Abstract 5137: The PI3K pathway regulates the actin cytoskeleton, tumor cell migration and metastasis via the atypical tumor suppressor p27

Abstract

Abstract The CDK inhibitor p27 plays a well-established role in cell cycle progression, regulating the G1-S phase transition. A growing body of evidence indicates a potential role for p27 as a mediator of tumor cell motility and invasiveness. The present data provides additional evidence for p27 as a mediator of cancer metastasis. These activities appear to require cytoplasmic mislocalization, and are independent of p27's action on CDKs and the cell cycle. p27 phosphorylation at T157 and T198 is mediated by the AGC kinases RSK, SGK, and AKT to promote cytoplasmic mislocalization and facilitate p27 binding to the RhoA cytoskeletal regulatory protein. p27-mediated inhibition of RhoA/ROCK signaling is associated with dynamic rearrangement of the actin cytoskeleton and increased cell migration and invasion. Overexpression of the PI3K effectors RSK, SGK, and mTOR all promote cytoplasmic mislocalization of p27, increased p27: RhoA binding, and all enhance directed cell migration as assayed by modified transwell assay. This increased tumor cell motility is dependent upon p27, as shRNA-mediated p27 knockdown reverts transwell invasion to parental levels. Overexpression of mTOR or these AGC kinases reduces cellular levels of RhoA-GTP and p-cofilin, both of which revert following shRNA p27 treatment. The increased p27: RhoA binding, decreased RhoA-GTP, and decreased p-cofilin correlate with dramatic loss of polymerized actin filaments as assayed by phalloidin staining, and all of these effects are rescued by shRNA p27. The in vivo relevance of these findings has been pursued in the highly metastatic human breast cancer sub-lines MDA-MB-231-1833 (bone-tropic) and MDA-MB-231-4175 (lung-tropic). Both highly metastatic variants demonstrate hyperactivation of the PI3K effectors SGK, RSK, and AKT relative to the parental MDA-MB-231 line and show markedly elevated p27 levels, with a shift in p27 protein localization to the cytoplasm, increased p27: RhoA binding, decreased p27-dependent levels of RhoA-GTP and p-cofilin, and increased p27-dependent invasiveness by modified transwell assay. The cell cycle profiles of MDA-MB-231-4175 and 1833 variants were unchanged by shRNA p27 supporting the notion that cytoskeletal effects of p27 in these lines are independent of cell cycle/Cdk regulatory activity. Of particular interest, the increase in lung tumor formation by MDA-MB-231-4175 following tail vein injection was strikingly reduced to near-parental levels after shRNA-mediated knockdown of p27. These results support a model whereby PI3K activation promotes p27 phosphorylation, mislocalization to the cytoplasm, enhanced RhoA binding and subsequent remodeling of the actin cytoskeleton. This cascade of events may be a major route whereby oncogenic PI3K activation mediates tumor cell acquisition of the invasive potential necessary for overt metastasis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5137.