Modulation of Glioma Growth and Invasion by HCMV Glycoprotein B

描述（由申请人提供）：胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤，并且是一致致命的。我们的实验室是第一个表明人巨细胞病毒（HCMV）核酸和蛋白质存在于超过90%的人类恶性胶质瘤中，这些发现现在已经被三个独立的小组证实。最近的研究表明，HCMV感染水平与患者的生存率呈负相关，这表明HCMV可能诱导向更具侵袭性和侵袭性的胶质瘤表型转变。我们最近发表了两篇报道，证明HCMV感染和GBM细胞中的基因表达可以诱导对肿瘤生长至关重要的细胞酪氨酸激酶信号通路（Akt，PLC 3，FAK），抑制p53和Rb肿瘤抑制活性，改变细胞周期检查点控制，并促进细胞增殖。我们还发现HCMV在GBM细胞中激活了强大的PDGFR 1信号传导，并且PDGFR 1激活实际上是HCMV感染所必需的。这一发现与神经胶质瘤生物学特别相关，因为PDGFR 1在高百分比的GBM细胞中扩增/过表达，并且正常NPC中PDGFR 1的激活涉及神经胶质瘤发生的最早阶段。我们发表的和初步的数据表明，HCMV糖蛋白B（gB，最丰富的病毒包膜蛋白）是结合和酪氨酸磷酸化PDGFR 1并激活胶质瘤细胞中下游PI 3 K-Akt信号传导的病毒部分。最近，我们有证据表明，gB诱导的趋触性胶质瘤细胞迁移和HCMV诱导的胶质瘤transwell迁移抑制PDGFR 1阻断抗体或gB中和抗体。综上所述，我们的数据表明，HCMV gB和PDGFR 1之间的相互作用，表达在胶质瘤细胞表面上可能发挥关键作用，在调节胶质瘤的生长和侵袭。为了研究这些假设，我们建议使用体外，离体和体内试验，将测试是否HCMV感染和gB表达的人神经胶质瘤细胞表现出增强的生长和侵袭力。我们的实验室有独特的访问人类原发性胶质母细胞瘤衍生的文化，内源性表达几个HCMV基因产物，包括gB。我们计划使用这些原代神经胶质瘤培养物，用临床HCMV分离株进行超感染，并设计异位表达gB，用于transwell迁移，脑切片侵袭和体内肿瘤生长测定。如果我们发现HCMV和gB确实促进胶质瘤生长和侵袭，我们计划研究这种调节的具体机制，包括细胞增殖，细胞侵袭和肿瘤血管生成。这些实验的结果对于理解HCMV在人类神经胶质瘤发病机制中的作用至关重要，并可能发现基于抗病毒策略的针对这种无法治愈的人类癌症的新治疗方法。 公共卫生相关性：我们的实验室发现，一种常见的人类病毒，巨细胞病毒（HCMV）存在于超过90%的恶性胶质瘤中，这是一种均匀致命的高度侵袭性癌症，没有已知的病因或治疗方法。本研究拟探讨HCMV包膜糖蛋白B在促进胶质瘤生长和侵袭中的作用。本文提出的实验结果可能会导致基于神经胶质瘤患者的抗病毒治疗的新的治疗方法。

DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor in adults and is uniformly fatal. Our laboratory was the first to show that human cytomegalovirus (HCMV) nucleic acids and proteins are present in over 90% of human malignant gliomas, and these findings have now been confirmed by three independent groups. Recent studies have shown that HCMV infection levels negatively correlate with patient survival, suggesting that HCMV may induce a shift towards a more aggressive and invasive glioma phenotype. We recently published two reports demonstrating that HCMV infection and gene expression in GBM cells can induce cellular tyrosine kinase signaling pathways critical for tumor growth (Akt, PLC3, FAK), inhibit p53 and Rb tumor suppressor activity, alter cell cycle checkpoint controls, and promote cell proliferation. We have also discovered that HCMV activates robust PDGFR1 signaling in GBM cells, and that PDGFR1 activation is actually required for HCMV infection. This finding is particularly relevant to glioma biology since PDGFR1 is amplified/overexpressed in a high percentage of GBM cells and activation of PDGFR1 in normal NPCs is implicated in the earliest stages of gliomagenesis. Our published and preliminary data indicate that HCMV glycoprotein B (gB, the most abundant viral envelope protein) is the viral moiety that binds and Tyrosine phosphorylates PDGFR1 and activates downstream PI3K-Akt signaling in glioma cells. Most recently, we have evidence that gB induces haptotactic glioma cell migration and that HCMV-induced glioma transwell migration is inhibited both by PDGFR1 blocking antibodies or gB neutralizing antibodies. Taken together, our data suggest that the interaction between HCMV gB and PDGFR1 expressed on the surface of glioma cells may play a critical role in modulating glioma growth and invasion. To investigate these hypotheses, we propose to use in vitro, ex vivo, and in vivo assays which will test whether HCMV-infected and gB -expressing human glioma cells exhibit enhanced growth and invasiveness. Our laboratory has unique access to human primary glioblastoma-derived cultures which endogenously express several HCMV gene products, including gB. We plan to use these primary glioma cultures, super-infected with a clinical HCMV isolate and engineered to ectopically express gB for transwell migration, brain slice invasion, and in vivo tumor growth assays. If we find that HCMV and gB do promote glioma growth and invasiveness, we plan to investigate specific mechanisms underlying this modulation, including cell proliferation, cell invasion, and tumor angiogenesis. Results from these experiments will be critical in understanding the role that HCMV plays in the pathogenesis of human gliomas and may uncover novel therapeutic approaches against this incurable human cancer, based on antiviral strategies. PUBLIC HEALTH RELEVANCE: Our laboratory has found that a common human virus, cytomegalovirus (HCMV) is present in over 90% of malignant gliomas, a uniformly fatal, highly aggressive cancer, with no known etiology or treatment. This proposal will investigate the role of HCMV envelope glycoprotein B in promoting glioma growth and invasion. Results from experiments proposed herein may lead to novel therapeutic approaches based on antiviral treatments for glioma patients.