RhoA and GPCR mediated transcriptional activation regulates glioblastoma

摘要 我们过去的研究对 G 蛋白偶联引发的信号提供了相当深入的了解 激活星形胶质细胞和 1321N1 胶质母细胞瘤细胞上的 RhoA 的受体 (GPCR)。途径 通过 PAR1 和 S1P 受体通过 Gα12/13 参与进行调节，包括 RhoA 激活以增加细胞 增殖、存活和侵袭——癌症的标志。我们最近的研究表明， 转录共激活因子 MRTF-A 和 YAP 通过胶质母细胞瘤细胞中的 RhoA 信号传导，并暗示 增殖和迁移反应中基因表达的改变。该提案的目的是 证明通过 RhoA 信号传导引发的强大转录基因程序对于 多形性胶质母细胞瘤 (GBM) 肿瘤的生长和胶质母细胞瘤干细胞 (GSC) 的维持，具有长期 长期目标是确定这种毁灭性疾病的新治疗靶点。目标 #1 使用人类 1321N1 胶质母细胞瘤细胞系，用于定义细胞事件并识别特定靶基因的变化，GPCR 可以通过这些变化 RhoA 参与有助于癌症相关细胞反应的转录途径。 S1P-和 在 WT 中评估凝血酶诱导的增殖、存活、粘附、迁移、侵袭和血管生成 和 MRTF-A 或 YAP CRISPR/Cas9 KO 细胞。 RNA seq 分析的数据用于识别关键的 调节基因，并测试它们在细胞反应中的功能重要性。靶基因的作用， 考虑了放大对 GPCR 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物（PDX）作为胶质母细胞瘤干细胞的模型。多条PDX线 将作为神经球或贴壁培养物在体外无血清培养基中生长，然后植入 作为原位（脑）异种移植物...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、细胞反应和体内肿瘤生长进行评估。目标 #3 检验 RhoA 介导的转录信号传导导致星形胶质细胞去分化和 由激活的 Ras 驱动的神经胶质瘤发生）。原理实验验证检验分离的去分化 感染编码致癌 Ras 的慢病毒的小鼠星形胶质细胞，其中 RhoA 中的分子 信号通路被基因删除或被 shRNA 敲除。胶质瘤发生的体内研究是 通过慢病毒感染将癌基因递送至 GFAP-Cre 小鼠的海马，然后 分析肿瘤生长、侵袭以及靶基因和干细胞标志物表达的变化。的 这些研究的总体结果表明 GPCR 和 RhoA 介导的转录 激活可以引发遗传和功能反应，导致胶质母细胞瘤失调，并且 RhoA 信号传导有助于已确定的 GBM 肿瘤驱动因素的致癌作用。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焦点从 确定了 GBM 中 GPCR 和 RhoA 调节信号通路的疾病驱动因素。

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.