Profiling and targeting micro-RNA expression during oncolytic virotherapy

描述（由申请人提供）：恶性神经胶质瘤患者的预期寿命只有15个月，因为治疗后肿瘤会迅速复发。因此，正在进行大量的努力来表征和靶向导致这些癌症复发的癌症起始细胞。 在肿瘤细胞中选择性复制并将其后代扩散到整个肿瘤中的病毒[溶瘤病毒（OV）]具有有效治疗恶性胶质瘤的强大潜力。然而，临床试验的结果并没有提供预期的结果，因此强调了确定限制OV在临床中疗效的因素的重要性。我们已经观察到，即使单纯疱疹病毒（HSV）衍生的OV有效地杀死原代神经胶质瘤细胞，它们的复制在源自相同肿瘤的癌症起始细胞中受到损害。因此，我们假设肿瘤起始细胞的OV抗性是这些病毒临床无效的部分原因。为了提高这种治疗的疗效，我们计划确定在胶质瘤起始细胞中诱导OV抗性的机制。 微小RNA（micro-RNAs，miRNAs）是一类非编码RNA分子，通过沉默特定的mRNA来调节靶基因的表达，在细胞病理生理学调控中起着重要作用。它们的表达在肿瘤发生和癌症治疗期间改变。哺乳动物的miRNAs也被发现在病毒感染后发生改变，并调节它们的向性。由于miRNA可以同时抑制属于同一本体途径的不同mRNA的翻译，我们相信，OV感染后的miRNA表达分析是一种有效的策略，以确定诱导胶质瘤起始细胞中OV抗性的机制。此外，由于很容易用改变特定miRNA活性的序列来设计OV，我们认为这种分析将为设计更有效的OV用于癌症治疗提供工具。 我们的目标是鉴定胶质瘤起始细胞中OV抗性的miRNA特征，并测试是否可以通过改变该特征来增强病毒治疗。为了实现这一目标，我们将首先使用人miRNA RT-PCR阵列来分析OV感染癌症起始细胞和来源于我们部门切除的GBM的已建立的原代癌细胞后的miRNA表达变化（目标1）。然后，我们将尝试通过改变目标1（目标2）中鉴定的miRNA的表达来增加OV在体外和体内的抗癌功效。这将首先通过在用miRNA抑制剂或放大剂瞬时转染胶质瘤细胞后分析OV的体外功效，然后通过用所选miRNA靶向序列工程化OV并在体内测试它们来进行。 因此，这些研究旨在表征脑肿瘤起始细胞对OV治疗的反应，并确定治疗干预的新靶点。如果成功，它们将提供新的手段来产生在临床应用中表现出增强功效的OV。 公共卫生相关性：通过这个项目，我们计划确定在癌细胞中诱导对病毒治疗产生抗性的机制。为此，我们将分析新鉴定的分子的存在，这些分子具有非常强大的调节肿瘤生物发生和病毒嗜性机制的能力。因此，我们将测试这些分子是否会干扰病毒杀死肿瘤细胞，然后构建可以靶向这些分子的新病毒。我们预计这类病毒将具有更强的治疗潜力。

DESCRIPTION (provided by applicant): Patients with malignant gliomas have a life expectancy of only 15 months due to rapid recurrence of the tumor after treatment. A large effort is therefore ongoing to characterize and target the cancer-initiating cells that cause recurrence of these cancers. Viruses that replicate selectively in tumor cells and spread their progeny throughout the tumor [oncolytic viruses (OVs)] present a strong potential for efficient treatment of malignant gliomas. However, results from clinical trials did not provide the expected results, thus highlighting the importance of identifying the factors that limit the efficacy of OVs in the clinics. We have observed that even though herpes simplex virus (HSV)-derived OVs efficiently kill primary glioma cells their replication is compromised in the cancer-initiating cells derived from the same tumor. Thus, we hypothesize that OV resistance of tumor-initiating cells is partially responsible for the clinical inefficacy of these viruses. To improve the efficacy of this treatment we plan to identify the mechanisms that induce OV resistance in glioma-initiating cells. Micro-RNAs (miRNAs) are non-coding RNA molecules that regulate expression of target genes by silencing specific mRNAs and are critical in controlling cellular pathophysiology. Their expression is altered during tumorigenesis and cancer treatment. Mammalian miRNAs were also found to be altered upon infection of viruses and to regulate their tropism. Because miRNAs can simultaneously inhibit translation of different mRNAs that belong to the same ontologic pathway, we believe that analysis of miRNA expression following OV infection is an efficient strategy to identify the mechanisms that induce OV resistance in glioma initiating cells. Moreover, because it is easy to engineer OVs with sequences that alter the activity of specific miRNAs, we think that this analysis will provide the tools to engineer more efficient OVs for cancer therapeutics. Our goal is to identify a miRNA signature of OV resistance in glioma initiating cells and to test if viral treatment can be enhanced by altering this signature. To accomplish this goal we will first use a human miRNA RT-PCR array to analyze miRNA expression changes following OV infection of cancer initiating cells and of established primary cancer cells derived from GBMs resected in our department (Aim 1). Then, we will attempt to increase OVs anti-cancer efficacy in vitro and in vivo by altering the expression of the miRNAs identified in aim 1 (Aim 2). This will be initially performed by analyzing OVs efficacy in vitro after transient transfection of glioma cells with miRNAs inhibitors or amplifiers, and then by engineering OVs with the selected miRNA targeting sequences and testing them in vivo. These studies are therefore designed to characterize brain tumor initiating cells in response to OV treatment and to identify new targets for therapeutic intervention. If successful, they will provide new means to generate OVs that exhibit enhanced efficacy in clinical applications. PUBLIC HEALTH RELEVANCE: With this project we plan to identify the mechanisms that induce resistance to viral therapy in cancer cells. For this purpose we will analyze the presence of newly identified molecules that have a very powerful capacity to regulate the mechanisms responsible of tumor biogenesis and viral tropism. We will thus test if these molecules interfere with virus killing of tumor cells and then build new viruses that can target these molecules. We expect that such viruses will have a stronger therapeutic potential.