Regulation of p21waf1/cip1 by Rho and Ras signaling

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DESCRIPTION (provided by applicant): Ras GTPase proteins (K-Ras, H-Ras, N-Ras) were first identified because of their association with oncogenic transformation. Activating oncogenic Ras mutations have been found in a variety of tumor types with an overall incidence of approximately 30% in human cancers. Despite the robust transforming ability of oncogenic Ras, additional signaling pathways influence the efficiency of Ras-induced oncogenesis. One signaling protein that contributes to oncogenic transformation by Ras is the Rho GTPase. Inhibition of Rho significantly impairs Ras-driven transformation, while active Rho synergistically co-operates with Ras or with the Ras effector protein Raf to promote transformation. In characterizing the basis of Rho and Ras cooperation, we found that Rho suppresses Ras-induction of the cyclin dependent kinase inhibitor p21Waf1/Cip1 (p21), thereby facilitating cell cycle progression. One mechanism of p21 suppression by Rho is through repression of Ras-induced transcriptional activation. While acute high intensity Ras signaling leads to cell cycle arrest due to the transcriptional up-regulation of p21, cells stably transformed by Ras have high levels of active Rho which suppresses p21 transcription thereby permitting cell cycle progression, p21 protein levels are significantly higher in Ras-transformed cells than in untransformed parental cells, despite comparable levels of p21 mRNA due to Rho-mediated transcriptional suppression, suggesting that posttranscriptional mechanisms are responsible for the elevation in protein levels. These findings raise several key questions. 1) What is the post-transcriptional mechanism that raises p21 protein levels in Ras transformed cells? 2) Does Rho activity squelch p21 levels by post-transcriptional means in addition to transcriptional suppression? 3) What is the pathway downstream of Rho that leads to p21 transcriptional suppression? To answer these questions, we will use cell biological and in vitro biochemical methods. Unlike the p16 cyclin dependent kinase inhibitor which is frequently deleted in tumors with Ras mutations, p21 has not been reported to be lost, suggesting that inhibiting Rho function to elevate p21 levels and arrest cell cycle progression potentially would be an effective anti-cancer therapeutic strategy.