cell lines with LAT2 knockdown or overexpression.Then,the molecular mechanism of LAT2 regulating glycolysis,glutamine metabolism and chemoresistance of pancreatic cancer was verified by immunoprecipitation,immunofluorescence,and glutamine deprivation assay.Finally,the role and mechanism of LAT2 in inducing chemoresistance of pancreatic cancer by regulating glycolysis and glutamine metabolism were clarified in vivo by establishing subcutaneous xenograft tumor models in nude mice.Based on the Pdxl-Cre;LSL-KrasG12D;Trp53flox/+(KPC)mice,we explored the therapeutic effect of targeting LAT2 downstream signaling pathway in combination with gemcitebine and PD-LI monoclonal antibody on pancreatic cancer in KPC mice.The proportion of immune cells in peripheral blood mononuclear cells and tumor tissue were detected by flow cytometry technology,and the expression levels of Ki67,PD-L1,LDHB and PKM2 in pancreatic cancer tissue were detected by immunohistochemical technique.ResultsThe expression level of LAT2 in the pancreatic cancer tissues was higher than that in the paracancerous tissues,and a high level of LAT2 was an independent risk factor for poor prognosis in pancreatic cancer.We demonstrated that LAT2 emerged as an oncogenic protein and could decrease the gemcitabine sensitivity of pancreatic cancer cells in vitro and in vivo.Furthermore,we found that LAT2 could promote proliferation,inhibit apoptosis,activate glycolysis and alter glutamine metabolism.LAT2 could inhibit apoptosis of pancreatic cancer cells and activates glycolysis to induce chemoresistance by activating mTOR signaling pathway.Mechanistically,we demonstrated that LAT2 could regulate a positive feedback loop(the glutamine/p-mTOR/glutamine synthetase loop)to upregulate LDHB and promote glycolysis,decreasing gemcitabine sensitivity in pancreatic cancer.Meanwhile,we clarified the inhibition of apoptosis and increased glycolysis by LAT2-mTOR-LDHB pathway were dependent on glutamine.The results of pancreatic cancer tissue microarray suggested that the expression level of LDHB in the pancreatic cancer tissues was higher than that in the paracancerous tissues,and the overall survival time of patients with high level of both LAT2 and LDHB was significantly shortened.Finally,we confirmed that the mTOR inhibitor in combination with PD-L1 monoclonal antibody and gemcitabine showed an obvious anti-tumor effect on pancreatic cancer in KPC mice.The proportion of CD68 positive cells in peripheral blood mononuclear cells of KPC mice in combination group was higher than other groups;The proportion of CD8 positive cells and Ki67,PD-L1,LDHB and PKM2 expression levels in pancreatic cancer tissues were significantly lower than other groups.ConclusionTaken together,our data reveal that LAT2 functions as an oncogenic protein and could regulate glutamine-dependent mTOR activation to promote glycolysis and decrease chemosensitivity in pancreatic cancer.The LAT2-mTOR-LDHB pathway might be a promising prognostic biomarker and therapeutic target in pancreatic cancer.