Synergistic innate immune activation and cell killing by RIG-I ligands in HCV-HCC

描述（由申请人提供）：肝细胞癌（HCC）是丙型肝炎病毒（HCV）感染的不幸和可预测的后果，将其定义为一个重要的全球人类健康问题。没有针对HCV的疫苗，目前的治疗方法无法治愈近一半的HCV感染病例。结果是未经治疗的感染细胞继续其向肝硬化和HCC的过程。该项目的目标是通过使用RNA分子激活肝脏中的先天免疫和细胞死亡反应途径来测试针对HCC的协同双重打击的前景。这一目标是基于最近的证据表明，这些途径共享共同的蛋白质，并共同组装在peption。我们实验室先前的工作已经鉴定了来源于丙型肝炎基因组RNA的3'非翻译区或其反义序列的RNA，它们是RIG-I介导的先天免疫信号传导途径的有效激活剂。这些RNA具有5'三磷酸，长度约为100个核苷酸，并被描述为polyU/UC或polyAG/A。与膜结合的toll样受体不同，RIG-I是一种细胞质RNA结合蛋白和模式识别受体，其识别具有5'三磷酸的RNA，启动信号级联，其在I型干扰素的表达中达到高潮，建立抗病毒环境。到目前为止，我们的工作主要集中在了解RIG-I如何与病毒RNA结合以及信号通路如何被激活。我们发现，含有修饰的核糖基团（例如2 '-F脱氧尿苷）的RNA与RIG-I结合，但不激活信号传导，与其未修饰的对应物相反。我们小组最近的证据表明，修饰的RNA是独特的工具，可用于剖析信号转导途径中的步骤，以了解机制并可能确定治疗干预点。在这个提议中，我们将我们的工作扩展到专注于确定RIG-I激动剂RNA（polyU/UC和polyAG/A）是否激活先天免疫信号传导和细胞死亡途径。该方法的一部分集中在一种新的原代人肝培养细胞系统的创新使用上，该系统可将许多生理标志物维持在正常水平长达两周，从而允许在生理相关模型中进行实验。我们的方法的意义在于RIG-I激动剂RNA可以作为佐剂来增强先天免疫应答以限制HCV感染，同时还刺激细胞死亡途径以杀死正在向肿瘤形成发展的细胞。事实上，最近的研究表明，RIG-I激动剂激活细胞死亡信号，并且黑素瘤细胞比正常细胞更容易受到杀伤作用的影响。虽然先天免疫系统的toll样受体配体被用作免疫刺激剂来治疗临床疾病，但现在可以考虑激活RIG-I样解旋酶受体的相关方法。这一提议有可能促进关于如何治疗或预防HCV感染和肝癌的新想法。 公共卫生相关性：丙型肝炎病毒感染在全球范围内持续增加，并且没有保护性疫苗。许多丙型肝炎病毒（HCV）感染的不幸后果是，疾病进展为肝硬化和肝癌，生存率很低。我们已经表征了增强免疫反应以对抗病毒感染的RNA分子，这项提案的目标是确定相同的分子是否也会激活杀死癌细胞的细胞反应。我们认为这种协同攻击机制有可能成为治疗进展为癌症的病毒感染的新方法。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is an unfortunate and predictable consequence of hepatitis C virus (HCV) infections, defining it as a significant global human heath problem. There is no vaccine against HCV, and current treatments fail to cure HCV infections in nearly half of the cases. The consequence is that the untreated infected cells continue on their course toward cirrhosis and HCC. The goal of this project is to test the promise of a synergistic double hit against HCC by using RNA molecules to activate both innate immune and cell death response pathways in liver. This goal is based on recent evidence demonstrating that these pathways share common proteins and jointly assemble at the mitochondrion. Prior work from our laboratory has identified RNAs derived from the 3' untranslated region of hepatitis C genomic RNA or its antisense sequence that are potent activators of the RIG-I mediated innate immune signaling pathway. These RNAs have a 5' triphosphate, are about 100 nucleotides in length, and are described as polyU/UC or polyAG/A. Unlike the membrane-bound toll-like receptors, RIG-I is a cytoplasmic RNA binding protein and pattern recognition receptor that recognizes RNAs with a 5' triphosphate, initiating a signaling cascade that culminates in the expression if Type I interferons, establishing an antiviral environment. To date, our work has focused primarily on understanding how RIG-I binds to viral RNA and how the signaling pathway is activated. We discovered that RNAs containing modified ribose groups (2'-F deoxyuridine, for example) bind to RIG-I, but do not activate signaling, in contrast to their unmodified counterparts. Recent evidence from our group shows that the modified RNAs are unique tools that can be used to dissect the steps in the signal transduction pathway to understand mechanisms and potentially identify points of therapeutic intervention. In this proposal, we extend our work to focus on determining if the RIG-I agonist RNAs (polyU/UC and polyAG/A) activate both innate immune signaling and cell death pathways. Part of the approach focuses on the innovative use of a novel primary human liver culture cell system that maintains many physiologic markers at normal levels for up to two weeks, permitting experimentation in a physiologically relevant model. The significance of our approach is that RIG-I agonist RNAs may serve as adjuvants to boost an innate immune response to limit HCV infection, while also stimulating cell death pathways to kill cells that are progressing toward tumor formation. Indeed, recent studies have shown that RIG-I agonists activate cell death signaling, and that melanoma cells are more susceptible than normal cells to the killing effects. Although toll-like receptor ligands of the innate immune system are being used a immunostimulants to treat clinical disease, related approaches that activate the RIG-I- like helicase receptors can now be considered. This proposal has the potential to foster new ideas about how both HCV infection and the liver cancer that often follows can be treated or prevented. PUBLIC HEALTH RELEVANCE: Hepatitis C virus infections continue to increase globally, and a protective vaccine is not available. An unfortunate consequence of many hepatitis C virus (HCV) infections is that the disease progresses to cirrhosis and liver cancer that have poor survival statistics. We have characterized RNA molecules that boost the immune response to fight virus infections, and the goal of this proposal is to determine if the same molecules will also activate a cell response that kills cancer cells. We propose that this synergistic attack mechanism has potential as a novel approach for treating viral infections that progress to cancers.