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: 8763706
• 负责人: Giovanna Tosato
• 金额: $ 44.32万
• 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/8763706
• 关键词: AIDS related cancer; Acquired Immunodeficiency Syndrome; Adhesions; Amino Acids; Angiogenic Factor; Area; Attenuated; B-Lymphocytes; Binding; Biochemical; Biological Assay; Blood; Blood Vessels; C-terminal; CD19 gene; CXCL10 gene; Cause of Death; Cell Proliferation; Cells; Cessation of life; Characteristics; Clinical; Clinical Trials; Collaborations; Complex; Contracts; DNA; Dermal; Development; Disease; Drug Targeting; Effector Cell; Endothelial Cells; Exhibits; FADD protein; Fibrosis; Frequencies; Gene Expression Microarray Analysis; Gene Expression Regulation; Gene Proteins; Genes; Goals; HIV; HIV Infections; Hepatomegaly; High Dose Chemotherapy; Hodgkin Disease; Homologous Gene; Human; Human Herpesvirus 8; Immunity; Immunodeficient Mouse; In Vitro; Individual; Infection; Inflammatory; Integral Membrane Protein; Interferons; Interleukin-10; Interleukin-6; Kaposi Sarcoma; Lesion; Link; Lymphangiogenesis; Lymphatic Diseases; Lymphocytosis; Lymphoproliferative Disorders; Lytic; MS4A1 gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mesenchymal; Molecular; Morbidity - disease rate; Morphology; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; NF-kappa B; Natural History; Notch Signaling Pathway; Nuclear; Organ; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Plasma Cells; Plasmablast; Play; Process; Property; Proteins; Reporter; Reporting; Research; Resistance; Role; STAT3 gene; Serum; Signal Pathway; Signal Transduction; Sirolimus; Spleen; Splenomegaly; Symptoms; T-Lymphocyte; TGF Beta Signaling Pathway; Tissues; Transgenes; Transgenic Mice; Tumor Angiogenesis; Uncertainty; Viral; Viral Load result; Work; angiogenesis; base; cancer type; cardiogenesis; cell growth; cell type; chemokine; cytokine; cytopenia; effusion; genetic regulatory protein; global health; in vivo; interest; leukemia/lymphoma; lymph nodes; mTOR protein; mortality; neoplastic cell; notch protein; novel; primary effusion lymphoma; programs; receptor; research study; slug; success; therapeutic target; trait; transcription factor; tumor; tumor progression; tumorigenic

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. Peculiar clinical presentations of MCD; 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. MCD is characterized by inflammatory symptoms, lymphadenopathy, splenomegaly and cytopenia. KSHV is the cause of virtually all cases of MCD occurring in HIV-infected patients. MCD lesions characteristically contain HHV-8-infected polyclonal IgMl plasmablasts. 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 homolog 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 IgMl B-cell lymphocytosis. At onset, this lymphoproliferative disorder may mimick plasmablastic leukemia/lymphoma. 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.

