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: 8350079
• 负责人: Giovanna Tosato
• 金额: $ 50.05万
• 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/8350079
• 关键词: Acquired Immunodeficiency Syndrome; Adhesions; Amino Acids; Angiogenic Factor; Angiolymphoid hyperplasia; Area; Ascites; Attenuated; Binding; Biochemical; Biological Assay; Blood Vessels; C-terminal; CXCL10 gene; CXCL11 gene; CXCL12 gene; CXCR4 Receptors; CXCR4 gene; Cause of Death; Cell Line; Cell Proliferation; Cells; Cessation of life; Characteristics; Complex; Dermal; Development; Disease; Drug Delivery Systems; Effector Cell; Endothelial Cells; Environment; Exhibits; FADD protein; Fibrosis; Frequencies; G-Protein-Coupled Receptors; Gene Expression Microarray Analysis; Gene Expression Regulation; Gene Proteins; Genes; Goals; Growth Factor; HIV; HIV Infections; Herpesviridae Infections; High Dose Chemotherapy; Hodgkin Disease; Human; Human Herpesvirus 8; Hypoxia; Immunity; Individual; Inflammatory; Interferons; Interleukin-10; Kaposi Sarcoma; Laboratories; Lesion; Ligands; Link; Location; Lymphangiogenesis; Lymphoid; Lymphoma; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mesenchymal; Modeling; Molecular; Morphology; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; NF-kappa B; Notch Signaling Pathway; Nuclear; Organ; Pathogenesis; Patients; Peritoneal; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Play; Pre-Clinical Model; Process; Property; Proteins; Reporter; Research; Resistance; Role; Signal Pathway; Signal Transduction; Sirolimus; T-Lymphocyte; Testing; Tissues; Transplant Recipients; Tumor Angiogenesis; Tumorigenicity; Uncertainty; Vascular Endothelial Growth Factors; Vascular Permeabilities; Viral; Work; angiogenesis; autocrine; base; cancer type; cardiogenesis; cell growth; chemokine; chemokine receptor; cytokine; effusion; genetic regulatory protein; in vivo; interest; mTOR protein; neoplastic cell; notch protein; novel; overexpression; prevent; programs; receptor; research study; response; slug; subcutaneous; 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; 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.

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 病中表达； 2.宿主细胞中KSHV诱导的G蛋白偶联受体CXCR7及其非KSHV诱导的相关受体CXCR4的研究； 3. 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进行微阵列分析，检测到促炎细胞因子、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。突变分析确定 C 端结构域（残基 427-790）对于 A20 调节 NF-kB 至关重要。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。 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，但仍不清楚 CXCR7 是否可以响应这些配体或其他信号而发出信号，或者其功能是否是将配体与其受体隔离。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 PEL（原发性渗出性淋巴瘤）细胞系中过度表达或沉默 CXCR7，并在小鼠中测试了它们的致瘤性。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 是人类致命的病毒性恶性肿瘤，通常表现为恶性渗出液，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，特别是淋巴源性肿瘤显示出疗效。我们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可以有效对抗小鼠皮下 PEL。然而，这种临床前模型与患者疾病的位置和进展相距甚远。最近，雷帕霉素治疗的移植后受者体内 PEL 的发展引发了对该药物抗 PEL 活性的质疑。我们开发并使用了渗出性 PEL 发展为腹膜肿瘤的小鼠模型来研究雷帕霉素的抗 PEL 活性。我们发现雷帕霉素显着减少腹水积聚并延长小鼠的存活时间。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的 VEGF 水平显着降低，VEGF 可促进血管通透性，从而导致积液形成。 IL-10（PEL 的主要自分泌生长因子）的表达最初在雷帕霉素治疗小鼠的 PEL 中降低，但尽管进行了治疗，但仍迅速增加。我们发现腹水的缺氧环境和雷帕霉素协同刺激PEL中IL-10的表达。这些结果并不支持使用雷帕霉素作为 PEL 的治疗方法，但确定雷帕霉素是减少恶性积液积聚的有效药物。实验室目前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。我们发现卡波西斯肉瘤相关疱疹病毒 (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 TGFb 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。