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Ikb激酶（IKK）复合物结合以激活NFkB。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。在进一步的实验中，我们发现KSHV K13诱导原代人微血管内皮细胞中NF-kB调控蛋白A20、ABIN-1和ABIN-3的表达，KS梭形细胞在KS组织中表达A20。在报告者实验中，A20强烈破坏了k13诱导的293T细胞中NF-kB的激活，但ABIN-1和ABIN-3没有。突变分析表明，c端结构域（427-790残基）对NF-kB的A20调制至关重要。在功能实验中，A20抑制k13诱导的IP-10分泌，降低k13诱导的细胞增殖。因此，我们证明了A20负调控KSHV K13直接诱导的NF-kB激活。通过减弱K13诱导的对kshv感染细胞有害的过度和延长的NF-kB激活，A20可能在kshv相关疾病的发病机制中发挥重要作用，其中K13表达。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。在其他实验中，我们研究了kshv感染的KS细胞表型多样性的生化基础。这种多样性给KS肿瘤细胞的起源带来了尚未解决的不确定性。我们研究了KSHV感染内皮细胞并将其转化为间充质细胞的可能性。内皮细胞向间充质细胞转化（EndMT）是内皮细胞向间充质细胞转化的过程，在心脏发育过程中起着关键作用，是某些形式的病理性器官纤维化和组织骨化的基础。我们发现Kaposis sarcoma-associated herpesvirus （KSHV）是一种末端mt的诱导剂。KSHV感染后，原代真皮微血管内皮细胞失去内皮标志物的表达，获得间充质标志物的表达，表现出新的侵袭性和迁移性，并表现出更高的存活率。我们发现KSHV利用Notch信号通路和Notch诱导的转录因子Slug和ZEB1诱导EndMT的激活，而先前与EndMT相关的TGFb信号通路没有被利用。kshv感染的KS病变梭形细胞表现出具有内皮细胞和间充质细胞特征的复杂表型，显示Notch活性并表达核ZEB1，这些特征与kshv诱导的体内EndMT相一致。这些结果表明，KSHV利用EndMT程序赋予内皮细胞侵袭性和抗死亡能力，这些特性可能有助于KS的进展和KSHV的持久性。靶向Notch信号是治疗KS的一种新的实验方法。

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 Ikb kinase (IKK) complex to activate NFkB. 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. In additional experiments, we found that that KSHV K13 induces the expression of the NF-kB regulatory proteins A20, ABIN-1 and ABIN-3 in primary human microvascular endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired K13-induced NF-kB activation in 293T cells, but ABIN-1 and ABIN-3 did not. Mutational analysis established that the C-terminal domain (residues 427-790) is critical for A20 modulation of NF-kB. In functional assays, A20 inhibited K13-induced secretion of IP-10, and reduced K13-induced cell proliferation. Thus, we demonstrate that A20 negatively regulates NF-kB activation directly induced by KSHV K13. By attenuating excessive and prolonged NF-kB activation induced by K13 that could be harmful to KSHV-infected cells, A20 likely plays an important role in the pathogenesis of KSHV-associated diseases, in which K13 is expressed. 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. In other experiments we have examined the biochemical basis for diversity of phenotype within KS cells that are KSHV-infected. Such diversity has created unresolved uncertainties as to the origin of KS tumor cells. We have examined the possibility that KSHV infects endothelial cells and turns them into mesenchymal cells. Endothelial to mesenchymal transition (EndMT), the process by which endothelial cells convert into mesenchymal cells, plays critical roles during development of the heart, and underlies certain forms of pathological organ fibrosis and tissue ossification. We found that Kaposis sarcoma-associated herpesvirus (KSHV) is an inducer of EndMT. Upon KSHV infection, primary dermal microvascular endothelial cells lose expression of endothelial markers, acquire expression of mesenchymal markers, display new invasive and migratory properties, and exhibit increased survival. We discovered that the canonical Notch signaling pathway and the Notch-induced transcription factors Slug and ZEB1 are deployed by KSHV to induce activation of EndMT, whereas the TGFb signaling pathway previously linked to EndMT, is not utilized. The KSHV-infected spindle cells within KS lesions display a complex phenotype with features of endothelial and mesenchymal cells, display evidence of Notch activity and express nuclear ZEB1, features compatible with KSHV-induced EndMT in vivo. These results show that KSHV utilizes the EndMT program to endow endothelial cells with invasiveness and resistance to death, traits that likely contribute to KS progression and KSHV persistence. Targeting Notch signaling emerges as a novel experimental approach to the treatment of KS.