NFkB-dependent antiviral pathways in VSV-resistant cancer cells

抗病毒反应在许多人类肿瘤中是有缺陷的，使它们容易被“溶瘤”病毒感染。 病毒如水泡性口炎病毒（VSV）。相比之下，正常细胞不会被感染，因为它们 先天免疫反应研究表明，一些癌症对VSV感染有抵抗力，因为它们 保持这些抗病毒反应。例如，许多抗VSV的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不。因此，重要的是要划定 癌症对VSV的敏感性与抗性的机制。野生型M蛋白抑制NFκB 激活，IFN应答和宿主基因表达，但不同的M蛋白突变可以选择性地 消除这些功能。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-介导的对全局宿主转录的抑制（第一个 抑制因子）和抑制NFκB活化（第二抑制因子）。 我们初步的体外和建模数据支持我们的中心假设，即VSV使用多种策略， 响应VSV感染控制抗病毒基因表达，包括全面宿主转录抑制， 靶向RIG-I途径中IKK上游的步骤，并抑制由 NFκB。本研究的目的在于增进我们对主持人能力之间平衡的认识 激活NFκ B依赖的抗病毒反应和病毒逃避这些防御的能力;以及这是如何实现的。 影响了溶瘤病毒治疗组成型表达抗病毒基因的肿瘤的应用。 本研究的目的是确定M蛋白突变对NFκ B依赖性反应的影响， 使用创新组合的VSV敏感（LNCaP）与VSV耐药（PC 3）前列腺癌细胞系 的体外和计算机模拟研究。在目的1中，我们将确定NFκB的活化和NFκB的表达。 病毒感染的LNCaP和PC 3细胞中的依赖性抗病毒基因（如干扰素、IL-6和TNF-α） 在M蛋白（Aim 1A）中携带不同突变。确定NFκ B依赖性通路在细胞凋亡中的作用 在对VSV的抗性中，感染的LNCaP和PC 3细胞的转录组将通过RNA-聚合酶链反应（PCR）进行比较。 seq（目标1B）。我们开发了一个可执行的细胞内信号通路网络模型 受小鼠细胞中野生型和M蛋白突变体VSV的影响。我们将使用特定于以下方面的数据来调整此网络： VSV感染人前列腺癌细胞系（在Aim 1中产生）的背景并进行模拟 确定关键的NFκ B依赖性信号分子和负责VSV敏感性的相互作用， 前列腺癌细胞的耐药性（Aim 2A）。最后，将进行新的体外实验以验证 这些预测（目标2B）。除了这些科学价值，该项目将提供本科和 硕士生具有高质量的生物医学研究经验，促进合作，并显着 改善罗切斯特理工学院的研究环境。

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.