Mechanisms of KSHV-Induced Cellular Transformation

描述（由申请人提供）：卡波西肉瘤（KS）是最常见的与艾滋病相关的恶性肿瘤，也是非洲部分艾滋病流行地区最常见的癌症。感染因子卡波西肉瘤疱疹病毒（KSHV，也称HHV8）存在于梭形细胞、其EC前体和KS病变中的单核细胞中，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）在该疾病中起作用。KS肿瘤表现出深刻的血管生成和广泛的炎症浸润，这两个特征都表明趋化因子/CR活性。此外，KS的多灶性表明，kshv感染的梭形细胞前体或白细胞回到允许位点，以启动或维持肿瘤的发展。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。我们已经证明，最近表征的SDF-1的替代受体RDC1（也称为CXCR7）在kshv感染的EC中被强烈诱导。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的EC中表达良好。我们最近发现RDC1在KS肿瘤组织中高表达，我们和其他人已经证明，通过RDC1的SDF-1信号传导促进了几种细胞类型的迁移、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中的任何一个发出信号，在KS肿瘤的播种和随后的发展中发挥重要作用。我们假设，在EC/梭形细胞中，rdg1和CXCR4是由KSHV病理诱导或激活的，而肿瘤微环境中的SDF-1和/或由EC/梭形细胞共同表达的SDF-1激活下游信号通路，导致促血管生成、促生长、促存活的表型，并促进细胞运输到病变发展部位。为了了解SDF-1/CXCR4和SDF-1/RDC1在KS肿瘤萌发、发生和发展中的作用，我们提出了三个具体目标。在Aim 1中，我们提出表征SDF-1、CXCR4和RDC1的表达模式(i)在体内，在KS肿瘤中，（ii）在体外，在不同类型的kshv感染的内皮细胞中。在目的2中，我们将确定kshv感染EC中(a) SDF-1/CXCR4和(b) SDF-1/RDC1信号的性质和功能。在Aim 3中，我们将使用一组KSHV血清阳性患者（有或没有KS和HIV）的样本来枚举CEC数量，并确定SDF-1、CXCR4和RDC1在这些细胞上的表达和功能。正常和队列CEC在体外扩增为血源性CEC后，也将进行功能研究。CXCR4拮抗剂目前正处于治疗白血病、肺癌和乳腺癌的临床前和临床开发阶段。特异性的RDC1拮抗剂最近被开发出来，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。

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.