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.

我们专注于三个相关领域：1。vFlip的研究是一种以KSHV感染的细胞靶标表达的KSHV潜在基因产物，以及Kaposi的肉瘤（KS），一级积液淋巴瘤（PEL）和多中心Castleman病； 2。对宿主细胞中KSHV诱导的G蛋白偶联受体的CXCR7的研究及其相关的受体CXCR4，该受体并非由KSHV诱导；和3。艾滋病患者发生的KSHV引起的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，并通过促进产生亲血管生成因子的细胞募集并促进细胞感染细胞的亲抗血管生成基因的表达，从而间接促进血管生成和淋巴管生成。 ORFK13/VFLIP编码188个氨基酸蛋白，该蛋白与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，我们将逆转录病毒转导的ORFK13/VFLIP转移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诱导了KS细胞的纺锤形态，并促进了典型的KS脉管系统典型的异常血管网络的形成。 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性细胞因子，趋化因子和干扰素反应性基因的表达增加。这项研究代表了KSHV-VFLIP对基因调节的首次综合分析。正如人们对促炎性细胞因子和趋化因子刺激可能期望的那样，我们发现ORFK13/VFLIP刺激KS病变特征的炎性细胞的粘附。 在其他实验中，我们发现KSHV K13在原代人微血管内皮细胞中诱导NF-KB调节蛋白A20，ABIN-1和ABIN-3的表达，并且KS纺锤体在KS组织中表达A20。在报告基因测定中，A20在293T细胞中严重损害了K13诱导的NF-KB激活，但是ABIN-1和ABIN-3没有。突变分析表明，C末端结构域（残基427-790）对于NF-KB的A20调节至关重要。在功能测定中，A20抑制了K13诱导的IP-10分泌，并降低了K13诱导的细胞增殖。因此，我们证明A20对KSHV K13直接诱导的NF-KB激活负调节。通过减轻K13诱导的过度和延长的NF-KB激活，可能对KSHV感染的细胞有害，A20可能在表达K13的KSHV相关疾病的发病机理中起重要作用，其中表达K13。 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结合了趋化因子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，或者其功能是否用于将配体隔离远离受体。最近，CXCR7显示与CXCR4的寡聚，该受体可以响应SDF1发出信号。我们对KSHV感染中CXCR7的功能感兴趣。我们已经在PEL（一级积液淋巴瘤）细胞系中过表达或沉默的CXCR7，并在小鼠中测试了其肿瘤性。最初的观察结果表明，CXCR7促进了PEL诱导的肿瘤进展。我们目前正在探索CXCR7在PEL恶性肿瘤中的这种促肿瘤效应的基础机制。 PEL是人类的致命病毒恶性肿瘤，通常将后来传播为恶性积液。尽管接受了高剂量化疗或其他疗法的治疗，但PEL还是一种迅速致命的恶性肿瘤。雷帕霉素靶向MTOR（雷帕霉素的哺乳动物靶标），这是一种经常在癌症中放松管制的细胞信号通路的效应子，对多种肿瘤，尤其是淋巴样的肿瘤表现出疗效。我们已经研究了雷帕霉素对实验PEL治疗的潜在效用。先前的研究表明，雷帕霉素可以在小鼠中对皮下PEL有效。但是，这种临床前模型与患者在其位置和进展的患者中被远离疾病。最近，经过雷帕霉素治疗的移植后接受者的PEL发育提出了有关该药物抗PEL活性的问题。我们已经开发并使用了一种鼠模型的鼠模型，该模型的脑肿瘤发展为腹膜肿瘤，以研究雷帕霉素的抗PEL活性。我们发现雷帕霉素可显着降低腹水的积累并延长小鼠的存活。最初，与对照小鼠相比，雷帕霉素降低了PEL载荷，但大多数小鼠迅速表现出PEL进展。与对照组相比，在雷帕霉素处理的小鼠的腹水中，VEGF的水平促进了导致积液形成的血管通透性的水平显着降低。 IL-10的表达是PEL的主要自分泌生长因子的表达，最初从雷帕霉素治疗的小鼠的PEL中降低了PEL的表达，但尽管治疗了，但仍迅速增加。我们发现，腹水和雷帕霉素的低氧环境在刺激PEL中的IL-10表达方面合作。这些结果不支持使用雷帕霉素作为PEL的治疗方法，而是鉴定雷帕霉素是减少恶性积液积累的有效药物。目前在实验室中的努力旨在进一步表征PEL对雷帕霉素的耐药性以及如何预防它的发展。特别是，我们正在测试将雷帕霉素结合到中和IL-10的潜在功效。 在其他实验中，我们研究了受KSHV感染的KS细胞内表型多样性的生化基础。这种多样性对KS肿瘤细胞的起源产生了未解决的不确定性。我们已经检查了KSHV感染内皮细胞并将其变成间质细胞的可能性。内皮到间质转变（EndMT），内皮细胞转化为间质细胞，在心脏发育过程中起着关键作用，并构成了某些形式的病理器官纤维化和组织骨化。我们发现Kaposis肉瘤相关的疱疹病毒（KSHV）是EndMT的诱导剂。 KSHV感染后，原发性皮肤微血管内皮细胞失去了内皮标记的表达，获得间充质标记的表达，显示出新的侵入性和迁移特性，并表现出增加的存活率。我们发现，KSHV部署了规范凹口信号通路和Notch诱导的转录因子SLUG和ZEB1，以诱导ENDMT的激活，而先前与EndMT链接的TGFB信号通路，并未利用。 KS病变中的KSHV感染的纺锤体细胞显示出复杂的表型，具有内皮和间质细胞的特征，显示了Notch活性的证据并表达核Zeb1，特征与KSHV诱导的EndMT In Vivo兼容。这些结果表明，KSHV利用EndMT程序将内皮细胞具有侵入性和对死亡的抵抗力，可能导致KS进展和KSHV持久性的特征。靶向Notch信号作为KS治疗的一种新型实验方法。