Abstract 4025: The retinoblastoma protein regulates cell adhesion: Implications for lung cancer progression

Abstract

The retinoblastoma (Rb) protein is a tumor suppressor commonly inactivated in human cancers. Our work has uncovered a role for Rb as a regulator of adherens junction assembly and cell-to-cell adhesion. This is a novel function since Rb is predominantly known as a cell cycle repressor. Our objective is to characterize the molecular mechanism by which Rb performs this function. We hypothesized that Rb controls the activity of known regulators of adherens junction assembly. Using qRT-PCR, immunoblots, and transcriptional assays with promoter-luciferase constructs, we found that Rb represses the expression of Pak1, which is a known effector of the small Rho GTPase Rac1. Notably, Rac1 is a well known regulator of adherens junction assembly whose increased activity in cancer is linked to tumor metastasis. We found that Pak1 repression by Rb is transcriptional and is E2F-dependent. Our chromatin immunoprecipitation assays showed that an Rb-E2F complex binds to an Rb-responsive element in the Pak1 promoter that is rich in E2F binding sites. This suggests that Pak1 is an E2F target and that Rb's repressive effect on Pak1 consists in blocking E2F activity. Further supporting a role for Rb in cell adhesion, microarray analyses comparing Rb-expressing with Rb-deficient cells showed that Rb transcriptionally regulates a variety of cell adhesion genes, including those coding for adherens junction cadherins. Pak1 also appeared among the transcript repressed by Rb, validating Rac1 and Pak1 as links between Rb and adherens junctions. Importantly, we have evidence suggesting that the de-regulation in cell adhesion-related gene expression due to Rb loss may be related to lung cancer development. We examined the Director's Challenge lung cancer database and found that numerous Rb-regulated cell adhesion genes correlate with overall survival in lung adenocarcinoma patients. For this analysis we arbitrarily defined an RB1 signature as all RB1-regulated genes with >2 activation or repression with statistically significant p-values. We found that a total of 1,154 RB1-regulated genes correlated with overall survival. This analysis suggests that a subset of RB1-regulated cell adhesion genes may be critical in the development of lung cancer. Three examples of RB1-activated cell adhesion genes that correlate with overall survival are E cadherin (Cdh 1), Integrin alpha 1 (Itga1) and Integrin alpha 10 (ItgA10). In summary, three main conclusions emerge from our studies. First, Rb transcriptionally controls expression of cadherins and other adherens junction-related genes. Second, Rb also controls adherens junction assembly by repressing Rac1 and its effector Pak1. Third, global de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated to lung cancer progression. 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 4025. doi:10.1158/1538-7445.AM2011-4025