ROLE OF Rac AND REACTIVE OXYGEN SPECIES IN KAPOSI'S SARCOMA VIRAL ONCOGENESIS

描述（由申请人提供）：卡波西肉瘤（KS）由卡波西肉瘤相关疱疹病毒（KSHV）引起，是一种与艾滋病相关的主要癌症，也是全球健康挑战。肿瘤的特点是血管生成强烈，梭形细胞增生，可影响皮肤、粘膜和内脏，引起显著的发病率。了解导致KS癌变的病毒和细胞基因的作用对于开发合理设计的KS治疗方法至关重要。Mesri和Goldschmidt实验室之间的合作鉴定了Rac1 GTPase，一种通过非吞噬性nadph氧化酶（NOX）触发活性氧（ROS）产生的信号介质，是KS的潜在主要参与者。我们发现，在转基因小鼠中，由-平滑肌肌动蛋白（-SMA）启动子驱动的组成型活性Rac1突变体（RacCA）的表达导致了与卡波西肉瘤非常相似的病变的形成。值得注意的是，RacCA- SMA肿瘤与KS肿瘤活检显示主要的转录组重叠。RacCA肿瘤发生与男性有关，并涉及血管生成和细胞增殖的ROS激活。此外，我们发现，在我们的KS小鼠模型（小鼠内皮细胞KSHV Bac36- mECK36）中，艾滋病-KS病变和KSHV感染的肿瘤在所有KSHV感染的（LANA+ve）细胞中过表达Rac1。此外，我们发现KS病变和mECK36病变过度表达NOX家族的关键成员，并且mECK36肿瘤以KSHV依赖的方式上调NOX成员。因此，我们测试了n -乙酰半胱氨酸（NAC），一种特性良好的抗氧化剂，在小鼠体内抑制mECK36肿瘤的能力。我们发现NAC可以阻止kshv诱导的肿瘤形成。有趣的是，我们还发现NAC通过血小板衍生生长因子（PDGF）受体和配体下调的机制抑制mECK36肿瘤中VEGF、c-myc和病毒基因的表达。这些数据表明，Rac1、NOX、ROS及其下游效应物是积极参与KS病毒癌变的分子，表明Rac1信号传导和氧化应激可能是KS化学预防和治疗的重要靶点。我们提出：研究Rac1激活在KSHV肿瘤发生中的机制和作用（Aim 1），研究NADPH氧化酶诱导ROS在KSHV肿瘤发生中的作用（Aim 2），测试药理学抑制ROS对RacCA和KSHV诱导肿瘤的预防和治疗效果（Aim 3）。

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS), caused by the Kaposi's sarcoma-associated herpes virus (KSHV), is a major cancer associated with AIDS and a global health challenge. The tumor is characterized by intense angiogenesis and the proliferation of spindle cells that can affect the skin, mucosa and viscera, causing significant morbidity. Understanding the role of viral and cellular genes leading to KS carcinogenesis is paramount to developing rationally designed therapies for KS. A collaboration between the Mesri and Goldschmidt labs has led to the identification of the Rac1 GTPase, a signaling mediator that triggers production of reactive oxygen species (ROS) by non-phagocytic NADPH-oxidase (NOX), as a potential major player in KS. We have found that expression of a constitutively-active Rac1 mutant (RacCA) driven by -smooth muscle actin (-SMA) promoter in transgenic mice led to the formation of lesions that strongly resemble those of Kaposi's sarcoma. Significantly, RacCA--SMA tumors revealed major transcriptome overlap with KS tumor biopsies. RacCA tumorigenesis was linked to male gender, and involved ROS activation of angiogenesis and cell proliferation. Furthermore, we found that AIDS-KS lesions and KSHV-infected tumors from our KS mouse model (mouse endothelial cell KSHV Bac36- mECK36) over-express Rac1 in all KSHV-infected (LANA+ve) cells. Moreover, we found that KS lesions and mECK36 lesions over-express key members of the NOX family and that mECK36 tumors upregulate NOX members in a KSHV dependent fashion. This led us to test the ability of N-acetyl cysteine (NAC), a well characterized antioxidant, to suppress mECK36 tumors in mice. We found that NAC prevented KSHV-induced tumor formation. Interestingly, we also found that NAC inibited VEGF, c-myc and viral gene expression in the mECK36 tumors through a mechanism involving platelet derived growth factor (PDGF) receptor and ligand downregulation. These data indicate that Rac1, NOX, ROS, and their downstream effectors are molecules actively involved in KS viral oncogenesis, and suggest that Rac1 signaling and oxidative stress could be attractive KS chemopreventive and therapeutic targets. We proposse to: Study mechanisms and role of Rac1 activation in KSHV oncogenesis (Aim 1), study the role of NADPH oxidase induction of ROS in KSHV oncogenesis (Aim 2) and to test the efficacy of pharmacologic ROS inhibition on prevention and treatment of RacCA and KSHV-induced tumors (Aim 3). PUBLIC HEALTH RELEVANCE: Kaposi's sarcoma (KS) is the most common type of cancer associated with AIDS (AIDS-KS). KS arises as multifocal lesions in the skin, lungs and gastrointestinal tract characterized by intense blood microvessel and cellular proliferation. Early KS lesions are treated with local and non-toxic therapies; however, advanced KS generally characterized as an advanced disseminated cancer with increased morbidity and mortality is treated with systemic chemotherapy. This treatment is difficult to tolerate for AIDS patients who are receiving Anti Retroviral regimes such as HAART. Thus, the development of rational therapies based on KS pathogenesis is critical to fill this gap. The identification of human and viral genes that are the cause of KS carcinogenesis is important because it could lead to the identification of new therapeutic targets. Essential to this work is the creation of animal models of the disease. Two laboratories of UM/SCCC we have succeeded in generating such a models and identifying a host gene named Rac that by itself can cause in mice many aspects of Kaposi's sarcoma. These models and insights are a very good combination to study mechanisms of Kaposi's sarcoma carcinogenesis. We will study how the Rac gene may participate in cell transformation and tumorigenesis. One of the ways this gene may participate in carcinogenesis is by promoting oxidative stress that can lead to cellular damage and genetic damage that could lead to carcinogenic mutations that may contribute to cancer. So we are going to explore ways of preventing this oxidative genetic damage as a means of preventing and possibly curing Kaposi's sarcoma. So our work will allow us to not only to identify mechanisms of KS carcinogenesis but also will help to identify potential new target and new drugs for anti-KS therapy.