Mechanisms of KSHV-Induced Cellular Transformation

描述（由申请人提供）：卡波西肉瘤（KS）是最常见的艾滋病相关恶性肿瘤，也是艾滋病毒流行的非洲部分地区最常见的癌症。感染因子卡波西肉瘤疱疹病毒（KSHV;也称为HHV 8）存在于KS病变中的梭形细胞、其EC前体和单核细胞以及循环EC祖细胞和PBMC中。KS的几个特征表明趋化因子及其受体（CR）在疾病中发挥作用。KS肿瘤显示出深刻的血管生成和广泛的炎性浸润，这两个特征指示趋化因子/CR活性。此外，KS的多灶性性质表明，KSHV感染的梭形细胞前体或白细胞归巢到允许位点以启动或维持肿瘤发展。SDF-1/CXCR4轴是与肿瘤生长、血管生成和转移相关的主要趋化因子/CR相互作用之一。然而，SDF-1/CXCR4在KS中的作用仍然不清楚;迄今为止进行的有限研究仅显示这些分子存在于KS肿瘤中。我们已经表明，最近表征的SDF-1，RDC1（也CXCR7）的替代受体，强烈诱导KSHV感染的EC。虽然RDC1在非恶性细胞中的表达有限，但它在几种癌症和肿瘤相关EC中表达良好。我们最近发现RDC1在KS肿瘤组织中高度表达，并且我们和其他人已经表明SDF-1通过RDC1信号传导促进几种细胞类型中的迁移，血管生成，生长和促生存活性。这些发现表明，SDF-1通过其受体CXCR4和RDC1中的任一种进行信号传导，在KS肿瘤的接种和随后的发展中起着重要作用。我们假设RDC1和CXCR4在EC/梭形细胞中由KSHV病理诱导或激活，并且肿瘤微环境中和/或由EC/梭形细胞共表达的SDF-1激活下游信号传导途径，导致促血管生成、促生长、促存活表型，并促进细胞向病变发展部位的运输。为了了解SDF-1/CXCR4和SDF-1/RDC1在KS肿瘤种植、起始和发展中的作用，我们提出了三个具体目标。在目的1中，我们提出了表征SDF-1，CXCR4和RDC1的表达模式（i）在体内，在KS肿瘤和（ii）在体外，在不同类型的KSHV感染的内皮细胞。在目标2中，我们将确定KSHV感染的EC中（a）SDF-1/CXCR 4和（B）SDF-1/RDC1信号传导的性质和功能。在目标3中，我们将使用来自一组KSHV血清阳性患者的样本（有或无KS和HIV）来计数CEC数量，并确定这些细胞上SDF-1、CXCR4和RDC1的表达和功能。还将对正常和队列CEC在体外扩增为血液生长EC后进行功能研究。CXCR4拮抗剂目前处于临床前和临床开发阶段，用于治疗白血病、肺癌和乳腺癌。最近已经开发出特异性RDC1拮抗剂，并在动物模型中显示出抗肿瘤活性。因此，确定SDF-1/CXCR4和SDF-1RDC1轴在KS中的作用可能为治疗干预提供途径。 公共卫生相关性：趋化因子是通过与应答细胞上的特异性趋化因子受体（CR）结合来调节细胞功能的分泌蛋白，其通过刺激新血管生长（肿瘤血管生成）和指导恶性或癌前细胞的迁移在人类肿瘤的发展、生长和转移中发挥重要作用。KS肿瘤细胞（梭形细胞）表达参与肿瘤转移和血管生成的关键趋化因子（SDF-1/CXCL 12）的CR（CXCR 4和RDC1/CXCR 7），我们的初步数据显示，KSHV诱导这些趋化因子/CR对的表达和活性。在本申请中，我们将研究KSHV诱导SDF-1、CXCR4和RDC1的机制和功能结果，这些信息将提高我们对KS发病机制的理解，并表明阻断趋化因子/CR相互作用的药物是否值得探索用于KS治疗。

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS) is the most common AIDS-associated malignancy and the most frequent cancer in parts of Africa where HIV is endemic. The infectious agent, Kaposi's sarcoma herpesvirus (KSHV; also HHV8) is found in spindle cells, their EC precursors and monocytes in KS lesions, as well as in circulating EC progenitors and PBMC. Several features of KS suggest that chemokines and their receptors (CR) play a role in the disease. KS tumors display profound angiogenesis and an extensive inflammatory infiltrate, both features indicative of chemokine/CR activity. Also, the multi-focal nature of KS suggests that KSHV-infected spindle cell precursors or leucocytes home to permissive sites to initiate or sustain tumor development. The SDF-1/CXCR4 axis is one of the main chemokine/CR interactions associated with tumor growth, angiogenesis and metastasis. The role of SDF-1/CXCR4 in KS however remains obscure; the limited studies performed to date reveal only that these molecules are present in KS tumors. We have shown that the recently characterized alternate receptor for SDF-1, RDC1 (also CXCR7), is strongly induced in KSHV-infected EC. While RDC1 expression is limited in non-malignant cells, it is well expressed by several cancers and tumor-associated EC. We recently found that RDC1 