Fanconi Anemia and HPV Transformation

摘要 范可尼贫血（FA）是一种基因组不稳定综合征，与13个突变之一有关。 基因. FA患者表现出进行性骨髓衰竭和对血液和 实体瘤在实体瘤中，大多数是鳞状细胞癌，其产生于肿瘤细胞。 表皮内的主要细胞类型，人角质形成细胞。FA患者的骨髓衰竭 可以通过成功的骨髓移植治愈然而，患有FA的患者 接受骨髓移植的人仍然处于肛门生殖器和头部的高风险之中， 颈部鳞状细胞癌（SCC），表明FA通路的功能，以防止 上皮细胞转化感染高危型HPV，如HPV 16， 与一般人群中的此类SCC相关。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， 用于病毒DNA复制。我们在这里证明，FA的损失导致显着上调 在没有其他病毒基因产物的情况下，E7蛋白水平。此外，FA损失增加 HPV 16阳性器官型上皮筏中的增生和病毒DNA复制， 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，至少部分通过E7 activation.我们将使用FA患者来源的和敲低的细胞来检验这一假设， 体外器官型筏模型和体内异种移植。 目的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E7结构域和反式作用细胞因子 将识别和功能性测试FA依赖性E7调节所需的功能。目标2将 定义调节E7的FA途径的特定成员，并将测量 FA损失对细胞和病毒DNA复制以及体外HPV 16载量的影响。 目的3将检查HPV驱动的FA SCC是否通过增加E7活性、病毒 在体内的扩增、细胞存活和/或增殖。HPV特异性将通过以下方法确定： 比较FA缺陷型HPV阳性和阴性细胞的恶性潜能。这些 研究将揭示DNA损伤信号通路控制病毒和 细胞DNA复制，并将确定其对HPV依赖性和- 独立SCC。

ABSTRACT 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.