TFEB Mediates Immune Evasion And Resistance To MTOR Inhibition Of Renal Cell Carcinoma Via Induction Of PD-L1

Purpose:Numerous genes related to mTOR pathway are associated with RCC occurrence and development,including VHL,PTEN,TSC1,TSC2,FLCN and mTORC1.Despite the FDA approval of mTOR inhibitors including Everolimus and Temsirolimus as second-line treatments for poor-prognosis RCC,the benefits are relatively modest and the responders usually develop resistance.mTOR can directly phosphorylate TFEB at two residues,Ser142 and Ser211 to prevent nuclear translocation of TFEB.Like mTOR,TFEB has recently been shown to be functionally associated with tumor development,autophagy,lysosomal biosynthesis and cell metabolism.As an important co-inhibitory ligand,PD-L1 can inhibit cytotoxic function of TILs and mediate immune escape of tumor cells.In this thesis,we are intended to investigate the roles of TFEB and PD-L1on RCC immune escape and the underlying molecular mechanism of mTOR inhibitors resistance.Methods:(1)786-O cells were transduced with a scramble sh RNA(con sh RNA)or TFEB sh RNA(sh TFEB-1,sh TFEB-2)lentiviral particles.769-P cells were transfected with either an empty vector(EV)or TFEB-S211A(TFEB)pc DNA3.1 plasmids.The effects of TFEB on RCC proliferation,apoptosis and migration were evaluated using CCK8,Ed U,Annexin V-PI or Transwell assay in vitro.(2)To study the potential function of TFEB in RCC,we generated a mouse-derived Renca cell line that overexpressed TFEB-S211A mutant.(1)We hypodermically inoculated WT and TFEB-S211A Renca cells to BALB/c nude mice,measured tumor volume daily,determined the percentages of Annexin V~+as well as Ki-67~+tumor cells using flow cytometry.(2)We repeated the same experiment in BALB/c wild type mice.Tumor volume were measured daily,TFEB and PD-L1 protein of tumor cells within Renca tumors were determined by immunohischemstry.CTLs function,NK,TAMs and MDSCs numbers were detected by flow cytometry.(3)To explore the correlation between TFEB and PD-L1 in RCC,expression and association of TFEB and PD-L1 were obtained using immunoblotting analysis in RCC cells lines.We compared immune reactive scores(IRS)of TFEB in PD-L1~-and PD-L1~+areas in human RCC tissues and explored possible link between PD-L1 expression and tumor progeression(grades,stages).(4)To investigate the regulatory mechanism of TFEB on PD-L1 in RCCs,we downregulated or overexpressed TFEB,detected the PD-L1 m RNA and protein level change by q PCR and flow cytometry.The binding of TFEB to the PD-L1 promoter was detected by CHIP-PCR and luciferase reporter assay.(5)To study the effect of mTOR inhibitors on RCC,786-O and 769-P cells were stimulated with rapamycin and Torin-1.The levels of TFEB and PD-L1 were determined by western blot or flow cytometry.(6)To elucidate the mechanism of mTOR inhibitors upregulating PD-L1 in RCC,we stimulated 786-O and 769-P cells with Torin-1,detected the binding of TFEB to the PD-L1 promoter using CHIP-PCR and luciferase reporter assay.WT and TFEB knockout 786-O cells were subjected to mTOR inhibitors,TFEB and PD-L1 protein levels changes were measured by immunoblotting.(7)To explore the effect of mTOR inhibitors on human renal cancer patients,we isolated primary renal cancer cells from freshly surgically removed tumor tissues and identified cell purity using immunofluorescence staining of CAIX.The changes of TFEB and PD-L1 protien were detected by western blot,flow cytometry and immunofluorescence after mTOR inhibitors stimulation.(8)To determine whether combination of antibodies against PD-L1 could potentiate the efficacy of Temsirolimus on cc RCC growth,mice were xenografted with tumor cells and were injected Temsirolimus alone,anti-PD-L1 alone or concomitant use of Temsirolimus and anti-PD-L1 intraperitoneally.Tumor volumes were measured daily,Ki-67~+tumor cells were determined by flow cytometry and immunohistochemistry.TFEB and PD-L1 expression were determined by western blot and immune-histochemistry staining.CTLs function were detected by flow cytometry.Results:(1)Knockdown of TFEB in 786-O cells or enhanced TFEB expression in 769-P cells did not affect cell proliferation,apoptosis and migratory potential.(2)(1)There was no significant difference in tumor growth between WT and TFEB-S211A Renca tumors in BALB/c nude mice.The percentages of Annexin V~+and Ki-67~+tumor cells in TFEB-S211A groups isolated from nude mice were comparable to the control group.(2)WT BALB/c mice that received Renca cells overexpressing TFEB-S211A showed significantly greater tumor burdens,enhanced expression of TFEB and PD-L1,reduced frequencies of CD107a,GZMB,IL-2 and TNF-?-producing CD8~+cytotoxic cells within tumor tissues compared to control group.The percentages of NK,TAMs and MDSCs were comparable between two groups.(3)Expression of TFEB was positively correlated with the levels of PD-L1 in RCC cell lines.Within individual RCC tumors,the PD-L1~+areas had higher expression and enhanced nuclear localizations of TFEB.Patients with PD-L1 high expression had higher tumor grades and more advanced tumor stages.(4)Knockdown of TFEB led to reduced PD-L1 m RNA and protein expression in 786-O cells,which was accompanied with reduced TFEB binding to the PD-L1 promoter.Overexpression of TFEB in the 769-P cells significantly enhanced TFEB binding to the PD-L1 promoter and PD-L1 expression.(5)mTOR inhibition by rapamycin and Torin-1 significantly induced TFEB and PD-L1expression in 786-O cells and 769-P cells.(6)Torin-1 treatment significantly enhanced TFEB binding to the PD-L1 promoter in both 786-O and 769-P cells compared with control groups.Knockdown of TFEB abolished the enhanced PD-L1 expression upon inhibition of mTOR.(7)mTOR inhibition by rapamycin and Torin-1 led to nuclear location of TFEB in a time-dependent manner and enhanced PD-L1 expression in primary RCC cells.(8)Temsirolimus treatment upregulated TFEB and PD-L1 expression in tumor cells isolated from tumor tissues in a xenograft model.Combination of Temsirolimus and anti-PD-L1 therapy significantly decerased the percentages of Ki-67~+tumor cells,enhanced CD107a and GZMB expression in CTLs and resulted in a significant reduction in tumor size.Conclusion:TFEB expression was positively correlated with the levels of PD-L1 in RCC cell lines and human RCC tissues.The tumor-promoting effect of TFEB is dependent on the presence of tumor infiltrating T cells rather than intrinsic effects on tumor cells.Inhibition of mTOR in RCC enhances TFEB nuclear localization and expression that drives PD-L1 expression,subsequently inhibits CTLs function and induces immune evasion and resistance to mTOR inhibitors.Simultaneously targeting mTOR and PD-L1 enhanced the treatment efficacy in a mouse RCC xenograft model.