Abstract 1238: The role of Ras-mediated SGK-1 Signaling in the Survival of ECM-detached mammary epithelial cells

Abstract

Abstract Cancer cells utilize numerous signaling pathways to inhibit anoikis (detachment-induced apoptosis) and alter their metabolism to enable their survival in abnormal environments. Additional research on cancer cells’ ability to survive outside their normal niche has the potential to reveal mechanisms that may be targeted through the development of novel and more effective chemotherapeutics. One critical pathway that requires further understanding and has caught our interest is the Ras signaling pathway in mammary epithelial cells, and more specifically, the surprising role for SGK-1 in mediating cell death. We have engineered a mammary epithelial cell line (MCF-10A) that expresses constitutively active Ras (Ras G12V). To examine the ability of the Ras pathway to permit cell survival outside of the normal environment, we use an in vitro cell culture system that allows epithelial cells to grow in suspension and mimics the conditions when cells are detached from the extracellular matrix (ECM). Using this approach, we have found that the expression of Ras G12V causes cells to maintain their ATP levels when detached from the ECM and results in elevated levels of phosphorylated Akt. To further address the significance of the enhanced activation of Akt, we treated ECM-detached Ras G12V cells with varying dilutions of LY 294002, a PI(3)K inhibitor. As expected, we found that ATP levels decreased with elevated concentrations of this inhibitor, but surprisingly, phoshorylated Akt levels remained consistently high. This suggests the presence of a PI(3)K dependent but Akt independent signaling pathway that maintains ATP levels in detached cells expressing Ras G12V. We subsequently investigated the role of SGK-1, another kinase downstream of PI(3)K, as a possible player in the maintenance of ATP by Ras in detached cells. We found that treating suspended Ras G12V cells with GSK 650394, an inhibitor of SGK-1, decreased ATP levels in detached cells suggesting that SGK-1 is necessary for the Ras-mediated maintenance of ATP levels. To further investigate this result, we are using lentiviral-delivery of shRNA in our Ras G12V cells to obtain a cell line with stable knockdown of SGK-1. To continue to unveil a role for SGK-1 in Ras signaling, we are also making a constitutively active SGK-1 cell line in MCF-10A cells to examine the effects of active SGK-1 in the absence of Ras signaling. In summary, these data demonstrate an interesting role for SGK-1 in Ras-mediated maintenance of ATP levels when mammary epithelial cells are detached from the ECM. Our future research will continue to examine this important pathway to provide a better understanding of how cancer cells use the Ras pathway to survive outside their niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1238. doi:10.1158/1538-7445.AM2011-1238