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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

基本信息

批准号：
10356023
负责人：
JOAN HELLER BROWN
金额：
$ 37.68万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-09 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10356023
关键词：
Adherent Culture Adhesions Affect Animal Model Astrocytes Brain Brain Neoplasms CRISPR/Cas technology Cell Adhesion Cell Line Cell Proliferation Cell surface Cells DTR gene Data Dependence Disease Disease Progression Epidermal Growth Factor Receptor Event Feedback G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Gene Expression Gene Targeting Genes Genetic Genetic Transcription Glial Fibrillary Acidic Protein Glioblastoma Glioma Gliomagenesis Goals Health Hippocampus (Brain)Human Implant In Vitro Infection Invaded Knock-out Label Lentivirus Maintenance Malignant Neoplasms Mediating Modeling Mus Mutation Neurosphere Oncogenes Oncogenic PAR-1 Receptor Pathway interactions Patients Pharmacology Property Proteins Publishing Regulation Research Resistance Role Serum Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Molecule Sphingosine-1-Phosphate Receptor Testing The Cancer Genome Atlas Therapeutic Intervention Thrombin Thromboplastin Transcription Coactivator Transcriptional Activation Transcriptional Regulation Translating Work Xenograft procedure angiogenesis autocrine base cancer cell cell growth clinical practice experimental study genetic testing in vivo innovation insight interest knock-down migration neoplastic cell new therapeutic target novel programs response rhoA GTP-Binding Protein small hairpin RNA stem cell biomarkers stem cell genes stem cells stem-like cell therapeutic target transcriptome sequencing tumor tumor growth tumor initiation tumor progression

项目摘要

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。