RhoA and GPCR mediated transcriptional activation regulates glioblastoma

摘要 我们过去的研究为通过G蛋白偶联引起的信号提供了相当大的洞察力 激活星形胶质细胞和1321N1胶质母细胞瘤细胞上RhoA的受体(GPCRs)。小路 通过G12/13参与的PAR1和S1P受体调节包括激活RhoA以增加细胞 增殖、存活和侵袭--癌症的特征。我们最近的研究表明， 转录共激活因子MRTF-A和YAP通过RhoA信号在胶质母细胞瘤细胞中表达，并参与 增殖性和移行性反应中基因表达的改变。这项建议的目的是 证明通过RhoA信号诱导的强大的转录基因程序对 多形性胶质母细胞瘤(GBM)肿瘤生长和维持的胶质母细胞瘤干细胞(GSC)，具有长 为这种毁灭性疾病确定新的治疗靶点的长期目标。AIM#1使用人类1321N1 胶质母细胞瘤细胞系定义细胞事件并确定GPCRs所依据的特定靶基因的变化 而RhoA参与了有助于癌症相关细胞反应的转录途径。S1P-和 评估凝血酶诱导的增殖、存活、黏附、迁移、侵袭和血管生成。 MRTF-A或YAP CRISPR/Cas9 KO细胞。来自RNA序列分析的数据用于确定关键的 调节基因，并测试它们在细胞反应中的功能重要性。靶基因作用于， 考虑了放大对GPCRs和RhoA反应的自分泌和转录途径。目标2 使用患者来源的胶质母细胞瘤异种移植(PDX)作为胶质母细胞瘤干细胞的模型。几条PDX线路 将以神经球或贴壁培养物的形式在体外无血清培养中生长，然后植入 作为原位(脑)异种移植。YAP、MRTF-A、RhoA及其下游靶基因将被敲除 使用shRNA和体外干细胞标记物，对细胞反应和体内肿瘤生长进行评估。目标 #3测试了RhoA介导的转录信号导致星形胶质细胞去分化和 由激活的RAS驱动的胶质瘤形成)。原理证明实验检验孤立的去分化 编码致癌RAS的慢病毒感染小鼠星形胶质细胞及其RhoA分子 信号通路被shRNAs基因缺失或破坏。胶质瘤形成的体内研究是 通过慢病毒感染将癌基因导入GFAP-CRE小鼠的海马区，随后 分析肿瘤生长、侵袭以及靶基因和干细胞标记物表达的变化。这个 这些研究的总体结果应该证明GPCR和RhoA介导的转录 激活可以引发导致胶质母细胞瘤失调的遗传和功能反应，并且 RhoA信号有助于已建立的GBM肿瘤驱动因素的致癌效应。与健康相关的 重要的是，这些发现可能会将当前研究和临床实践的重点从 已确定的疾病驱动因素是考虑GPCR和RhoA在GBM中调节的信号通路。

Abstract Our past research has provided considerable insight into the signals elicited through G-protein coupled receptors (GPCRs) that activate RhoA on astrocytes and in 1321N1 glioblastoma cells. The pathways regulated through PAR1 and S1P receptors via G12/13 engagement include RhoA activation to increase cell proliferation, survival, and invasion-hallmarks of cancer. Our recent studies demonstrate robust activation of the transcriptional co-activators MRTF-A and YAP through RhoA signaling in glioblastoma cells, and implicate altered gene expression in proliferative and migratory responses. The objective of this proposal is to demonstrate that robust transcriptional gene programs elicited through RhoA signaling are critical to glioblastoma multiforme (GBM) tumor growth and maintenance of glioblastoma stem cells (GSC), with a long term goal of identifying new therapeutic targets for this devastating disease. Aim #1 uses the human 1321N1 glioblastoma cell line to define cellular events and identify changes in specific target genes by which GPCRs and RhoA engage transcriptional pathways that contribute to cancer-relevant cellular responses. S1P- and thrombin-induced proliferation, survival, adhesion, migration, invasion and angiogenesis are assessed in WT and MRTF-A or YAP CRISPR/Cas9 KO cells. Data from RNA seq analysis are used to identify critical regulated genes, and tested for their functional importance in cellular responses. Actions of target genes on, autocrine and transcriptional pathways that amplify response to GPCRs and RhoA are considered. Aim #2 uses patient-derived glioblastoma xenografts (PDX), as a model of glioblastoma stem cells. Several PDX lines will be grown in serum free medium in vitro as neurospheres or adherent cultures and subsequently implanted as orthotopic (brain) xenografts... YAP, MRTF-A, RhoA and their downstream target genes will be knocked down using shRNA and in vitro stem cell markers, cellular responses and in vivo tumor growth assessed. Aim #3 tests the hypothesis that RhoA-mediated transcriptional signaling leads to astrocyte dedifferentiation and gliomagenesis driven by activated Ras). Proof of principle experiments examine dedifferentiation of isolated mouse astrocytes infected with lentiviruses encoding oncogenic Ras and in which molecules in the RhoA signaling pathway are genetically deleted or knocked down with shRNAs. In vivo studies of gliomagenesis are carried out by delivery of oncogenes by lentiviral infection into the hippocampus of GFAP-Cre mice followed by analysis of tumor growth, invasion, and changes in expression of target genes and stem cell markers. The overall findings from these studies should demonstrate that GPCR- and RhoA-mediated transcriptional activation can elicit genetic and functional responses that contribute to dysregulation in glioblastoma, and that RhoA signaling contributes to the oncogenic effect of established GBM tumor drivers. The health-related significance is that these findings could shift the focus of current research and clinical practice from the established disease drivers towards consideration of GPCR- and RhoA-regulated signaling pathways in GBM.