Combined inhibition of autophagy with mTOR inhibitor to enhance cell death in renal cell carcinoma.

Abstract

450 Background: mTOR (mammalian target of rapamycin) and autophagy are increasingly recognized as being a central cellular and pathological process for numerous human diseases, including renal cell carcinoma (RCC). Depending on the cellular context, autophagy may promote cancer cell survival or cell death. However, little is known about the mechanisms of regulating mTOR activity and autophagic function in RCC. We hypothesize that autophagy promotes cell survival via mTOR mediated-phosphatidylinositol 3-kinase (PI3K)/AKT pathway and is regulated by the von Hippel-Lindau (VHL) tumor suppressor. Methods: RCC cells were stably lentiviral transduced with expression of VHL or mCherry-EGFP tandemfluorescent-tagged LC3B for studying autophagic flux. Cell viability was evaluated by cytotoxic XTT and clonogenic assays and flow cytometry. The efficacy of PI3K/AKT/mTOR pathway inhibition by RAD001, PI-103, MK2206, AZD8055, and/or lysosomotropic inhibitors were evaluated by immunoblots and immunofluorescence for autophagy process of autophagosome and lysosome. Results: We show that mTOR is hyperactive in VHL-deficient cells compared to cells with wild-type VHL or VHL-expressing cells. AZD8055-induced toxicity occurs in a VHL-independent manner via cell cycle arrest and clonogenic senescent cell death, but results in significantly increased expression of autophagic marker LC3-II and the formation of autophagic vacuoles. Pharmacologic inhibition or siRNA silencing of autophagy pathway components promotes AZD8055-induced cell death in VHL-deficient cells. Interestingly, defective autophagy marked by the presence of sustained p62 expression in VHL-deficient cells appears to contribute to cell survival via mTOR signaling, which in turn influences autophagosome-lysosome fusion, and thus controls autophagic flux by acting at the termination stage of the process. Conclusions: These results support mTOR and autophagy pathways as potential targets of anticancer drugs and reveal VHL in control of the autophagic program in RCC. Further, this work suggests that combined inhibition of autophagy along with mTOR inhibitors could be a novel therapeutic strategy for the treatment of RCC.