A Role for KSHV in the Pathogenesis of Malignancies

KSHV 在恶性肿瘤发病机制中的作用

• 批准号: 7969830
• 负责人: Giovanna Tosato
• 金额: $ 44.42万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969830
• 关键词: Acquired Immunodeficiency Syndrome; Adhesions; Amino Acids; Angiogenic Factor; Area; Ascites; Binding; Blood Vessels; CCL2 gene; CXCL12 gene; CXCR4 gene; Cause of Death; Cells; Characteristics; Code; Complex; DNA; Deoxyuridine; Development; Disease; Drug Delivery Systems; Effector Cell; Endothelial Cells; Environment; Enzymes; Fluorouracil; G-Protein-Coupled Receptors; Gene Expression Microarray Analysis; Gene Expression Regulation; Gene Proteins; Genes; Goals; Growth Factor; HIV Infections; High Dose Chemotherapy; Hodgkin Disease; Human; Human Herpesvirus 8; Hypoxia; Immunity; Individual; Infection; Inflammatory; Interferons; Interleukin-10; Kaposi Sarcoma; Laboratories; Lesion; Ligands; Location; Lymphangiogenesis; Lymphoid; Lymphoma; Malignant - descriptor; Malignant Neoplasms; Microarray Analysis; Modeling; Molecular; Morphology; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Pathogenesis; Patients; Peritoneal; Pharmaceutical Preparations; Phenotype; Phosphorylase a; Phosphotransferases; Pre-Clinical Model; Process; Prodrugs; Proteins; RNA chemical synthesis; Research; Resistance; Role; Signal Pathway; Signal Transduction; Sirolimus; T-Lymphocyte; Thymidine; Thymidine Phosphorylase; Transplant Recipients; Tumor Angiogenesis; Vascular Endothelial Growth Factors; Vascular Permeabilities; Viral; Virus; Work; angiogenesis; autocrine; cancer type; cell growth; chemokine; chemokine receptor; cytokine; cytotoxicity test; effusion; inhibitor/antagonist; interest; killings; mTOR protein; neoplastic cell; prevent; receptor; response; subcutaneous; success; therapeutic target; treatment strategy; tumor; tumor progression; tumorigenic

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.

我们专注于三个相关领域：VFLIP的研究，这是一种在Kaposi的肉瘤（KS），一级积液淋巴瘤（PEL）和多中心Castleman病中表达的KSHV潜伏基因产物； CXCR7的研究是宿主细胞中KSHV诱导的G蛋白偶联受体的研究；以及在艾滋病患者中开发针对KSHV引起的恶性肿瘤的新疗法。 KSHV的特征之一是它促进血管生成和淋巴管生成的能力。与其他劫持了促进血管生成的细胞基因的病毒不同，KHSV不为促血管生成因子代码，而是通过其感染的细胞促进促血管生成基因的表达。 ORFK13/VFLIP编码188-氨基酸蛋白，该蛋白与I＆＃954; B激酶（IKK）复合物结合以激活NF＆＃954; b。我们检查了ORFK13/VFLIP对KS表型的贡献以及治疗靶向的潜力。为此，我们将逆转录病毒转导的ORFK13/VFLIP转移到原代人内皮细胞中，并检查了该基因对KS表型的贡献。我们发现ORFK13/VFLIP诱导了KS细胞的纺锤形态，并促进了典型的KS脉管系统典型的异常血管网络的形成。 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性细胞因子，趋化因子和干扰素反应性基因的表达增加。这项研究代表了KSHV-VFLIP对基因调节的首次综合分析。正如人们对促炎性细胞因子和趋化因子刺激可能期望的那样，我们发现ORFK13/VFLIP刺激KS病变特征的炎性细胞的粘附。微阵列分析发现，ORFK13/VFLIP促进了胸苷磷酸化酶的表达，胸苷磷酸化酶是一种可代谢前药5-氟-5-脱氧尿苷（5-DFURD）为5-氟岛（5-Fuouridine（5-FU））。有效的胸苷合成酶抑制剂，5-FU阻断DNA和RNA合成。当测试细胞毒性时，5-DFURD（0.1-1＆＃956; m）有选择地杀死了在保留控制细胞的同时杀死表达的内皮细胞的ORFK13/VFLIP表达内皮细胞。这些结果表明，ORFK13/VFLIP直接和间接地有助于KS的炎症和血管表型，并将5-DFURD鉴定为一种潜在的新药，该药物针对KSHV延迟KSHV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结合了趋化因子SDF1和MCP1，但尚不清楚CXCR7是否可以对这些配体或其他信号发出信号，或者其功能是否用于将配体隔离远离受体。最近，CXCR7显示与CXCR4的寡聚，该受体可以响应SDF1发出信号。我们对KSHV感染中CXCR7的功能感兴趣。小鼠的最初观察结果表明，CXCR7促进了KSHV诱导的肿瘤进展，但是目前正在研究此作用的机制。原发性积液淋巴瘤（PEL）是一种致命的病毒恶性肿瘤，通常以恶性积液形式呈现，后来散发出来。尽管接受了高剂量化疗或其他疗法的治疗，但PEL还是一种迅速致命的恶性肿瘤。雷帕霉素靶向MTOR（雷帕霉素的哺乳动物靶标），这是细胞信号通路的效应因子，通常在癌症中放松管制，对多种肿瘤，尤其是淋巴样起源的肿瘤表现出疗效。我们已经研究了雷帕霉素对实验PEL治疗的潜在效用。先前的研究表明，雷帕霉素可以在小鼠中对皮下PEL有效。但是，这种临床前模型与患者在其位置和进展的患者中被远离疾病。最近，经过雷帕霉素治疗的移植后接受者的PEL发育提出了有关药物抗PEL活性的问题。我们已经开发并使用了一种鼠模型的鼠模型，该模型的脑肿瘤发展为腹膜肿瘤，以研究雷帕霉素的抗PEL活性。我们发现雷帕霉素可显着降低腹水的积累并延长小鼠的存活。最初，与对照小鼠相比，雷帕霉素降低了PEL载荷，但大多数小鼠迅速表现出PEL进展。 与对照组相比，在雷帕霉素处理的小鼠的腹水中，VEGF的水平促进了导致积液形成的血管通透性的水平显着降低。 IL-10的表达是PEL的主要自分泌生长因子的表达，最初从雷帕霉素治疗的小鼠的PEL中降低了PEL的表达，但尽管治疗了，但仍迅速增加。我们发现，腹水和雷帕霉素的低氧环境在刺激PEL中的IL-10表达方面合作。这些结果不支持使用雷帕霉素作为PEL的治疗方法，而是鉴定雷帕霉素是减少恶性积液积累的有效药物。目前在实验室中的努力旨在进一步表征PEL对雷帕霉素的耐药性以及如何预防它的发展。