The ARF‐p16 Gene Locus in Carcinogenesis and Therapy of Head and Neck Squamous Cell Carcinoma

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Abstract
Objectives/Hypothesis We have identified families with a high incidence of tumors including head and neck squamous cell carcinoma (HNSCC). The occurrence of melanoma in these kindreds suggested that the ARF‐p16 gene may be involved in carcinogenesis. We wished to determine the gene defect associated with the familial predisposition to HNSCC and to determine whether restoration of the gene may have therapeutic benefit.Study Design Translational molecular research.Methods Molecular techniques were used to identify mutations of the ARF‐p16 gene from the affected families and to test the activity of p16 and ARF mutants. In additional, HNSCC tumor tissue was analyzed to determine whether the wild‐type p16 allele was lost or maintained. ARF‐expressing adenoviruses were created, and their effect on HNSCC cell lines and normal head and neck epithelial cells was determined.Results Mutation of the ARF‐p16 gene was found in two families with predisposition to develop HNSCC. Independent mutations detected in the germline DNA of both families inactivated p16, but not ARF, and the inactive mutant p16 allele segregated with disease within both families. The wild‐type p16 allele was lost in HNSCC tumor tissue from both families. The efficacy of ARF in treatment of HNSCC was found to depend on retention of p53 activity within HNSCC tumor cells. Remarkably, ARF expression was found to kill cells, depending on loss of retinoblastoma activity. Because loss of retinoblastoma activity is nearly universal in tumors, ARF killed tumor cells that retained p53, but ARF spared normal cells.Conclusions Our results support the recognition of a new clinical entity of familial head and neck cancer. We have shown that this syndrome is associated with inactivating mutations of the p16 gene that these mutations segregate with disease in two described families. Loss of the wild‐type p16 allele in HNSCC tissue from both families strongly supports the role of the mutant p16 in carcinogenesis. We have also investigated the therapeutic utility of the alternate reading frame product of the p16 gene, ARF. The finding that ARF kills cells depending on loss of retinoblastoma activity and retention of p53 suggests that ARF may be effective in treatment of roughly 50% of head and neck cancers while sparing normal cells. Recognition of p16 mutations as an etiological factor in familial HNSCC provides an accessible tool for diagnosis of this syndrome. Clinical acceptance of familial head and neck cancer will ensure that patients are appropriately diagnosed and managed.