Fanconi Anemia and HPV Transformation

摘要 Fanconi贫血(FA)是一种与13种基因突变之一相关的基因组不稳定综合征 基因。FA患者表现为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，它起源于 表皮内的主要细胞类型，即人类角质形成细胞。红斑狼疮患者的骨髓衰竭 可以通过成功的骨髓移植治愈。然而，患有FA的患者 接受骨髓移植对肛门和头部的风险仍然很高， 颈部鳞状细胞癌(SCC)，提示FA通路具有预防作用 上皮细胞转化。感染高危HPV类型，如HPV16，通常 与普通人群中的此类鳞状细胞癌有关。病毒E7基因产物是 主要癌基因，刺激分化的表皮中的细胞增殖，是必需的 用于病毒DNA复制。我们在这里证明，FA的损失导致显著的上调 在没有其他病毒基因产物的情况下，E7蛋白水平的变化。此外，FA损失增加 HPV16阳性器官型上皮筏中的增殖和病毒DNA复制，以及 免疫缺陷小鼠的肿瘤发生。假设是FA的损失刺激了HPV驱动 细胞和病毒的增殖以及恶性转化，至少部分通过E7 激活。我们将使用FA患者来源的和击倒的细胞来验证这一假设 体外器官型移植物模型，体内异种移植。 目标1将确定FA途径通过一种 特别强调蛋白质的稳定性。顺式作用E7结构域与反式作用细胞因子 将确定依赖FA的E7法规所需的内容并进行功能测试。目标2将 定义调节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是否是通过E7活动增加、病毒 体内扩增、细胞存活和/或增殖。HPV的特异性将由以下因素确定 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，并将确定它们对HPV依赖和- 独立的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.