PHLPP Is a Negative Regulator of RAF1, Which Reduces Colorectal Cancer Cell Motility and Prevents Tumor Progression in Mice

Abstract

Hyperactivation of the RAS-RAF signaling pathway in colorectal tumors is associated with metastasis and poor outcomes of patients. Little is known about how RAS-RAF signaling is turned off once activated. We investigated how the pH domain and leucine-rich repeat protein phosphatases (PHLPPs) control RAS-RAF signaling and colorectal cancer (CRC) development. We used co-immunoprecipitation assays to identify substrates of PHLPP1 and PHLPP2. We studied phosphorylation of RAF1 in CRC cells that express exogenous PHLPP1 or PHLPP2, or lentiviral-based small hairpin RNAs against their transcripts; we measured effects on cell motility, migration, and invasion invitro. Tumor progression and survival were analyzed in Phlpp1(-/-) Apc(Min) and Apc(Min)/Phlpp1(-/-) mice. Microarray datasets of colorectal tumor and nontumor tissues were analyzed for PHLPP gene expression. PHLPP1 and 2 were found to dephosphorylate RAF1 at S338, inhibiting its kinase activity invitro and in CRC cells. In cells, knockdown of PHLPP1 or PHLPP2 increased the amplitude and duration of RAF-MEK-ERK signaling downstream of epidermal growth factor receptor and KRAS, whereas overexpression had the opposite effect. In addition, knockdown of PHLPP1 or PHLPP2 caused CRC cells to express markers of the epithelial-mesenchymal transition, and increased cell migration and invasion. Apc(Min)/Phlpp1(-/-) mice had decreased survival and developed larger intestinal and colon tumors compared to Apc(Min) mice. Whereas Apc(Min) mice developed mostly low-grade adenomas, 20% of the tumors that developed in Apc(Min)/Phlpp1(-/-) mice were invasive adenocarcinomas. Normal villi and adenomas of Apc(Min)/Phlpp1(-/-) mice had significantly fewer apoptotic cells than Apc(Min) mice. Human CRC patient microarray data revealed that the expression of PHLPP1 or PHLPP2 is positively correlated with CDH1. PHLPP1 and PHLPP2 dephosphorylate RAF1 to reduce its signaling, increase the invasive and migratory activities of CRC cells, and activate the epithelial-mesenchymal transition. In Apc(Min) mice, loss of PHLPP1 promotes tumor progression.