The soluble frizzled receptor protein (SFRP) family encodes antagonists of the WNT pathway, and silencing of these genes, through promoter hypermethylation, leads to constitutive WNT signaling. In bladder cancers, hypermethylation of the SFRP genes occurs more often in current and former smokers and is a strong predictor of poor patient survival. Hence, we examined methylation of these genes in another tobacco-related epithelial cancer, head and neck squamous cell carcinoma (HNSCC), to determine if the pattern of tobacco exposure again predicts the epigenetic alteration of these genes. Using methylation-specific PCR, the prevalence of methylation of SFRP1, SFRP2, SFRP4 and SFRP5 was 35, 32, 35 and 29%, respectively among 350 HNSCC cases. Promoter methylation of SFRP1 occurred more often in both heavy (OR 3.5, 95% CI 0.9-13.7) and light drinkers (OR 3.7, 95% CI 1.0-14.3) compared to nondrinkers. SFRP4 promoter methylation, on the other hand, occurred at a higher prevalence in never smokers and former smokers than in current smokers, and also was independently associated with HPV16 viral DNA. A joint effects model of SFRP4 promoter methylation demonstrated that smoking status and HPV virus significantly interacted (p < 0.04) such that never smokers with HPV16 had an OR of SFRP4 methylation of 9.0 (95% CI 2.1-38.6). These results suggest that epigenetic alterations of the SFRP genes are highly prevalent in HNSCC, and that the clonal selection for these alterations is complex and may be related to the carcinogenic exposures that are known risk factors for this disease. ' 2006 Wiley-Liss, Inc.Key words: head and neck cancer; SFRP; hypermethylation; HPV; tobacco smoking; alcohol Head and neck squamous cell carcinoma (HNSCC) is the ninth most common cancer in men in the United States (U.S.) and in 2003 390,000 new cases of oral cancer and 160,000 cases of laryngeal cancer were diagnosed worldwide.1 In the U.S., it is estimated that in 2004 there were 28,260 new cases of oral and pharyngeal cancer (excluding cancers of the lip) and 10,270 laryngeal cancers, and the overall 5-year survival rate for oral and pharyngeal cancer is approximately 50%.2 HNSCC is causally linked to tobacco smoking and smokeless tobacco use, 3,4 as well as to alcohol consumption, which acts both independently and synergistically with tobacco smoking to contribute to disease.
5Human papillomavirus (HPV) infection and low dietary folate have also been implicated in the etiology of this disease. [6][7][8][9] The WNT signaling pathway, activated through the binding of WNT proteins to the membrane-bound Frizzled receptors, leads to the stabilization and increase in the levels of the transcription factor b-catenin protein. This, in turn, leads to the activation of an array of target genes involved in numerous cellular processes important in tumor development, such as cell growth and survival, intercellular adhesion and cellular morphogenesis.10 Disruption of this pathway, through activating mutations of CTNNB1 (encoding b-catenin) or loss-of-function...