is highly expressed on KS tumor tissue and we, and others, have shown that SDF-1 signaling through RDC1 promotes migratory, angiogenic, growth and pro-survival activity in several cell types. These findings suggest that SDF-1, signaling through either of both of its receptors CXCR4 and RDC1, plays an important role in the seeding and subsequent development of KS tumors. We hypothesize that RDC1 and CXCR4 are pathologically induced or activated by KSHV in EC/spindle cells and that SDF-1 in the tumor microenvironment and/or co-expressed by EC/spindle cells activates downstream signaling pathways that lead to a pro-angiogenic, pro-growth, pro-survival phenotype and promote trafficking of cells to sites of lesion development. To understand the role of SDF-1/CXCR4 and SDF-1/RDC1 in KS tumor seeding, initiation and development, we propose three Specific Aims. In Aim 1 we propose to characterize the expression patterns of SDF-1, CXCR4 and RDC1 (i) in vivo, in KS tumors and (ii) in vitro, in different types of KSHV-infected endothelial cells. In Aim 2, we will determine the nature and function of (a) SDF-1/CXCR4 and (b) SDF-1/RDC1 signaling in KSHV-infected EC. In Aim 3, we will use samples from a cohort of KSHV seropositive patients with and without KS and HIV to enumerate CEC numbers and determine the expression and function of SDF-1, CXCR4 and RDC1 on these cells. Functional studies will also be conducted on normal and cohort CEC following their expansion in vitro as blood outgrowth EC. CXCR4 antagonists are currently in pre-clinical and clinical development for the treatment of leukemia, lung and breast cancer. Specific RDC1 antagonists have recently been developed and have shown anti-tumor activity in animal models. Thus, defining the role of SDF-1/CXCR4 and SDF-1RDC1 axes in KS may provide an avenue for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Chemokines, secreted proteins that regulate cell function by binding to specific chemokine receptors (CR) on responsive cells, play an essential role in the development, growth and metastasis of human tumors by stimulating new blood vessel growth (tumor angiogenesis) and directing the migration of malignant or pre-malignant cells. KS tumor cells (spindle cells) express both of the CRs (CXCR4 and RDC1/CXCR7) for one of the key chemokines (SDF-1/CXCL12) involved in tumor metastasis and angiogenesis, and our preliminary data shows that KSHV induces the expression and activity of these chemokine/CR pairs. In this application we will investigate the mechanisms and functional outcomes of KSHV-induction of SDF-1, CXCR4 and RDC1, information that will both improve our understanding of KS pathogenesis and indicate if drugs that block chemokine/CR interactions are worth exploring for KS therapy.