Mechanisms of KSHV-Induced Cellular Transformation

描述（由申请人提供）：卡波西肉瘤（KS）是一种多灶性肿瘤，其特征为内皮细胞来源的梭形细胞增殖、红细胞填充的新生血管裂隙和炎性细胞浸润。KS是HIV感染者最常见的癌症，也是发展中国家儿童最常见的肿瘤之一。卡波西肉瘤相关疱疹病毒（KSHV或HHV-8）是KS的病原体，并通过直接感染病变内皮细胞和梭形细胞来驱动肿瘤的发展。KSHV体外感染的真皮微血管内皮细胞（DMVEC）主要潜伏感染一部分表达裂解病毒基因的细胞，这种模式反映了体内病毒基因的表达模式。KSHV感染的DMVEC形成梭形形态和转化的表型，细胞基因表达谱揭示了许多潜在的致瘤基因的病毒诱导。这些基因之一，原癌基因c-Kit，被证明在体外DMVEC转化中发挥重要作用，KS组织的初步分析证实了c-Kit在体内KS肿瘤中的表达。该项目的长期目标是阐明KS肿瘤中c-Kit表达的范围和时间动力学，并阐明导致c-Kit表达和c-Kit介导的细胞转化的病毒和细胞机制。具体目标1将使用免疫组织化学和RT-PCR分析，结合潜伏性或溶解性KSHV感染的血清学检测，评价KS肿瘤组织在早期（斑块和斑块）至晚期（结节）发育阶段的c-Kit表达。具体目标2将在体外使用感染的DMVEC来鉴定负责c-Kit诱导的KSHV基因以及涉及的分子机制。特异性目标3将在体外使用感染的DMVEC来阐明由c-Kit激活的最终导致KSHV诱导的转化的下游信号通路。对于目的2和3，腺病毒载体将用于过表达感兴趣的病毒和细胞基因。将使用反义寡核苷酸或药理学抑制剂和适当的功能测定来验证基因功能，c-Kit已被证明是治疗胃肠道间质瘤（GIST）的有效治疗靶点。阐明c-Kit在KSHV相关内皮转化中的作用可能提示类似的KS治疗方法。

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS) is a multifocal tumor characterized by proliferating spindle cells of endothelial cell origin, erythrocyte-filled neovascular slits and an inflammatory cell infiltrate. KS is the most frequent cancer arising in HIV-infected individuals and is one of the most common neoplasms of children in developing countries. Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiologic agent of KS, and drives tumor development via direct infection of lesional endothelial and spindle cells. Dermal microvascular endothelial cells (DMVEC) infected with KSHV in vitro harbor a primarily latent infection with a fraction of cells expressing lytic viral genes, a pattern that reflects viral gene expression patterns in vivo. KSHV-infected DMVEC develop a spindle morphology and a transformed phenotype and cellular gene expression profiling reveals viral induction of a number of potentially tumorigenic genes. One of these genes, the proto-oncogene c-Kit, was shown to play an essential role in DMVEC transformation in vitro and a preliminary analysis of KS tissue confirmed expression of c-Kit in KS tumors in vivo. The long-term goals of this project are to clarify the scope and temporal kinetics of c-Kit expression in KS tumors, and to clarify the viral and cellular mechanisms that lead to c-Kit expression and c-Kit-mediated cellular transformation. Specific Aim 1 will use immunohistochemical and RT-PCR analysis to evaluate c-Kit expression in KS tumor tissue at early (patch and plaque) through late (nodular) stages of development in conjunction with serologic detection of latent or lytic KSHV infection. Specific Aim 2 will use in vitro-infected DMVEC to identify the KSHV gene(s) responsible for c-Kit induction, and the molecular mechanisms involved. Specific Aim 3 will use in vitro-infected DMVEC to elucidate downstream signaling pathways activated by c-Kit that lead ultimately to KSHV-induced transformation. For Aims 2 and 3, adenovirus vectors will be used to overexpress viral and cellular genes of interest. Gene function will be validated with antisense oligonucleotides or pharmacologic inhibitors and appropriate functional assays, c-Kit has proven to be an effective therapeutic target for treatment of gastrointestinal stromal tumor (GIST). Clarification of the role of c-Kit in KSHV-associated endothelial transformation may suggest a similar approach for treatment of KS.