p190 RhoGAP signaling in epithelial oncogenesis

摘要 最近对人类癌症基因突变的分析意外发现编码p190A的ARHGAP35 RhoGAP(P190A)作为一种高度突变的基因，即前30名泛癌。突变尤其丰富，在 腺癌，包括肾细胞癌。ARHGAP35的突变谱与 作为肿瘤抑制者的角色。此外，ARHGAP35基因座位于基因组中经常 在癌症中失去了生命。然而，生物信息学数据还没有确定基因的功能结果。 突变。因此，这项提案的范围是定义与p190A缺失相关的致癌能力 在人类癌症中的表达和自然发生的ARHGAP35突变。 我们最近获得的结果表明，p190A和它的伴星p190B-统称为 P190在此介导了上皮细胞的接触性增殖抑制(CIP)。此外，我们还进行了一次 全基因组rna-seq分析，并确定p190调控受 YAP癌蛋白。YAP是TEAD家族转录因子的转录共激活因子。YAP的活动 是由河马途径控制的，该途径广泛参与CIP。 在目标1中，我们将定义p190影响河马信号和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信号通过典型和/或非典型河马通路；(Iii)确定p190是否影响YAP- 以及(Iv)确定p190A和p190B之间是否存在明显的冗余。 上皮细胞是上下文相关的。 在目标2中，我们将阐明p190在黏附连接(AJs)中的功能，它在 通过河马途径促进CIP。在这个目标中，我们将(I)测试p190在激活河马中的作用 在新生AJ形成时的途径；(Ii)确定E-钙粘蛋白是否需要p190来恢复河马 癌细胞中的信号转导；(Iii)确定p190在失巢凋亡中的作用，这是一种肿瘤抑制机制，是 受到AJs和河马途径的调节；以及(Iv)阐明p190和p120之间的相互作用是否- 河马信号和CIP需要连结蛋白。 在目标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 ARHGAP35突变导致(I)杂合性缺失；(Ii)YAP调控基因的表达；以及(Iii)共突变 和排他性数据。接下来，我们将确定自然发生的ARHGAP35突变对p190A的影响 RhoGAP活性，以及河马信号和CIP。最后，我们将在体外和体内测试是否表达 外源性p190A抑制无或极低水平p190A的人癌细胞株的致癌能力 内源性p190A，以及这种效应是否依赖于GAP活性和/或河马信号。这些努力 对于阐明靶向Rho信号在未来个体化肿瘤治疗中的作用至关重要。

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.