A Role for KSHV in the Pathogenesis of Malignancies

我们重点关注四个相关领域：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发病机制中的作用研究；3. MCD的特殊临床表现；4. 体腔微环境在PEL发病机制中的作用和5。艾滋病患者发生的kshv诱导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。因此，这些结果提供了证据，证明KSHV通过促进NF-kB激活（导致炎症细胞因子的表达）和通过促进NF-kB抑制因子A20的表达（导致细胞死亡的抑制）来精细调节宿主细胞中的NF-kB。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，其表达仅限于某些细胞类型。由于gp130是一种相当普遍的蛋白质，vIL-6与细胞细胞因子相结合，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，它们的肿瘤生成率明显高于对照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，il -6转基因小鼠与il -6缺失小鼠杂交后，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。在相关研究中，我们发现了3例出现严重MCD症状的患者，并且由于HHV-8阳性细胞共享MCD病变中HHV-8感染细胞的表型（IgMl, CD19+, CD20-, CD138-），循环b细胞迅速扩增（44-81%）。这些患者在血液中表现出非常高的HHV-8病毒载量（7 log HHV-8 DNA拷贝/ml）和高水平的血清il -6，人类白细胞介素6的病毒同源物。血清IL-6和IL-10也异常升高。因此，我们扩大了hiv感染患者中与HHV-8相关的浆母细胞性淋巴细胞增生性疾病的范围，包括HHV-8+多克隆b细胞淋巴细胞增多症。此外，我们还发现了一组临床表现和实验室参数与MCD相似的患者，但不符合MCD的组织学标准，他们没有淋巴结病变，也没有MCD的组织学证据。我们将这种新综合征称为KICS， kshv相关炎症细胞因子综合征。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感染促进内皮细胞向间质细胞转化（EndMT）的可能性。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至关重要的作用，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。此外，KSHV调节DLC1的表达，DLC1调节NFkB的激活及其下游靶标A20。靶向Notch信号是治疗KS的一种新的实验方法。PEL是一种与KSHV相关的毁灭性淋巴瘤，通常与EBV合并，EBV通常表现为体腔内的液体恶性肿瘤。这种特殊的位置通常归因于PEL细胞高水平的VEGF分泌，这促进了与积液发病机制相关的血管通透性。然而，我们已经考虑到PEL出现的特殊部位可能反映了这个小生境特殊的促肿瘤功能的可能性。目前的研究已经确定了间皮来源的因素，对PEL的发展和生长至关重要。针对这些因素是我们目前对PEL研究的重点。

We have focused in four 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 pathogenesis of MCD; 3. Peculiar clinical presentations of MCD; 4. The role of body cavity microenvironment in the pathogenesis of PEL; and 5. 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 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. Thus, these results provide evidence that KSHV finely modulates NF-kB in the host cells by both promoting NF-kB activation (resulting in expression of inflammatory cytokines) and tempering this activation by promoting expression of the NF-kB repressor A20 (resulting in inhibition of cell death). 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, whose expression is restricted to certain cell types. Since gp130 is a fairly ubiquitous protein, vIL-6, in contract to the cellular cytokine, can activate virtually all cells in the body. Previously, we have transduced vIL6 in NIH3T3 cells; when these cells were transferred into 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, hypergammaglobulinemia, and plasmacytosis. Interestingly, the vIL-6 transgenic mice crossed into 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. In related studies, we have identified three patients who presented with severe symptoms of MCD and had a rapid expansion of circulating B-cells (44-81%) attributable to HHV-8 positive cells sharing the phenotype (IgMl, CD19+, CD20-, CD138-) of HHV-8-infected cells from MCD lesions. These patients displayed a very high HHV-8 viral load in blood (7 logs HHV-8 DNA copies/ml) and high levels of serum vIL-6, the viral homologue of human interleukin 6. Serum IL-6 and IL-10 were also abnormally elevated. Thus, we have expanded the spectrum of HHV-8-related plasmablastic lymphoproliferative disorders in HIV-infected patients to include HHV-8+ polyclonal B-cell lymphocytosis. Additionally, we have identified a group of patients who resemble MCD patients in their clinical manifestations and laboratory parameters, but do not meet the histological criteria of MCD and they have no lymphadenopathy and no histologic evidence of MCD. We have labeled this new syndrome as KICS, KSHV-associated inflammatory cytokine syndrome. 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 infection promotes endothelial to mesenchymal transition (EndMT). This process of endothelial cell conversion 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 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. Additionally, KSHV regulates expression of DLC1, which modulates NFkB activation and its downtream targer A20. Targeting Notch signaling emerges as a novel experimental approach to the treatment of KS. PEL is a devastating lymphoma associated with KSHV, often in conjunction with EBV, which typically presents as a liquid malignancy in the body cavities. This peculiar location has often been attributed to high level VEGF secretion by PEL cells, which promotes vascular permeability associated with the pathogenesis of effusions. However, we have considered the possibility that the peculiar site of PEL presentation may reflect peculiar pro-tumorigenic functions of this niche. Current studies have identified mesothelial-derived factors that critically contribute to PEL development and growth. Targeting such factors is a current focus of our research on PEL.