In chemoprevention of head and neck squamous carcinoma (HNSCC), new approaches must consider thesustained epigenetic effects of tobacco use on host molecular mechanisms and identification of novel agentswith limited toxicity and acceptable therapeutic ratio. Smoking and nutrition have been identified assignificant factors that contribute to carcinogenesis and prognosis of these cancers. Epigenetic CpG islandmethylation in the tumor promoter gene p16 is frequent in HNSCC and in the mucosa of smokers,premalignant dysplastic tissue and in non-cancerous tissue adjacent to proven malignancies suggesting animportant role in early carcinogenesis and second primary malignancy. An important downstream target ofp16 that may play a central role in HNSCC carcinogenesis is COX-2. COX-2 over expression is common inpremalignant and malignant mucosa and is associated with smoking and chronic inflammation. Whenblocked, COX-2 decreases tumor growth and invasiveness. COX-2 is influenced by EGFR signaling andp16 inactivation which are commonly perturbed in HNSCC. We propose to identify frequency of genemethylation for p16 and 3 other candidate genes, gene expression alterations and related serum biomarkersassociated with clinical outcome using our extensive retrospective tissue and clinical data resource. We willprospectively validate and expand these data and determine specific effects of a tailored smoking cessationprogram on modulation of identified biomarkers. A Phase II trial is proposed to determine if preoperative soyisoflavones can modulate these markers. Soy isoflavones have been shown to reverse p16 methylation,decrease COX-2 expression and modulate VEGF, IL6, tumor proliferation, angiogenesis and apoptosis. Wehypothesize that suppressor gene hypermethvlation in tumor and adjacent non-tumor mucosa andassociated molecular changes in downstream targets such as COX-2. VEGF. EGFR and IL6 areassociated with clinical outcome and can be abrogated by soy isoflavone supplementation andsmoking cessation. We will use pyrosequencing to measure methylation of p16 and three other candidategenes in pretreatment samples from both retrospective and prospective patients and correlate with outcome.We will determine expression of EGFR, COX-2, VEGF, p53 , p65,a6p4 integrin, HPV-16, Bcl-xL in tissue andlevels of VEGF, IL6, HGF and 15-Ft2-isoprostane in serial serum and saliva samples from our prospectivepatients. This comprehensive proposal will investigate the molecular rationale for future soy isoflavone andsmoking cessation prevention trials and identify intermediate molecular endpoints. These data will helpdefine high risk patients according to biomarkers and smoking status who might benefit most fromchemoprevention. Studies of upregulated gene profiles associated with tumor suppressor genehypermethylation may identify new target genes for future study.
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