我们重点关注三个相关领域： 1. vFLIP 的研究，vFLIP 是一种 KSHV 潜伏基因产物，在 KSHV 感染的细胞靶标以及卡波西肉瘤 (KS)、原发性渗出性淋巴瘤 (PEL) 和多中心 Castleman 病 (MCD) 中表达； 2、与细胞IL-6结构相似的KSHV病毒产物vIL-6及其在MCD发展中作用的研究； 3.MCD特有的临床表现； 4. 开发针对艾滋病患者中 KSHV 诱发的恶性肿瘤的新疗法。 KSHV的特征之一是其能够感染内皮细胞，并主要通过促进产生促血管生成因子的细胞的募集以及促进其感染的细胞表达促血管生成基因来间接促进血管生成和淋巴管生成。 ORFK13/vFLIP 编码 188 个氨基酸的蛋白质，该蛋白质与 Ikb 激酶 (IKK) 复合物结合以激活 NFkB。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，我们通过逆转录病毒将 ORFK13/vFLIP 转导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 KS 细胞独特的纺锤体形态，并促进无序 KS 脉管系统典型的异常血管网络的形成。对 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、趋化因子和干扰素反应基因的表达增加。这项研究代表了 KSHV-vFLIP 基因调控的首次全面分析。正如人们对促炎细胞因子和趋化因子的刺激所预期的那样，我们发现 ORFK13/vFLIP 刺激 KS 病变特征性炎症细胞的粘附。在其他实验中，我们发现KSHV K13诱导原代人微血管内皮细胞中NF-kB调节蛋白A20、ABIN-1和ABIN-3的表达，并且KS梭形细胞在KS组织中表达A20。在报告基因检测中，A20 严重损害了 293T 细胞中 K13 诱导的 NF-kB 激活，但 ABIN-1 和 ABIN-3 却没有。突变分析表明，C 端结构域（残基 427-790）对于 A20 PEL 至关重要，A20 PEL 是人类致命的病毒恶性肿瘤，通常表现为恶性渗出物，随后扩散。尽管采用高剂量化疗或其他疗法进行治疗，PEL 仍是一种快速致命的恶性肿瘤。雷帕霉素以 mTOR（雷帕霉素的哺乳动物靶标）为靶点，mTOR 是一种在癌症中经常失调的细胞信号传导途径的效应器，它对多种肿瘤显示出功效，特别是对 NF-kB 的调节。在功能测定中，A20 抑制 K13 诱导的 IP-10 分泌，并减少 K13 诱导的细胞增殖。因此，我们证明 A20 负向调节 KSHV K13 直接诱导的 NF-kB 激活。通过减弱 K13 诱导的过度和长时间的 NF-kB 激活（可能对 KSHV 感染的细胞有害），A20 可能在 KSHV 相关疾病（K13 表达于其中）的发病机制中发挥重要作用。我们小组之前的研究将 vIL-6 描述为 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 可以直接结合 gp130/JAK-STAT 通路并发出信号，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，与细胞因子结合，vIL-6 几乎可以激活体内所有细胞。此前，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，它们产生肿瘤的速度明显高于对照 NIH3T3 细胞，而且重要的是，小鼠在许多组织中出现了脾肿大、肝脏肿大和浆细胞增多症。这些特征对于 MCD 患者来说很常见。我们现已培育出普遍表达 vIL-6 的转基因小鼠品系。研究发现，这些小鼠的 vIL-6 血清水平与 KSHV 感染患者中观察到的水平相当，在大量产生 vIL-6 的脾脏和淋巴结中磷酸化 STAT3 含量升高，并且自发地发展出人类的关键特征。浆细胞型 MCD，包括脾肿大、多灶性淋巴结病、高丙种球蛋白血症和浆细胞增多症。有趣的是，vIL-6转基因在细胞IL-6缺陷小鼠中的表达并没有产生在IL-6感受态小鼠中观察到的MCD样表型。这表明内源性细胞 IL-6 是 MCD 自然史中的关键辅助因子。我们之前报道过vIL-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vIL-6和细胞IL-6的水平异常高。这些观察结果表明人 IL-6 在 KSHV 相关 MCD 的发病机制中发挥重要作用。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。 MCD 的特征是炎症症状、淋巴结肿大、脾肿大和血细胞减少。 KSHV 是 HIV 感染者中几乎所有 MCD 病例的病因。 MCD病变特征性地含有HHV-8感染的多克隆IgM1浆母细胞。我们已经确定了三名出现严重 MCD 症状且循环 B 细胞快速扩增 (44-81%) 的患者，这是由于 HHV-8 阳性细胞具有以下表型（IgMl、CD19+、CD20-、CD138-）来自 MCD 病变的 HHV-8 感染细胞。这些患者的血液中显示出非常高的 HHV-8 病毒载量（7 个对数 HHV-8 DNA 拷贝/ml）和高水平的血清 vIL-6（人白细胞介素 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 HHV-8 相关的浆母细胞淋巴增殖性疾病的范围，包括 HHV-8+ 多克隆 IgM1 B 细胞淋巴细胞增多症。发病时，这种淋巴增殖性疾病可能类似于浆母细胞白血病/淋巴瘤。在其他实验中，我们检查了 KSHV 感染的 KS 细胞内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) 是内皮细胞转化为间充质细胞的过程，在心脏发育过程中发挥着关键作用，并且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记物的表达，获得间充质标记物的表达，表现出新的侵袭和迁移特性，并表现出生存增加。我们发现，KSHV 部署经典的 Notch 信号通路以及 Notch 诱导的转录因子 Slug 和 ZEB1 来诱导 EndMT 的激活，而之前与 EndMT 相关的 TGF-β 信号通路并未被利用。 KS 病变内感染 KSHV 的梭形细胞表现出复杂的表型，具有内皮细胞和间充质细胞的特征，显示出 Notch 活性的证据并表达核 ZEB1，这些特征与体内 KSHV 诱导的 EndMT 兼容。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，这些特征可能有助于 KS 进展和 KSHV 持续存在。靶向 Notch 信号传导是治疗 KS 的一种新颖的实验方法。