A Role for KSHV in the Pathogenesis of Malignancies

我们集中在三个相关领域：1。KSHV潜伏基因产物vFLIP在KSHV感染的靶细胞和卡波西肉瘤（KS）、原发性积液淋巴瘤（PEL）和多中心Castleman病中表达的研究；2. 宿主细胞中KSHV诱导的G蛋白偶联受体CXCR7及其不受KSHV诱导的相关受体CXCR4的研究；和3。kshv诱导的艾滋病患者恶性肿瘤的新疗法的发展。KSHV的特征之一是其感染内皮细胞的能力，主要通过促进产生促血管生成因子的细胞的募集和促进促血管生成基因的表达，间接促进血管生成和淋巴管生成。ORFK13/vFLIP编码一种188个氨基酸的蛋白，该蛋白与IKK复合物结合激活NFkappaB。我们研究了ORFK13/vFLIP对KS表型的贡献和治疗靶向的潜力。为此，我们将ORFK13/vFLIP逆转录到原代人内皮细胞中，并检测了该基因对KS表型的贡献。我们发现ORFK13/vFLIP诱导了KS细胞特有的纺锤体形态，并促进了KS血管系统紊乱的典型异常血管网络的形成。用ORFK13/vFLIP转导的内皮细胞中基因表达的微阵列分析检测到促炎细胞因子、趋化因子和干扰素应答基因的表达增加。该研究首次对KSHV-vFLIP基因调控进行了全面分析。正如人们对促炎细胞因子和趋化因子的刺激所期望的那样，我们发现ORFK13/vFLIP刺激了KS病变特征的炎症细胞的粘附。芯片分析发现ORFK13/vFLIP促进胸苷磷酸化酶的表达，胸苷磷酸化酶是一种细胞酶，可以将前药5-氟-5-脱氧尿嘧啶（5- dfur）代谢为5-氟吡啶（5-FU）。一种有效的胸腺嘧啶合成酶抑制剂，5-Fu阻断DNA和RNA的合成。在细胞毒性测试中，5- dfur （0.1-1muM）选择性杀死表达ORFK13/ vflip的内皮细胞，同时保留对照细胞。这些结果表明ORFK13/vFLIP直接和间接地参与了KS的炎症和血管表型，并确定5- dfur是一种潜在的靶向KSHV潜伏期的新药，可用于治疗KS和其他KSHV相关的恶性肿瘤。在进一步的研究中，我们发现来自表达K13/vFLIP的内皮细胞的条件培养基在对照细胞中诱导STAT1磷酸化，而在缺乏ifn α受体β亚基（Ifnar2） 190的U5A细胞中则没有，这表明K13/vFLIP诱导i型干扰素分泌以激活STAT1。K13/vFLIP表达后，我们在内皮细胞培养上清液中检测到IFNbeta1。因此，IFNbeta1至少是K13/vFLIP诱导的刺激STAT1的体液因子之一，这也可以解释为什么内皮细胞中K13/vFLIP的表达可以促进多种ifn诱导基因的转录189。I型ifn通过诱导p21，启动细胞周期阻滞和抑制病毒复制，在病毒感染的细胞中发挥抗病毒功能。IFN反应通常代表宿主对病毒和病毒感染细胞的免疫反应的一个组成部分。令人困惑的是，KSHV在进化过程中保持了K13/vFLIP的这种抗病毒功能，这可能会损害其持久性。我们现在正在分析这一观察结果的潜在意义，并验证K13/vFLIP诱导的IFN反应可以阻断KSHV复制，从而调节病毒复制的假设。KSHV高度诱导的细胞基因之一是趋化因子受体RDC1/CXCR7。最近的研究表明，CXCR7可以结合趋化因子SDF1和I-TAC，但目前尚不清楚CXCR7是否可以对这些配体或其他信号发出信号，或者其功能是否可以将配体与受体隔离开来。最近，CXCR7被证明与CXCR4寡聚，CXCR4是一种可以响应SDF1信号的受体。我们对CXCR7在KSHV感染中的功能感兴趣。我们在原发性积液性淋巴瘤（PEL）细胞系中过表达或沉默CXCR7，并在小鼠中测试了其致瘤性。初步观察表明，CXCR7促进pel诱导的肿瘤进展。我们目前正在探索CXCR7在PEL恶性肿瘤中促肿瘤作用的机制。PEL是人类中一种致命的病毒性恶性肿瘤，通常表现为恶性积液，随后扩散。尽管采用高剂量化疗或其他治疗方法，PEL仍是一种迅速致命的恶性肿瘤。雷帕霉素靶向mTOR（哺乳动物雷帕霉素靶点），这是一种在癌症中经常不受调节的细胞信号通路的效应物，对多种肿瘤，特别是淋巴细胞起源的肿瘤都有疗效。我们已经研究了雷帕霉素治疗实验性PEL的潜在效用。先前的研究表明，雷帕霉素对小鼠皮下肾小球可能有效。然而，这种临床前模型与患者的疾病位置和进展相去甚远。最近，在雷帕霉素治疗的移植后受者中，PEL的发展引起了对该药抗PEL活性的质疑。我们建立并使用小鼠腹腔肿瘤积液PEL模型来研究雷帕霉素的抗PEL活性。我们发现雷帕霉素能显著减少腹水积聚，延长小鼠存活时间。最初，与对照小鼠相比，雷帕霉素降低了PEL负荷，但大多数小鼠迅速表现出PEL进展。与对照组相比，雷帕霉素治疗小鼠腹水中促进血管通透性、促进积液形成的VEGF水平显著降低。IL-10 （PEL的主要自分泌生长因子）的表达在雷帕霉素处理小鼠的PEL中最初降低，但在治疗后迅速增加。我们发现腹水缺氧环境和雷帕霉素共同刺激PEL中IL-10的表达。这些结果不支持使用雷帕霉素作为PEL的根治性治疗，但确定雷帕霉素是减少恶性积液积累的有效药物。目前实验室的工作旨在进一步表征PEL对雷帕霉素耐药性的发展以及如何预防它。特别是，我们正在测试联合雷帕霉素中和IL-10的潜在功效。

We have focused in three related areas: 1. the study of vFLIP, a KSHV latent gene product expressed in KSHV-infected cell targets and in Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's disease; 2. the study of CXCR7, a G protein-coupled receptor induced by KSHV in the host cells, and its related receptor CXCR4, which is not induced by KSHV; and 3. the development of new therapies for KSHV-induced malignancies occurring in AIDS patients. One of the characteristic features of KSHV is its ability to infect endothelial cells,and to indirectly promote angiogenesis and lymphangiogenesis predominantly by promoting the recruitment of cells that produce pro-angiogenic factors and promoting the expression of pro-angiogenic genes by the cells it infects. ORFK13/vFLIP encodes a 188-amino acid protein, which binds to the Ikappab kinase (IKK) complex to activate NFkappaB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. To this end, we have retrovirally transduced ORFK13/vFLIP into primary human endothelial cells and examined the contribution of this gene to KS phenotype. We found that ORFK13/vFLIP induces the spindle morphology distinctive of KS cells and promotes formation of abnormal vascular networks typical of the disorderly KS vasculature. Microarray analysis of gene expression in endothelial cells transduced with ORFK13/vFLIP detected increased expression of pro-inflammatory cytokines, chemokines, and interferon-responsive genes. This study represents the first comprehensive analysis of gene regulation by KSHV-vFLIP. As one might expect from stimulation of pro-inflammatory cytokines and chemokines, we found that ORFK13/vFLIP stimulates adhesion of inflammatory cells characteristic of KS lesions. The microarray analysis found that ORFK13/vFLIP promotes the expression of thymidine phosphorylase, a cellular enzyme that can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU). A potent thymidine synthase inhibitor, 5-Fu blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1-1muM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS, and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies. In additional studies, we have found that the conditioned medium from K13/vFLIP-expressing endothelial cells induces STAT1 phosphorylation in control cells but not in U5A cells that are lacking the IFNalpha receptor beta subunit (Ifnar2) 190, suggesting that K13/vFLIP induces type-I interferon secretion to activate STAT1. By ELISA, we detected IFNbeta1 in the culture supernatants of endothelial cells only after K13/vFLIP was expressed. Thus,IFNbeta1 is at least one of the humoral factors induced by K13/vFLIP to stimulate STAT1, which could also explain why K13/vFLIP expression in endothelial cells promotes the transcription of a variety of IFN-inducible genes 189. Type I IFNs exert antiviral functions in virus-infected cells by inducing p21, initiating cell cycle arrest, and inhibiting viral replication. The IFN response usually represents an integral part of the hosts immune response against viruses and virus-infected cells. It is puzzling that KSHV has maintained during evolution this antiviral function of K13/vFLIP, which is potentially damaging to its persistence. We are now analyzing the potential significance of this observation and are testing the hypothesis that the IFN response induced by K13/vFLIP can block KSHV replication, thereby modulating viral replication. One of the cellular genes that are highly induced by KSHV is the chemokine receptor RDC1/CXCR7. Recent studies have shown that CXCR7 binds the chemokines SDF1 and I-TAC but it is still unclear whether CXCR7 can signal in response to these ligands or other signals, or whether its function is to serve to sequester ligands away from their receptors. Recently, CXCR7 was shown to oligomerize with CXCR4, a receptor that can signal in response to SDF1. We are interested in the function of CXCR7 in the context of KSHV infection. We have overexpressed or silenced CXCR7 in PEL (Primary Effusion Lymphoma) cell lines and tested their tumorigenicity in mice. Initial observations have shown that CXCR7 promotes PEL-induced tumor progression. We are currently exploring the mechanisms underlying this pro-tumorigenic effect of CXCR7 in the context of PEL malignancy. PEL is a fatal viral malignancy in humans, which typically presents as a malignant effusion that later disseminates. In spite of therapy with high-dose chemotherapy or other therapies, PEL is a rapidly fatal malignancy. Rapamycin, which targets mTOR (mammalian target of rapamycin), an effector of cell signaling pathways often deregulated in cancer, showed efficacy against a variety of tumors, particularly those of lymphoid origin. We have investigated the potential utility of Rapamycin for the treatment of experimental PEL. Previous studies have suggested that rapamycin could be effective against subcutaneous PEL in mice. However, this pre-clinical model is far removed from the disease in patients in its location and progression. Recently, PEL development in rapamycin-treated post-transplant recipients raised questions about the drug's anti-PEL activity. We have developed and used a murine model of effusion PEL progressing to peritoneal tumors to investigate the anti-PEL activity of rapamycin. We found that rapamycin significantly reduces ascites accumulation and extends mouse survival. Initially, rapamycin reduced PEL load compared to control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of rapamycin-treated mice compared to controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from rapamycin-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and rapamycin cooperate in stimulating IL-10 expression in PEL. These results do not support the use of rapamycin as a curative treatment for PEL, but identify rapamycin an effective drug to reduce accumulation of malignant effusions. Current efforts in the laboratory are intended to further characterize development of PEL resistance to rapamycin and how to prevent it. In particular, we are testing the potential efficacy of combining rapamycin to neutralization of IL-10.