Fanconi Anemia and HPV Transformation

描述(申请人提供)：Fanconi贫血(FA)是一种与13个基因中的一个基因突变有关的基因组不稳定综合征。FA患者表现为进行性骨髓衰竭，对血液和实体瘤易感性高。在实体瘤中，大多数是鳞状细胞癌，它起源于表皮内的主要细胞类型，即人类角质形成细胞。FA患者的骨髓衰竭可以通过成功的骨髓移植治愈。然而，接受了骨髓移植的FA患者仍然是肛门和头颈部鳞状细胞癌(SCC)的高危人群，这表明FA途径起到了防止上皮细胞转化的作用。在普通人群中，感染HPV16型等高危HPV型通常与此类鳞状细胞癌有关。病毒E7基因产物是主要的癌基因，刺激分化的表皮细胞增殖，是病毒DNA复制所必需的。我们在这里证明，在没有其他病毒基因产物的情况下，FA的丢失会导致E7蛋白水平的显著上调。此外，FA丢失增加了HPV16阳性器官型上皮筏的增殖和病毒DNA复制，并增加了免疫缺陷小鼠的肿瘤形成。假说是FA的丢失刺激了HPV驱动的细胞和病毒的增殖以及恶性转化，至少部分是通过激活E7。我们将使用FA患者来源的、击倒的细胞和器官类型的体外移植物模型以及体内的异种移植来验证这一假设。目标1将确定FA途径控制E7水平的机制，特别强调蛋白质的稳定性。顺式作用的E7结构域和反式作用的细胞因子所需的依赖FA的E7将被识别和功能测试。AIM 2将定义调控E7的FA途径的特定成员，并将测量FA丢失对细胞和病毒DNA复制以及体外HPV16载量的影响。AIM 3将检查HPV驱动的FA SCC是否通过体内E7活性增加、病毒扩增、细胞存活和/或增殖来实现。HPV的特异性将通过比较FA缺乏的HPV阳性和阴性细胞的恶性潜能来确定。这些研究将揭示DNA损伤信号通路控制病毒和细胞DNA复制的机制，并将确定它们对HPV依赖和独立的鳞状细胞癌的意义。 公共卫生相关性：鳞状细胞癌(SCC)是第二种最常见的皮肤癌形式，据估计，美国每年有超过25万新病例。感染人乳头瘤病毒(HPV)和表达两个病毒癌基因E6和E7是一个明确的原因。Fanconi贫血(FA)是一种基因组不稳定综合征，其表现包括极高的鳞癌风险。我们的数据表明，FA功能的丧失增加了HPV16E7蛋白水平，从而在实验室模型中揭示了FA途径和HPV转化活动之间的特定分子联系。此外，HPV阳性细胞中FA的丢失会刺激细胞和病毒的DNA复制以及体内肿瘤的形成。拟议的FA患者衍生和基因敲除模型的研究将确定潜在的分子机制，并可能为改进HPV依赖和非依赖的SCC的治疗定义新的细胞靶点。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a genome instability syndrome associated with mutations in one of 13 genes. FA patients exhibit progressive bone marrow failure and high susceptibility to blood and solid tumors. Of the solid tumors, most are squamous cell carcinomas, which arise from the main cell type within the epidermis, the human keratinocyte. Bone marrow failure in FA patients can be cured with a successful bone marrow transplant. However, FA patients that have undergone a bone marrow transplant continue to be at high risk for anogenital and head and neck squamous cell carcinoma (SCC), suggesting that the FA pathway functions to prevent epithelial cell transformation. Infection with high risk HPV types such as HPV16 is often associated with such SCCs in the general population. The viral E7 gene product is the predominant oncogene, stimulates cell proliferation in differentiated epidermis, and is required for viral DNA replication. We demonstrate here that FA loss results in the marked upregulation of E7 protein levels in the absence of other viral gene products. Additionally, FA loss increases hyperplasia and viral DNA replication in HPV16-positive organotypic epithelial rafts, and tumorigenesis in immunodeficient mice. The hypothesis is that FA loss stimulates HPV-driven cellular and viral proliferation as well as malignant transformation, at least in part through E7 activation. We will test this hypothesis using FA patient-derived and knockdown cell and organotypic raft models in vitro, and xenograft transplantations in vivo. Aim 1 will determine the mechanism by which the FA pathway controls E7 levels with a particular emphasis on protein stability. Cis-acting E7 domains and trans-acting cellular factors required for FA-dependent E7 regulation will be identified and functionally tested. Aim 2 will define specific members of the FA pathway which regulate E7, and will measure the consequences of FA loss on cellular and viral DNA replication, as well as HPV16 load in vitro. Aim 3 will examine whether HPV-driven FA SCC is through increased E7 activities, virus amplification, cell survival and/or proliferation in vivo. Specificity for HPV will be determined by comparing the malignant potential of FA deficient HPV-positive and -negative cells. These studies will uncover mechanisms by which DNA damage signaling pathways control viral and cellular DNA replication, and will determine their significance for HPV dependent and - independent SCC. PUBLIC HEALTH RELEVANCE: Squamous cell carcinoma (SCC) is the second most common form of skin cancer, with over 250,000 new cases per year estimated in the United States. Infection with the human papillomaviruses (HPVs) and expression of the two viral oncogenes E6 and E7 is one well defined cause. Fanconi anemia (FA) is a genome instability syndrome whose manifestations include an extreme risk of SCC. Our data suggest that loss of FA function increases HPV16 E7 protein levels, thus uncovering specific molecular links between the FA pathway and HPV transforming activities in laboratory models. Furthermore, FA loss in HPV positive cells stimulates cellular and viral DNA replication as well as tumorigenesis in vivo. The proposed studies of FA patient-derived and knockdown models will identify the underlying molecular mechanisms and may define new cellular targets for improved treatments of HPV-dependent and -independent SCC.