Role of an unusual GTPase in DNA damage response and carcinogenesis

描述（由申请人提供）：我们最近鉴定了一种新的p53促存活信号传导途径，其涉及p53靶基因或DNA损伤检查点基因的上调，其可以影响p53依赖性DNA损伤应答中细胞死亡与停滞/衰老之间的平衡。我们已经确定RhoE基因作为p53和DNA损伤的新的转录靶点。我们发现，DNA损伤触发肌动蛋白解聚，通过p53依赖性RhoE激活导致应力纤维解体。RhoE是小GTP酶超家族的新成员，与RhoA、B和C具有最强的同源性。尽管有这种高度的同源性，RhoE有几个不寻常的特性，使其区别于其他Rho蛋白。与其他Rho蛋白不同，RhoE似乎是Rho家族中唯一对DNA损伤有转录响应的蛋白，DNA损伤可以诱导肌动蛋白应力纤维的损失。我们假设RhoE蛋白通过抑制促凋亡蛋白的活性同时增强促存活蛋白对暴露的DNA损伤的响应的功能，对于具有wt-p53的癌细胞的存活是必需的。 这个建议的主要目标是了解一个不寻常的作用的小GTdR，RhoE，作为一种新的靶基因的p53依赖性DNA损伤和翻译我们的基本理解的细胞生存功能的RhoE到人类癌细胞，包括乳腺癌细胞的凋亡。虽然RhoE在DNA损伤反应途径中的生物学作用尚未确定，但我们的初步数据表明，RhoE基因通过p53依赖性DNA损伤的反式激活，其作为肌动蛋白细胞骨架的关键组织者的潜在作用，以及其在DNA损伤诱导的细胞存活中的特殊作用，都引起了我们的兴趣。越来越多的证据表明，通过Rho蛋白的异常信号传导与肿瘤发生和癌变的各个步骤密切相关。 在本申请中，生物化学和遗传学方法的组合将用于（i）描绘RhoE在p53依赖性细胞结果和DNA损伤应激反应中的功能;（ii）鉴定和表征RhoE相互作用伴侣，其将定义响应于遗传毒性应激影响RhoE介导的细胞存活和p53依赖性细胞死亡的分子途径; iii）确定ROCK 1是否是JNK介导的细胞死亡信号传导的必需上游调节因子;和（iv）使用小鼠敲除方法定义RhoE在DNA损伤检查点信号传导和细胞存活信号传导中的作用。

DESCRIPTION (provided by applicant): We have recently identified a novel p53 pro-survival signaling pathway involving up- regulation of p53 target genes or DNA damage checkpoint genes, which can influence the balance between cell death and arrest/senescence in a p53 dependent DNA damage response. We have identified the RhoE gene as a novel transcriptional target of p53 and DNA damage. We found that DNA damage triggers actin depolymerization, resulting in stress-fiber disassembly through p53-dependent RhoE activation. RhoE is a recent addition of the small GTPases superfamily and has the strongest homology to RhoA, B, and C. Despite this high homology, RhoE has several unusual properties which distinguish it from the other Rho proteins. Unlike other Rho proteins, RhoE seems to be only Rho family protein transcriptionally responsive to DNA damage that can induce loss of actin stress fibers. We hypothesize that RhoE protein are essential for survival of cancer cells with wt- p53 by suppressing the activity of proapoptotic proteins while enhancing the function of prosurvival proteins in response to the exposure of DNA damage. The main goal of this proposal is to understand the unusual roles of a small GTPase, RhoE, as a novel target gene of p53-dependent DNA damage and to translate our basic understanding of the cell survival function of RhoE into apoptosis for human cancer cells including breast cancer cells. Although the biological role of RhoE in DNA damage response pathway has not yet been defined, our preliminary data demonstrating transactivation of the RhoE gene by p53-dependent DNA damage, its potential role as a key organizer of the actin cytoskeleton, and its exceptional role in DNA damage-induced cell survival are intriguing to us. Accumulating evidence suggest that an aberrant signaling through Rho proteins is closely associated with various steps of tumorigenesis and carcinogenesis. In this application, a combination of biochemical and genetic approaches will be used to (i) delineate the functions of RhoE in p53 dependent cellular outcomes and DNA damage stress response; (ii) Identify and characterize RhoE Interacting partners, which will define a molecular pathway that affects RhoE-mediated cell survival and p53-dependent cell death in response to genotoxic stress; iii) determine if ROCK 1 is an essential upstream regulator for JNK-mediated cell death signaling; and (iv) define the role of RhoE in DNA damage checkpoint signaling and cell survival signaling using in mouse knockout approach.