Epigenetic Inactivation of RASSF 1 A in Head and Neck Cancer 1

Purpose: RASSF1A, a recently identified candidate tumor suppressor gene, was found to be inactivated in lung cancer and other tumor types. We sought to understand the role of RASSF1A in head and neck cancer. Experimental Design: We analyzed the status of RASSF1A and presence of high-risk human papilloma virus (HPV) in head and neck cancer squamous cell carcinoma (HNSCC) cell lines and primary tumors. We used methylation-specific PCR to detect promoter hypermethylation and direct sequence analysis to detect point mutations in primary tumors and cell lines. 5-aza-2-deoxycytidine was used to demethylate the RASSF1A promoter in cell lines. Results: Promoter methylation of RASSF1A was detected in 42.9% (3 of 7) cell lines and 15% (7 of 46) primary tumors but not in the normal control DNA. Direct sequence analysis revealed a point mutation in a cell line and another in a primary HNSCC. After treatment with 5-aza-2-deoxycytidine, re-expression and demethylation of RASSF1A gene were detected in cell lines with promoter hypermethylation. HPV DNA was detected in 34.7% (16 of 46) primary HNSCC. We found a significant inverse correlation between RASSF1A promoter methylation and HPV infection (P 0.038). Conclusions: Our results suggest that RASSF1A is inactivated in a subset of HNSCC primary tumors. Moreover, an inverse correlation between RASSF1A and HPV supports a biological mechanism in which both RASSF1A promoter methylation and HPV infection abrogate the same pathway in tumorigenesis. INTRODUCTION HNSCC occurs in 50,000 Americans each year, and the incidence of this type of tumor is expected to rise as a result of the increasing number of female and adolescent smokers. Despite the great emphasis on early diagnosis and the efforts to improve surgical and radiation treatment, 50% of HNSCC patients do not survive for 5 years after diagnosis (1). The growing epidemiological problem and lack of progress in head and neck oncology emphasizes the need for basic studies on the molecular biology of HNSCC. Over the past decade, molecular and cytogenetic studies have contributed significantly to the identification of genetic and epigenetic changes associated with this cancer. Tobacco and alcohol consumption are well-established risk factors of HNSCC (2). Loss of chromosomal areas and mutations in oncogenes are crucial events in tumor formation (3–7). In addition, inactivation of TSGs by promoter methylation is also common. However, a small proportion (15–20%) of HNSCC occurs in nonsmokers and nondrinkers, suggesting the presence of other risk factors. Recent epidemiological and molecular data suggest that HPV infection of the upper airway may promote head and neck tumorigenesis (8–10). HPV-positive oropharyngeal cancers display distinct pathological molecular features as well as a different clinical course from HPV-negative oropharyngeal cancers, whose etiology is linked to smoking and drinking (9). Allelic loss on chromosomal arm 3p is common in head and neck cancers (11–15) and other cancers, including lung, kidney, breast, and bladder (14–16). Dammann et al. (20) described the cloning and characterization of a human RAS effector homologue, RASSF1, located in the 120-kb region of minimal homozygous deletion at 3p21.3 in lung cancer (17). The RASSF1A isoform was frequently inactivated in several types of tumors (18–20). Huang et al. (21) also provided evidence that promoter hypermethylation and allelic loss are the major mechanisms for inactivation of RASSF1A in nasopharyn-