Abstract 3124: Smoking effects on CYP24A1 in lung adenocarcinoma: Epigenetic changes by smoking

Abstract

Abstract CYP24A1 is the rate limiting catabolic enzyme for 1α,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. We have previously demonstrated that the mRNA expression of CYP24A1 is an independent prognostic factor for survival in lung adenocarcinoma (AC) and that the antiproliferative effects of 1,25-D3 are inversely proportional to CYP24A1 expression. We then asked if smoking is associated with increased expression of CYP24A1 and a decrease in serum level of 1,25-D3; whether increased CYP24A1 expression was due to epigenetic changes caused by smoking. We measured CYP24A1 mRNA in resected lung AC (n=100) specimens and compared CYP24A1 in smokers (former/current) versus non-smokers. In parallel, we performed metabolism experiments in A549 (high CYP24A1) and SKLU-1 (low CYP24A1) cells to study the functional relevance of increased CYP24A1. We evaluated the promoter methylation status of CYP24A1 in the cancer cell lines and lung AC patients using pyrosequencing PCR analysis. Finally, quantitative chromatin immunoprecipitation PCR (ChIP-qPCR) analysis was performed to validate the CYP24A1 methylation was regulated by histone modifiers. Non-smokers had higher serum levels of 25-D3 compared with smokers (albeit small numbers of non-smokers, n=11). Smoking lung AC patients showed higher mRNA expression of CYP24A1 (P < 0.00374) and poorer 5-year survival compared to nonsmokers in Kaplan-Meir survival analysis (Logrank P < 0.0435). A549 cells catabolized 1,25-D3 to a greater extent compared to SKLU-1 at 48-h treatment of 100 nM 1,25-D3 (5.67 vs 103 pmole 1,25-D3 remained). Smoking increased CYP24A1 expression in lung AC patients through hypomethylation of CYP24A1 promoter. In the pyrosequencing analysis, A549 cells were unmethylated (1.01%) but SKLU-1 cells highly methylated (52.02%). Treatment with the DNA methyltransferase inhibitor 5-aza-2α-deoxycytidine (5-Aza) activates CYP24A1 expression in lung cancer cells. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in lung cancer cells. ChIP-qPCR reveals that treatment with 5-Aza and/or TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with 5-Aza and/or TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Taken together, smoking induced the expression of CYP24A1 and showed faster metabolism of 1,25-D and led to poorer survival in lung AC patients, This induction might be related to epigenetic changes in CYP24A1 gene. By increasing the substrate or silencing CYP24A1 gene, the possibility of CYP24A1 gene for chemoprevention will be investigated. Supported by NIH R21CA128193-01-A1 and VA Merit I01CX000333-02. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3124. doi:1538-7445.AM2012-3124