We previously found that the small GTPase Rheb regulates the cell-cycle inhibitor p27KIP1 (p27) in colon cancer cells by a mTORC1-independent mechanism. However, the biological function of the Rheb/p27 axis in cancer cells remains unknown. Here, we show that siRNA-mediated depletion of Rheb decreases survival of human colon cancer cells under serum deprivation. As autophagy can support cell survival, we analyzed the effect of Rheb on this process by detecting the modification of the autophagy marker protein LC3 by western blot and imunofluorescence. We found that Rheb promotes autophagy in several human cancer cell lines under serum deprivation. Accordingly, blocking autophagy inhibited the pro-survival effect of Rheb in colon cancer cells. We then analyzed whether p27 was involved in the biological effect of Rheb. Depletion of p27 inhibited colon cancer cell survival, and Rheb induction of autophagy. These results suggest that p27 has an essential role in the effect of Rheb in response to serum deprivation. In addition, we demonstrated that the role of p27 in autophagy stands on the N-terminal portion of the protein, where the CDK-inhibitory domain is located. Our results indicate that a Rheb/p27 axis accounts for the activation of autophagy that supports cancer cell survival. Our work therefore highlights a biological function of Rheb and prompts the need for future studies to address whether the mTORC1-independent Rheb/p27 axis could contribute to tumorigenesis and/or resistance to mTOR inhibitors.

Tuberous sclerosis complex (TSC) disease results from inactivation of the TSC1 or TSC2 gene, and is characterized by benign tumors in several organs. Because TSC tumorigenesis correlates with hyperactivation of mTORC1, current therapies focus on mTORC1 inhibition with rapamycin or its analogs. Rapamycin-induced tumor shrinkage has been reported, but tumor recurrence occurs on withdrawal from rapamycin. Autophagy has been associated with development of TSC tumors and with tumor cell survival during rapamycin treatment. mTORC1 and AMPK directly inhibit and activate autophagy, respectively. AMPK is hyperactivated in TSC cells and tumors, and drives cytoplasmic sequestration of the cell-cycle inhibitor p27KIP (p27). Whether AMPK and p27 are involved in rapamycin-induced autophagy and survival of TSC cells remain unexplored. Here, we show that inhibition of AMPK by compound C or by shRNA-mediated depletion of LKB1 reduces activation of autophagy by rapamycin in Tsc2-null cells. Similarly, shRNA-mediated depletion of p27 inhibited rapamycin-induced autophagy. In support of p27 lying downstream of AMPK on the activation of autophagy in Tsc2-null cells, a p27 mutant that preferentially localizes in the cytosol recovered the effect of rapamycin on autophagy in both p27- and LKB1-depleted cells, but a nuclear p27 mutant was inactive. Finally, we show that p27-dependent activation of autophagy is involved in Tsc2-null cell survival under rapamycin treatment. These results indicate that an AMPK/p27 axis is promoting a survival mechanism that could explain in part the relapse of TSC tumors treated with rapamycin, exposing new avenues for designing more efficient treatments for TSC patients.

A fish meal supply shortage is limiting aquaculture development. Currently, plant‐based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti‐inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract−1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL−1, the P. radiata extract showed no cytotoxicity in the CHSE‐214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL−1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK‐1 salmon cell line challenged with an oxidant stimulus (H2O2), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti‐inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) and of the inducible cyclooxygenase‐2 (COX‐2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.

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.