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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cancer Etiology Study Section (CE)
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Blair, Donald G
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Cincinnati Children's Hospital Medical Center
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