p190 RhoGAP signaling in epithelial oncogenesis

摘要 最近对人类癌症中基因突变的分析意外地鉴定出编码p190 A的ARHGAP 35 RhoGAP（p190 A）作为高度突变的基因，即前30个泛癌症。突变尤其丰富， 腺癌，包括肾细胞癌。ARHGAP 35的突变谱与一个突变谱一致。 作为肿瘤抑制剂。此外，ARHGAP 35基因座位于基因组的一个区域，该区域经常 在癌症中消失了。然而，生物信息学数据未能确定基因的功能后果， 突变因此，本提案的范围是定义与p190 A缺失相关的致癌能力 表达和人类癌症中天然存在的ARHGAP 35突变。 我们最近获得的结果表明，p190 A和它的parasitic p190 B-统称为 p190在此介导上皮细胞中的接触增殖抑制（CIP）。此外，我们进行了一项 全基因组RNA-seq分析，并确定p190调节基因的表达调控的基因， 雅普癌蛋白。雅普是TEAD家族转录因子的转录共激活因子。雅普的活性 由Hippo途径控制，该途径广泛涉及CIP。 在目标1中，我们将定义p190影响Hippo信号传导和CIP的机制。为此，我们将（一） 测试Rho蛋白是否需要和/或足以促进p190下游的CIP;（ii）确定 p190通过经典和/或非经典Hippo途径发出信号;（iii）确定p190是否影响雅普- TEAD介导的基因表达;和（iv）确定在TEAD介导的基因表达中p190 A和p190 B之间是否存在明显的冗余。 上皮细胞是环境依赖性的。 在目的2中，我们将阐明p190在粘附连接（AJs）中的功能，它在以下方面发挥重要作用： 通过Hippo途径促进CIP。在这个目标中，我们将（i）测试p190激活海马的作用 （ii）确定E-cadherin是否需要p190来恢复Hippo （iii）确定p190在失巢凋亡中的作用，失巢凋亡是一种肿瘤抑制机制， 由AJs和Hippo通路调节;和（iv）阐明p190和p120- 连环蛋白是Hippo信号传导和CIP所必需的。 在Aim 3中，我们将与布罗德研究所的Gad Getz博士合作，分析人类肿瘤， ARHGAP 35突变，用于（i）杂合性缺失;（ii）雅普调节基因的表达;和（iii）共突变 和排他性数据。接下来，我们将确定自然发生的ARHGAP 35突变对p190 A的影响。 RhoGAP活性以及Hippo信号传导和CIP。最后，我们将在体外和体内测试， 外源性p190 A减弱了人癌细胞系的致癌能力， 内源性p190 A，并且如果这种作用依赖于GAP活性和/或Hippo信号传导。这些努力 对于阐明靶向Rho信号传导在未来个性化肿瘤治疗中的效用至关重要。

ABSTRACT Recent analyses of gene mutations in human cancer unexpectedly identified ARHGAP35 encoding p190A RhoGAP (p190A) as a highly mutated gene, i.e. top 30 pan-cancer. Mutations were particularly abundant in adenocarcinomas, including renal cell carcinoma. The mutation spectrum for ARHGAP35 is consistent with a role as tumor suppressor. Moreover the ARHGAP35 locus is located in a region of the genome that frequently is lost in cancer. However, bioinformatics data stop short of establishing the functional consequences of gene mutations. The scope of this proposal is therefore to define oncogenic capacities associated with loss of p190A expression and with naturally occurring ARHGAP35 mutations in human cancer. We have recently obtained results demonstrating that p190A and its paralog p190B - collectively termed p190 here - mediate contact inhibition of proliferation (CIP) in epithelial cells. We moreover conducted a genome wide RNA-seq analysis, and determined that p190 modulates expression of genes regulated by the YAP oncoprotein. YAP is a transcriptional co-activator of TEAD family transcription factors. The activity of YAP is controlled by the Hippo pathway, which is widely implicated in CIP. In Aim 1 we will define mechanisms whereby p190 impacts Hippo signaling and CIP. To this end, we will (i) test whether Rho proteins are required and/or sufficient to promote CIP downstream of p190; (ii) determine if p190 signals through the canonical and/or non-canonical Hippo pathways; (iii) establish if p190 impacts YAP- TEAD-mediated gene expression; and (iv) determine if apparent redundancy between p190A and p190B in epithelial cells is context-dependent. In Aim 2 we will elucidate the function of p190 at adherens junctions (AJs), which play essential roles in promoting CIP through the Hippo pathway. In this aim, we will (i) test a role for p190 to activate the Hippo pathway upon formation of nascent AJs; (ii) establish whether p190 is required for E-cadherin to restore Hippo signaling in carcinoma cells; (iii) define a role for p190 in anoikis, a tumor suppressor mechanism that is modulated by both AJs and the Hippo pathway; and (iv) elucidate if an interaction between p190 and p120- catenin is required for Hippo signaling and CIP. In Aim 3 we will in collaboration with Dr. Gad Getz of the Broad Institute, analyze human tumors with ARHGAP35 mutation for (i) loss-of-heterozygosity; (ii) expression of YAP regulated genes; and (iii) co-mutation and exclusivity data. Next, we will determine the effects of naturally occurring ARHGAP35 mutations on p190A RhoGAP activity, as well as Hippo signaling and CIP. Finally, we will test in vitro and in vivo if expression of exogenous p190A attenuates oncogenic capacities of human cancer cell lines with no or very low levels of endogenous p190A, and if such effects are dependent GAP activity and/or on Hippo signaling. These efforts are essential to elucidate the utility of targeting Rho signaling in future personalized oncology therapy.