The Atypical Tumor Suppressor P27 Regulates Cellular Proliferation, Invasion, And Metastasis Via Subcellular Localization In Distinct Microenvironments

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL While mutational loss of the p27 cell cycle regulatory protein is rare, oncogenic deregulation of p27 via accelerated degradation or mislocalization to the cytoplasm is commonly observed in human tumors. A growing body of evidence indicates that, when mislocalized to the cytoplasm, p27 promotes cell motility via RhoA-ROCK signaling inhibition. The present data explores the role of p27 in the regulation of both orthotopic mammary tumor growth and ectopic pulmonary metastasis in the MDA-MB-231-4175 model of malignant human breast cancer. The MDA-MB-231-4175 cell line (hereafter 4175) shows much greater metastasis to the lungs in murine xenograft models. Relative to the parental MDA-MB-231 cells, the metastatic 4175 derivative demonstrates activation of PI3K/mTOR signaling. These cells have elevated pmTOR, pAKTS473, pSGK1, and pRSK1. This results in increased total p27 and, when equal levels of total p27 are titrated, the highly metastatic 4175 cell line demonstrates extensive phosphorylation of p27 at the T157 and T198 sites. These sites have previously been implicated in the modulation of p27 localization and in the binding of p27 to RhoA. Thus, in the 4175 line, we demonstrate increased cytoplasmic p27, increased p27:RhoA binding, and impaired RhoA-ROCK signaling. This attenuation of RhoA activity correlates with increased migration (as measured by scratch assay) and increased invasion (as measured by modified transwell assay). Importantly, this hypermotility was abrogated by lentiviral knockdown of p27. Knockdown of p27 did not alter in vitro cell cycle profiles of either cell line as assessed by flow cytometry. The enhanced pulmonary tropism of the 4175 cell line was also abrogated in vivo following p27 knockdown: shRNAp27 reverts the lung metastatic phenotype of these cells back toward parental levels following tail vein injection in Balb/c nude female mice as assessed by IVIS imaging. Interestingly, orthotopic injection of these same lines, treated with the same lentivirus, into the mammary fat pad of Balb/c nude female mice produced a dramatically different result: shRNAp27 resulted in a significant increase in parental MDA-MB-231 tumor growth, while the metastatic derivative 4175 cells did not demonstrate a statistically meaningful change in orthotopic growth. These results indicate that, within the same cellular background, and in the same murine species, loss of p27 results in substantially different phenotypes depending upon the microenvironmental context: after tail vein injection cytoplasmic p27 plays a crucial role in promoting extravasation and colonization in the lung tissue; loss of p27 in this context impairs the metastatic process. Conversely, in the orthotopic environment, p27's pro-invasive role is not necessary to establish productive microenvironmental interactions and loss of p27 does not attenuate tumor growth. 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 2333. doi:10.1158/1538-7445.AM2011-2333