Role of an unusual GTPase in DNA damage response and carcinogenesis

描述(申请人提供)：我们最近发现了一个新的P53促生存信号通路，涉及P53靶基因或DNA损伤检查点基因的上调，它可以影响依赖于P53的DNA损伤反应中细胞死亡和停滞/衰老之间的平衡。我们发现RhoE基因是P53和DNA损伤的一个新的转录靶点。我们发现DNA损伤触发肌动蛋白解聚，通过P53依赖的RhoE激活导致应力纤维解体。RhoE是最近增加的一个小GTP酶超家族，与RhoA、B和C具有最强的同源性。尽管有如此高的同源性，RhoE仍具有一些不同于其他Rho蛋白的特殊性质。与其他Rho蛋白不同，RhoE似乎是唯一一种对DNA损伤有转录反应的Rho家族蛋白，DNA损伤会导致肌动蛋白应激纤维的丢失。我们推测，RhoE蛋白对于携带wt-p53的癌细胞的存活是必不可少的，因为它抑制了促凋亡蛋白的活性，同时增强了原存活蛋白的功能，以应对DNA损伤的暴露。 这个建议的主要目的是了解一个小的GTPase，RhoE，作为一个新的P53依赖的DNA损伤的靶基因的不同寻常的作用，并将我们对RhoE的细胞生存功能的基本理解转化为包括乳腺癌细胞在内的人类癌细胞的凋亡。虽然RhoE在DNA损伤反应通路中的生物学作用尚未确定，但我们的初步数据表明，RhoE基因在依赖于P53的DNA损伤中反式激活，它作为肌动蛋白细胞骨架的关键组织者的潜在作用，以及它在DNA损伤诱导的细胞生存中的特殊作用，对我们来说是有趣的。越来越多的证据表明，通过Rho蛋白的信号异常与肿瘤发生和癌变的各个步骤密切相关。 在这一应用中，将结合生物化学和遗传学方法来(I)描述RhoE在p53依赖的细胞结果和DNA损伤应激反应中的功能；(Ii)识别和表征RhoE相互作用的伙伴，这将定义影响RhoE介导的细胞存活和p53依赖的细胞在遗传毒性应激反应中死亡的分子通路；iii)确定ROCK 1是否是JNK介导的细胞死亡信号的重要上游调节因子；以及(Iv)确定RhoE在DNA损伤检查点信号和细胞存活信号中的作用。

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.