141 PI3K REGULATES P27 THROUGH MTORC2 IN RENAL CELL CARCINOMA

Abstract

INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most lethal of all urologic malignancies. Moreover, its incidence is rising and it is among the ten most common malignancies in both men and women. Dysregulation of the cell cycle regulator p27, particularly reduced nuclear expression has been shown to inversely correlate with RCC tumor stage and grade. The PI3K/AKT signaling axis has been implicated in the molecular pathogenesis of RCC and has been previously shown to regulate p27 through a variety of mechanisms. For example, PI3K signaling may regulate AKT-mediated phosphorylation of p27 which can impact p27 localization and steadystate levels. In addition PI3K/ AKT signaling may regulate levels of SKP2, a component of the SCFSKP2 ubiquitin ligase complex that mediates p27 proteolysis. Recent evidence suggests that the mTOR signaling complex 2 (mTORC2), in conjunction with PI3K signaling, regulates AKT activation. Based on these data, we investigated the role of mTORC2 in p27 regulation in the context of RCC. METHODS: Both pharmacologic and genetic techniques were utilized to determine the effects of mTORC2 signaling on p27. Effects on p27 steady state levels were analyzed through the combined use of real time RT-PCR as well as immunoblotting. Effects on localization were determined through cell fractionation studies in addition to immunofluorescence. The impact of mTORC2 on cell proliferation was determined through cell cycle analysis in addition to thymidine incorporation assays. RESULTS: Similar to inhibition of PI3K signaling, inhibition of mTORC2, either via genetic or pharmacologic means, led to an increase in both total as well as nuclear p27 protein levels. mTORC2 signaling, through AKT activation, mediates p27 levels in part through the regulation of SKP2. Finally, mTORC2 blockade led to inhibition of cell cycle progression with concomitant reduced cellular proliferation in RCC cells. CONCLUSIONS: Our data demonstrate a role for mTORC2 in the regulation of p27 levels as well as localization in RCC cells. Furthermore, our data demonstrate that these effects mediate the proliferation of RCC cells. Given the established role of p27 as a prognostic biomarker for RCC, our data suggest a role for targeting mTORC2 in the treatment of kidney cancer.