Regulation of p21waf1/cip1 by Rho and Ras signaling

描述(由申请人提供)： RAS GTP酶蛋白(K-RAS、H-RAS、N-RAS)因与致癌转化相关而首次被鉴定。激活致癌基因RAS突变在多种肿瘤类型中被发现，在人类癌症中的总发生率约为30%。尽管RAS具有很强的转化能力，但额外的信号通路影响RAS诱导的肿瘤发生的效率。参与RAS致癌转化的一个信号蛋白是Rho GTP酶。抑制Rho显著削弱RAS驱动的转化，而活性Rho与RAS或RAS效应蛋白Raf协同促进转化。在描述Rho和Ras协同作用的基础上，我们发现Rho抑制了细胞周期蛋白依赖性激酶抑制因子p21Waf1/Cip1(P21)的RAS诱导，从而促进了细胞周期的进展。Rho抑制p21的一个机制是通过抑制RAS诱导的转录激活。虽然急性高强度RAS信号导致p21转录上调导致细胞周期停滞，但经RAS稳定转化的细胞具有高水平的活性Rho，从而抑制p21转录，从而允许细胞周期进展，尽管由于Rho介导的转录抑制，转化细胞的p21蛋白水平显著高于未转化的亲本细胞，这表明转录后机制是蛋白质水平上升的原因。这些发现提出了几个关键问题。1)RAS转化细胞中p21蛋白水平升高的转录后机制是什么？2)除了转录抑制外，Rho活性是否还通过转录后途径抑制p21水平？3)Rho下游导致p21转录抑制的途径是什么？为了回答这些问题，我们将使用细胞生物学和体外生化方法。与p16细胞周期蛋白依赖的激酶抑制物不同，p21在RAS突变的肿瘤中经常被缺失，这表明抑制Rho功能以提高p21水平并潜在地阻止细胞周期进展将是一种有效的抗癌治疗策略。

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.