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）、原发性积液性淋巴瘤（PEL）和多中心Castleman病（MCD）中表达的研究；2. 与细胞IL-6结构相似的KSHV病毒产物IL-6及其在MCD发病中的作用研究；3. 宿主细胞中受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；和4。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某些促炎细胞因子、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（残基427-790）对a20pel至关重要，它是人类中一种致命的病毒恶性肿瘤，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失调的细胞信号通路的效应物，对多种肿瘤，特别是NF-kB的调节都有疗效。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。本课题组先前的研究表明，IL-6是KSHV表达的早期裂解基因，与细胞IL-6细胞因子同源。与细胞IL-6不同，我们发现vIL-6可以直接通过gp130/JAK-STAT途径结合并发出信号，而不需要细胞IL-6受体。Gp130是一种相当普遍的跨膜蛋白；因此，在细胞细胞因子的作用下，vIL-6可以激活体内几乎所有的细胞。之前，我们已经在NIH3T3细胞中转导了vIL6；当这些细胞被转移到t细胞免疫缺陷小鼠体内时，它们产生肿瘤的比率明显高于对照NIH3T3细胞，重要的是，小鼠在许多组织中出现脾肿大、肝肿大和浆细胞增多。这些特征在MCD患者中很常见。我们现在已经生产了转基因小鼠系，在这些小鼠系中，vIL-6无处不在地表达。研究发现，这些小鼠的血清il -6水平与kshv感染患者相当，脾脏和淋巴结中磷酸化STAT3含量升高，其中大量产生了il -6，并且自发发展为人类浆细胞型MCD的关键特征，包括脾肿大、多灶性淋巴结病、高γ球蛋白血症和浆细胞增多症。有趣的是，在细胞性il -6缺陷小鼠中表达il -6转基因并没有产生在il -6正常小鼠中观察到的mcd样表型。这表明内源性细胞IL-6在MCD的自然史中是一个关键的辅助因子。我们之前报道过IL-6可以在体外诱导细胞IL-6的表达，并且在MCD患者中经常检测到异常高水平的IL-6和细胞IL-6。这些观察结果表明，人IL-6在kshv相关MCD的发病机制中起重要作用。因此，我们与NCI的HAMB小组合作，启动了一项临床试验，探索阻断细胞IL-6在MCD治疗中的效用。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与趋化因子SDF1和I-TAC结合，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/CXCL12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。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KSHV是EndMT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tgf - β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 (MCD); 2. the study of vIL-6, a KSHV viral product with structural similarity to cellular IL-6, and its role in the athogenesis of MCD; 3. 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 4. 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 certain 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 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 thomodulation 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. Previous studies from our group characterized vIL-6 as an early lytic gene expressed by KSHV, homologous to the cellular IL-6 cytokine. Unlike cellular IL-6, we found that vIL-6 can directly bind and signal through the gp130/JAK-STAT pathway without a requirement for the cellular IL-6 receptor. gp130 is a fairly ubiquitous transmembrane protein; thus, in contract to the cellular cytokine, vIL-6 can activate virtually all cells in the body. Previously, we have transduced vIL6 in NIH3T3 cells; when these cells were transferred to T-cell immunodeficient mice, they generated tumors at a significantly higher rate than control NIH3T3 cells and, importantly, the mice developed splenomegaly, hepatomegaly and plasmacytosis in many tissues. These features are common to patients with MCD. We have now produced transgenic mouse lines in which vIL-6 is ubiquitously expressed. These mice were found to exhibit vIL-6 serum levels comparable with those observed in KSHV-infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was abundantly produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulin-emia, and plasmacytosis. Interestingly, expression of the vIL-6 transgene in cellular IL-6-deficient mice did not yield the MCD-like phenotype observed in IL-6-competent mice. This indicated that endogenous cellular IL-6 is a critical co-factor in the natural history of MCD. We have previously reported that vIL-6 can induce the expression of cellular IL-6 in vitro, and that vIL-6 and cellular IL-6 are often detected at abnormally high levels in patients with MCD. These observations suggest that human IL-6 plays an important role in the pathogenesis of KSHV-associated MCD. Thus, in collaboration with the HAMB group at NCI we have initiated a clinical trial exploring the utility of blocking cellular IL-6 in the treatment of MCD.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 sequester ligands away from their receptors. Recently, CXCR7 was shown to oligomerize with CXCR4, a receptor that can signal in response to SDF1/CXCL12. 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. In other experiments we have examined the biochemical basis for diversity of phenotype within KS cells that are KSHV-infected. Such diversity has created uncertainties on 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 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 TGF-beta 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.