Abstract 1068: Downregulation of PH domain leucine-rich repeat protein phosphatases (PHLPPs) identifies a subgroup of human hepatocellular and colorectal carcinomas with elevated levels of activated AKT and low apoptosis rate

Abstract

Abstract Dysregulation of the phosphoinositide-3-kinase (PI3K)/AKT axis has been implicated in human hepatic and colorectal carcinogenesis. Although point mutations of the p110 alpha subunit of PI3K (PIK3CA), the upstream inducer of AKT, have been previously detected in these tumor types, the molecular mechanisms responsible for unrestrained activity of AKT remain largely unknown. Since it has been recently shown that PH domain leucine-rich repeat protein phosphatase 1 and 2 (PHLPP1 and PHLPP2) proteins are able to directly dephosphorylate AKT in vivo, we assessed the expression pattern of both PHLPP isoforms in a collection of human liver and colorectal cancer tissues and corresponding non-neoplastic surrounding counterparts using western blot, reverse-transcription real-time PCR, and immunohistochemical analyses. In liver specimens, we found that the expression of PHLPP1 and PHLPP2 was significantly decreased in a subset of human hepatocellular carcinomas (HCCP) (22/40, 55%; 15/40, 37.5%, respectively) characterized by a clinically aggressive phenotype (patient's survival shorter than 3 years following partial liver resection), whereas strong expression of the two proteins was detected in non-neoplastic surrounding liver tissues. Downregulation of PHLPP1 (36/50, 72%) and PHLPP2 (38/50, 76%) was frequent in colorectal tumors, whereas a remarkable upregulation of PHLPP1 and PHLPP2 occurred in the respective, adjacent normal colonic mucosa. In both liver and colorectal tumors, PHLPP1 and PHLPP2 levels inversely correlated with those of activated AKT and directly with the apoptosis rate. In human Hep3B and PLC HCC cell lines, displaying low PHLPP1 and PHLPP2 levels, overexpression of either PHLPP1 or PHLPP2 gene decreased the rate of cell proliferation and induced apoptosis. These effects were paralleled by decrease in AKT activation, upregulation of BAD, BAX, FOXO1, and active caspase 9 pro-apoptotic proteins, and downregulation of XIAP and Bcl-2 anti-apoptotic proteins. Conversely, silencing of either PHLPP1 or PHLPP2 gene by siRNA in the WRL-68 hepatoblast cell line (displaying high levels of PHLPP1 and PHLPP2) resulted in increased cell proliferation, reduced apoptosis, downregulation of BAD, BAX, FOXO1, and active caspase 9, and upregulation of XIAP and Bcl-2 proteins. In the same cell line, overexpression of a constitutively active form of AKT induced downregulation of PHLPP1 and PHLPP2, implying a negative modulation by AKT over PHLPP1 and PHLPP2 expression. Altogether, our results suggest that PHLPP1 and PHLPP2 phosphatases play a tumor suppressive role both in human hepatic and colorectal carcinogenesis via their ability to suppress AKT activation and to induce apoptosis 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 1068. doi:10.1158/1538-7445.AM2011-1068