RhoA and GPCR mediated transcriptional activation regulates glioblastoma

RhoA 和 GPCR 介导的转录激活调节胶质母细胞瘤

• 批准号: 9905280
• 负责人: JOAN HELLER BROWN
• 金额: $ 6.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-09 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905280
• 关键词: Adherent Culture; Adhesions; Affect; Animal Model; Astrocytes; Brain; Brain Neoplasms; CRISPR/Cas technology; Cell Line; Cell Proliferation; Cell surface; Cells; Data; Disease; Event; G-Protein-Coupled Receptors; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic screening method; Glial Fibrillary Acidic Protein; Glioblastoma; Gliomagenesis; Goals; Health; Hippocampus (Brain); Human; Implant; In Vitro; Infection; Invaded; Maintenance; Malignant Neoplasms; Mediating; Modeling; Mus; Oncogenes; Oncogenic; PAR-1 Receptor; Pathway interactions; Patients; Proteins; Research; Serum; Signal Pathway; Signal Transduction; Sphingosine-1-Phosphate Receptor; Stem cells; Subfamily lentivirinae; Testing; Thrombin; Transcription Coactivator; Transcriptional Activation; Translating; Xenograft procedure; angiogenesis; autocrine; clinical practice; experimental study; in vivo; insight; knock-down; migration; neoplastic cell; new therapeutic target; novel; programs; response; rhoA GTP-Binding Protein; small hairpin RNA; transcriptome sequencing; tumor; tumor growth

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.

摘要 我们过去的研究提供了相当多的洞察信号引起的G蛋白偶联 在星形胶质细胞和1321 N1胶质母细胞瘤细胞中，RhoA受体（GPCR）激活RhoA。的途径 通过PAR 1和S1 P受体经由G β 12/13接合调节包括RhoA活化以增加细胞增殖， 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和雅普在胶质母细胞瘤细胞中通过RhoA信号传导， 在增殖和迁移反应中改变基因表达。这项建议的目的是 证明了通过RhoA信号传导引发的强大的转录基因程序对于 多形性胶质母细胞瘤（GBM）肿瘤生长和胶质母细胞瘤干细胞（GSC）的维持，具有长时间的 长期目标是为这种毁灭性疾病确定新的治疗靶点。目标1使用人类1321 N1 胶质母细胞瘤细胞系定义细胞事件，并确定GPCR通过的特定靶基因的变化 和RhoA参与有助于癌症相关细胞反应的转录途径。S1 P-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或雅普CRISPR/Cas9 KO细胞。来自RNA seq分析的数据用于鉴定关键的 调节基因，并测试其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的应答的自分泌和转录途径。目标2 使用源自患者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生产线 将在体外无血清培养基中作为神经球或贴壁培养物生长，随后植入 作为原位异种移植物将敲除雅普、MRTF-A、RhoA及其下游靶基因 使用shRNA和体外干细胞标记物，评估细胞应答和体内肿瘤生长。目的 #3检验了RhoA介导的转录信号传导导致星形胶质细胞去分化的假设， 由激活的Ras驱动的胶质瘤发生）。原理证明实验检查分离的 用编码致癌Ras的慢病毒感染小鼠星形胶质细胞，其中RhoA中的分子 信号传导通路被基因删除或被shRNA敲低。胶质瘤形成的体内研究是 通过将癌基因通过慢病毒感染递送到GFAP-Cre小鼠的海马体中，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应该证明GPCR和RhoA介导的转录 激活可以引起遗传和功能反应，导致胶质母细胞瘤的失调， RhoA信号传导有助于已建立的GBM肿瘤驱动因子的致癌作用。与卫生有关的 重要的是，这些发现可以将当前研究和临床实践的重点从 在GBM中考虑GPCR和RhoA调节的信号传导途径的疾病驱动因素。