A Role for KSHV in the Pathogenesis of Malignancies

我们主要集中在三个相关领域：KSHV潜伏基因产物vFlIP的研究，它在Kaposi肉瘤(KS)、原发性渗出性淋巴瘤(PEL)和多中心Castleman病中的表达；CXCR7的研究，它是KSHV在宿主细胞中诱导的G蛋白偶联受体；以及针对KSHV诱导的艾滋病患者恶性肿瘤的新疗法的开发。KSHV的特征之一是其促进血管生成和淋巴管生成的能力。与其他劫持促进血管生成的细胞基因的病毒不同，KHSV不编码促血管生成因子，而是促进其感染的细胞表达促血管生成基因。ORFK13/vFLIP编码一个188个氨基酸的蛋白质，它与I&B激酶(IKK)复合体结合，激活NF&B。我们研究了ORFK13/vFLIP对KS表型的贡献和治疗靶向的可能性。为此，我们将ORFK13/vFLIP逆转录病毒导入原代人内皮细胞，并检测该基因对KS表型的影响。我们发现ORFK13/vFLIP可以诱导KS细胞特有的纺锤体形态，并促进异常血管网络的形成，这是KS血管系统紊乱的典型特征。对转导ORFK13/vFLIP的内皮细胞基因表达的微阵列分析发现，促炎症细胞因子、趋化因子和干扰素反应基因的表达增加。本研究首次对KSHV-vFLIP的基因调控进行了全面分析。正如人们可能预期的那样，在促炎细胞因子和趋化因子的刺激下，我们发现ORFK13/vFLIP刺激了KS皮损特有的炎症细胞的黏附。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能将前药5-氟-5-脱氧尿苷(5-dFUrd)代谢为5-氟尿苷(5-FU)的细胞酶。5-FU是一种有效的胸苷合成酶抑制剂，可以阻止DNA和RNA的合成。当进行细胞毒性测试时，5-dFUrd(0.1-1；M)选择性地杀死表达ORFK13/vFLIP的内皮细胞，而保留对照细胞。这些结果表明，ORFK13/vFLIP直接和间接参与了KS的炎症和血管表型，并证实5-dFUrd是一种潜在的针对KSHV潜伏期的新药，用于治疗KS和其他KSHV相关的恶性肿瘤。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MCP1，但目前还不清楚CXCR7是否可以响应这些配体或其他信号，或者它的功能是将配体从受体上隔离出来。最近，CXCR7被证明与CXCR4寡聚，CXCR4是一种可以对SDF1做出反应的受体。我们感兴趣的是CXCR7在KSHV感染中的作用。在小鼠身上的初步观察表明，CXCR7促进了KSHV诱导的肿瘤进展，但这种效应背后的机制目前正在研究中。原发性渗出性淋巴瘤(PEL)是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。尽管有大剂量化疗或其他治疗方法，但PEL是一种迅速致命的恶性肿瘤。雷帕霉素针对的是mTOR(哺乳动物雷帕霉素的靶点)，mTOR是一种在癌症中经常被解除调控的细胞信号通路的效应器，它对多种肿瘤，特别是淋巴来源的肿瘤显示出疗效。我们已经研究了雷帕霉素治疗实验性PEL的潜在用途。以前的研究表明，雷帕霉素可以有效地对抗小鼠皮下PEL。然而，这种临床前模型在其位置和进展方面与患者的疾病相去甚远。最近，雷帕霉素治疗的移植后受者的PEL的发展引发了对药物抗PEL活性的质疑。我们建立并使用了积液性PEL进展为腹膜肿瘤的小鼠模型来研究雷帕霉素的抗PEL活性。我们发现，雷帕霉素显著减少腹水积聚，延长小鼠的存活时间。最初，与对照小鼠相比，雷帕霉素降低了PEL负荷，但大多数小鼠迅速出现PEL进展。与对照组相比，接受雷帕霉素治疗的小鼠腹水中的血管内皮生长因子水平显著降低，血管通透性促进渗出形成。IL-10是PEL的主要自分泌生长因子，在雷帕霉素治疗的PEL小鼠中，IL-10的表达最初减少，但尽管治疗后仍迅速增加。我们发现腹水的低氧环境和雷帕霉素协同刺激PEL中IL-10的表达。这些结果不支持使用雷帕霉素作为PEL的根治疗法，但确认雷帕霉素是一种减少恶性积液的有效药物。目前在实验室的努力旨在进一步表征PEL对雷帕霉素的耐药性的发展以及如何预防它。

We have focused in three related areas: the study of vFLIP, a KSHV latent gene product expressed in Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and multicentric Castleman's disease; the study of CXCR7, a G protein-coupled receptor induced by KSHV in the host cells; and the development of new therapies for KSHV-induced malignances occurring in AIDS patients. One of the characteristic features of KSHV is its ability to promote angiogenesis and lymphangiogenesis. Unlike other viruses that have hijacked cellular genes that promote angiogenes, KHSV does not code for pro-angiogenic factors but rather promotes the expression of pro-angiogenic genes by the cells it infects. ORFK13/vFLIP encodes a 188-amino acid protein, which binds to the Iκb kinase (IKK) complex to activate NFκB. 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 (0.1-1μM) 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. 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 MCP1 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 signals in response to SDF1. We are interested in the function of CXCR7 in the context of KSHV infection. Initial observations in mice have shown that CXCR7 promotes KSHV-induced tumor progression, but the mechanisms underlying this effect are currently being investigated. Primary effusion lymphoma (PEL) is a fatal viral malignancy, 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 originin. 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 drugs 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.