Fanconi Anemia and HPV Transformation

描述（由申请人提供）：范可尼贫血（FA）是一种基因组不稳定综合征，与13个基因中的一个突变相关。FA患者表现出进行性骨髓衰竭和对血液和实体肿瘤的高易感性。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，人类角化细胞。骨髓衰竭可以通过成功的骨髓移植治愈。然而，接受骨髓移植的FA患者继续处于肛门生殖器和头颈部鳞状细胞癌（SCC）的高风险中，这表明FA途径具有阻止上皮细胞转化的功能。在一般人群中，感染高危型HPV（如HPV16）通常与此类SCCs相关。病毒E7基因产物是主要的致癌基因，刺激分化表皮细胞增殖，并且是病毒DNA复制所必需的。我们在这里证明，在缺乏其他病毒基因产物的情况下，FA损失导致E7蛋白水平显著上调。此外，FA缺失增加了hpv16阳性器官型上皮筏的增生和病毒DNA复制，以及免疫缺陷小鼠的肿瘤发生。假设FA缺失刺激hpv驱动的细胞和病毒增殖以及恶性转化，至少部分是通过E7激活。我们将在体外使用FA患者来源的和敲低的细胞和器官型筏模型，以及体内的异种移植物移植来验证这一假设。目的1将确定FA途径控制E7水平的机制，特别强调蛋白质稳定性。将对fa依赖性E7调控所需的顺式作用E7结构域和反式作用细胞因子进行鉴定和功能测试。目的2将定义调控E7的FA通路的特定成员，并将测量FA损失对细胞和病毒DNA复制的影响，以及体外HPV16负载。目的3将检验hpv驱动的FA SCC是否通过增加E7活性、病毒扩增、细胞存活和/或体内增殖而发生。HPV特异性将通过比较FA缺陷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，并将确定其对HPV依赖性和非依赖性SCC的意义。

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.