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NFkB-dependent antiviral pathways in VSV-resistant cancer cells

VSV 耐药癌细胞中 NFkB 依赖性抗病毒途径

基本信息

批准号：
10209637
负责人：
MAUREEN C FERRAN
金额：
$ 45.12万
依托单位：
ROCHESTER INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10209637
关键词：
Address Antiviral Agents Antiviral Response Biomedical Research Cancer cell line Cell Line Cells Collaborations Computer Models Data Defect Development Environment Equilibrium Experimental Models Fostering Gene Expression Genes Genetic Transcription Genomics Goals Human Immune Evasion In Vitro Infection Innate Immune Response Institutes Interferons Interleukin-6 LNCaP Malignant Neoplasms Mediating Modeling Mus Mutation Normal Cell Oncolytic Oncolytic viruses Outcome PC3 cell line Pathway interactions Production Prostate Research Research Personnel Resistance Role Scientist Signal Pathway Signal Transduction Signaling Molecule Students Study models TNF gene Technology Testing Training Tropism Vesicular stomatitis Indiana virus Vesicular stomatitis virus M protein Virus Virus Diseases Work cancer cell cytokine data modeling experience experimental study improved in silico in vitro Model inhibitor/antagonist innovation insight multiple myeloma M Protein mutant network models next generation oncolysis predictive modeling prostate cancer cell prostate cancer cell line refractory cancer response simulation transcriptome transcriptome sequencing transcriptomics tumor undergraduate student viral resistance virus host interaction

项目摘要

Antiviral responses are defective in many human tumors, leaving them susceptible to infection by “oncolytic” viruses such as vesicular stomatitis virus (VSV). In contrast, normal cells are not infected because they mount an innate immune response. Studies show that some cancers are resistant to VSV infection because they retain these antiviral responses. For example, many VSV-resistant prostate cell lines have constitutively active NFκB, while VSV-sensitive prostate cancer cell lines do not. Therefore it is important to delineate the mechanisms of sensitivity versus resistance of cancers to VSV. The wild-type M protein inhibits NFκB activation, the IFN response, and host gene expression, but different M protein mutations can selectively eliminate each of these functions. These findings have led us to conclude that the M protein uses at least two mechanisms to limit expression of antiviral genes: M-mediated inhibition of global host transcription (the first suppressor) and inhibition of NFκB activation (the second suppressor). Our preliminary in vitro and modeling data support our central hypothesis that VSV uses multiple strategies to control antiviral gene expression in response to VSV infection, including global host transcription inhibition, targeting of steps upstream of IKK in the RIG-I pathway, and suppression of antiviral genes controlled by NFκB. The objectives of this study are to enhance our understanding of the balance between the host’s ability to activate an NFκB-dependent antiviral response and the virus’s ability to evade these defenses; and how this impacts the use of oncolytic viruses to treat tumors that constitutively express antiviral genes. The goal of this study is to determine the effects of M protein mutations on NFκB-dependent responses in VSV-sensitive (LNCaP) versus VSV-resistant (PC3) prostate cancer cell lines using the innovative combination of in vitro and in silico modeling studies. In Aim 1, we will determine NFκB activation and expression of NFκB- dependent antiviral genes (e.g. interferon, IL-6 and TNF-α) in LNCaP and PC3 cells infected with viruses bearing different mutations in the M protein (Aim 1A). To determine the role of NFκB-dependent pathway activation in resistance to VSV, the transcriptomes of infected LNCaP and PC3 cells will be compared by RNA- seq (Aim 1B). We have developed an executable network model of the intracellular signaling pathways impacted by wildtype and M protein mutant VSV in mouse cells. We will tune this network using data specific to the context of VSV infection of human prostate cancer cell lines (generated in Aim 1) and perform simulations to identify key NFκB-dependent signaling molecules and interactions responsible for VSV sensitivity or resistance in prostate cancer cells (Aim 2A). Finally, new in vitro experiments will be performed to validate these predictions (Aim 2B). In addition to these scientific merits, this project will provide undergraduate and Master’s students with a quality biomedical research experience, foster collaborations, and significantly enhance the research environment at The Rochester Institute of Technology.

许多人类肿瘤的抗病毒反应存在缺陷，使它们容易受到“溶瘤”感染病毒，例如水疱性口炎病毒（VSV）。相反，正常细胞不会被感染，因为它们会安装先天免疫反应。研究表明，一些癌症对 VSV 感染具有抵抗力，因为它们保留这些抗病毒反应。例如，许多 VSV 抗性前列腺细胞系具有组成型活性 NFκB，而 VSV 敏感的前列腺癌细胞系则不然。因此，划定范围很重要癌症对 VSV 的敏感性与耐药性机制。野生型 M 蛋白抑制 NFκB 激活、IFN 反应和宿主基因表达，但不同的 M 蛋白突变可以选择性地消除这些功能中的每一个。这些发现使我们得出结论，M 蛋白至少使用两个限制抗病毒基因表达的机制：M介导的全局宿主转录抑制（第一个抑制子）和抑制 NFκB 激活（第二个抑制子）。我们的初步体外和建模数据支持我们的中心假设，即 VSV 使用多种策略来控制针对 VSV 感染的抗病毒基因表达，包括全局宿主转录抑制，靶向 RIG-I 途径中 IKK 上游的步骤，并抑制由 NFκB。这项研究的目的是增强我们对宿主能力之间平衡的理解激活 NFκB 依赖性抗病毒反应以及病毒逃避这些防御的能力；以及这如何影响使用溶瘤病毒治疗组成型表达抗病毒基因的肿瘤。本研究的目的是确定 M 蛋白突变对 NFκB 依赖性反应的影响使用创新组合的 VSV 敏感 (LNCaP) 与 VSV 耐药 (PC3) 前列腺癌细胞系体外和计算机模拟研究。在目标 1 中，我们将确定 NFκB 的激活和 NFκB- 的表达感染病毒的 LNCaP 和 PC3 细胞中依赖的抗病毒基因（例如干扰素、IL-6 和 TNF-α） M 蛋白具有不同的突变（目标 1A）。确定 NFκB 依赖性途径的作用 VSV 抗性激活后，感染的 LNCaP 和 PC3 细胞的转录组将通过 RNA- seq（目标 1B）。我们开发了细胞内信号通路的可执行网络模型受小鼠细胞中野生型和 M 蛋白突变体 VSV 的影响。我们将使用特定于的数据来调整该网络人类前列腺癌细胞系（在目标 1 中生成）的 VSV 感染背景并进行模拟识别负责 VSV 敏感性的关键 NFκB 依赖性信号分子和相互作用，或前列腺癌细胞的耐药性（目标 2A）。最后，将进行新的体外实验来验证这些预测（目标 2B）。除了这些科学优点外，该项目还将提供本科生和硕士生拥有优质的生物医学研究经验，促进合作，并显着改善罗彻斯特理工学院的研究环境。