Abstract B08: Rheb regulation of p27KIP promotes metabolic stress-induced autophagy in colon cancer cells

Abstract

Abstract Rheb is a small GTPase that couples growth factor signaling to activation of the mammalian Target of Rapamycin Complex 1 (mTORC1), thereby regulating key processes of cellular metabolism. The activity of Rheb/mTORC1 is controlled by the TSC2 tumor suppressor, whose deficiency causes pathology characterized by tumors in many organs. TSC2 functions as a complex with the TSC1 tumor suppressor, and inhibits Rheb by acting as a GTPase-activating protein (GAP). We recently reported that activation of Rheb induces mTORC1-independent cytoplasmic localization of the cell-cycle inhibitor p27KIP (p27) in TSC2-null and colon cancer cells (Lacher et al. Oncogene 2010). However, the relevance of this function of Rheb in colon cancer cells is unknown. Since cytoplasmic p27 is associated with poor prognosis in cancer, the aim of this work was to address the relevance of this function of Rheb independently of mTORC1 in colon cancer cells. Cytoplasmic p27 has been associated with autophagy and survival of cells under metabolic stress conditions. Thus, we hypothesized that Rheb, through regulation of p27, enhances survival of colon cancer cells under stress conditions. We analyzed Rheb function in colo320HSR and SW620 colon cancer cells, in which we previously demonstrated that Rheb regulates p27 function. By using two specific Rheb siRNAs, we found that depletion of Rheb decreased survival of colon cancer cells under serum starvation. We then measured whether Rheb affects autophagy under these conditions. Autophagy was analyzed by detecting modification of the autophagy marker protein LC3 by western blot and immunofluorescence. We found that the effect of Rheb on the survival of colon cancer cells was correlated with Rheb induction of autophagy. This result was in principle surprising as Rheb regulation of mTORC1 is expected to inhibit autophagy. However, our results are consistent with a recent report showing that Rheb induces mitochondrial autophagy through an unclear mTORC1-independent mechanism. Since Rheb regulates p27 in colon cancer cells, we analyzed whether this regulation was associated with the biological effects of Rheb in these cells under serum starvation conditions. We demonstrated that shRNA-mediated depletion of p27 decreased survival of colon cancer cells, and reduced the levels of autophagy in response to serum deprivation. In addition, we found that Rheb regulation of autophagy depends on the expression of p27. Altogether, our results suggest that Rheb regulation of p27 is important for autophagy and survival of cancer cells under stress conditions. In light of the use of mTORC1 inhibitors for the treatment of cancer, our work highlights biological consequences of mTORC1-independent responses driven by Rheb in cancer cells, which could help to identify mechanisms of tumor cells resistance to mTORC1 inhibitors. Funded by DIUC 210.037.011-1.0 and FONDECYT 1120923 grants to AFC (arcastro@udec.cl). TC is funded by a PhD fellowship from CONICYT (Chile). Citation Format: Tania Campos, Javiera Ziehe, Ariel Fernando Castro. Rheb regulation of p27KIP promotes metabolic stress-induced autophagy in colon cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B08.