Ramnath Nithya, Nadal Ernest, Jeon Chae Kyung, Sandoval Juan, Colacino Justin, Rozek Laura S, Christensen Paul J, Esteller Manel, Beer David G, Kim So Hee
*Division of Medical Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; †Ann Arbor Veterans Administration Medical Center, Ann Arbor, Michigan; ‡Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan; §Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, Barcelona, Spain; ‖Department of Environmental Health Sciences, School of Public Health, Ann Arbor, Michigan; ¶Division of Pulmonary Medicine, Department of Medicine, University of Michigan, Ann Arbor, Michigan; #Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Spain; **Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain; and ††College of Pharmacy and Research Institute of Pharmaceutical Science and Technology, Ajou University, Suwon, Republic of Korea.
J Thorac Oncol. 2014 Apr;9(4):473-82. doi: 10.1097/JTO.0000000000000114.
1α,25-Dihydroxyvitamin D3 (1,25-D3) is antiproliferative in preclinical models of lung cancer, but in tumor tissues, its efficacy may be limited by CYP24A1 expression. CYP24A1 is the rate limiting catabolic enzyme for 1,25-D3 and is overexpressed in human lung adenocarcinoma (AC) by unknown mechanisms.
The DNA methylation status of CYP24A1 was determined by bisulfite DNA pyrosequencing in a panel of 30 lung cell lines and 90 surgically resected lung AC. The level of CYP24A1 methylation was correlated with CYP24A1 expression in lung AC cell lines and tumors. In addition, histone modifications were assessed by quantitative chromatin immunoprecipitation-polymerase chain reaction (ChIP-qPCR) in A549, NCI-H460, and SK-LU-1.
Bisulfite DNA pyrosequencing analysis revealed that CYP24A1 gene was heterogeneously methylated in lung AC. Expression of CYP24A1 was inversely correlated with promoter DNA methylation in lung AC cell lines and tumors. Treatment with 5-aza-2'-deoxycytidine (5-Aza) and trichostatin A (TSA) increased CYP24A1 expression in lung AC. We observed that CYP24A1 promoter hypermethylation decreased CYP24A1 enzyme activity in vitro, whereas treatment with 5-Aza and/or TSA increased CYP24A1 enzyme affinity for its substrate 1,25-D3. In addition, ChIP-qPCR analysis revealed specific histone modifications within the CYP24A1 promoter region. Treatment with TSA increased H3K4me2 and H3K9ac and simultaneously decreased H3K9me2 at the CYP24A1 promoter and treatment with 5-Aza and/or TSA increased the recruitment of vitamin D receptor (VDR) to vitamin D response elements (VDRE) of the CYP24A1 promoter.
The expression of CYP24A1 gene in human lung AC is in part epigenetically regulated by promoter DNA methylation and repressive histone modifications. These findings should be taken into consideration when targeting CYP24A1 to optimize antiproliferative effects of 1,25-D3 in lung AC.
1α,25-二羟基维生素D3（1,25-D3）在肺癌临床前模型中具有抗增殖作用，但在肿瘤组织中，其疗效可能受CYP24A1表达的限制。CYP24A1是1,25-D3的限速分解代谢酶，在人肺腺癌（AC）中通过未知机制过度表达。
通过亚硫酸氢盐DNA焦磷酸测序法测定30种肺癌细胞系和90例手术切除的肺AC组织中CYP24A1的DNA甲基化状态。CYP24A1甲基化水平与肺AC细胞系和肿瘤中CYP24A1的表达相关。此外，通过定量染色质免疫沉淀-聚合酶链反应（ChIP-qPCR）在A549、NCI-H460和SK-LU-1细胞中评估组蛋白修饰。
亚硫酸氢盐DNA焦磷酸测序分析显示，CYP24A1基因在肺AC中存在异质性甲基化。肺AC细胞系和肿瘤中CYP24A1的表达与启动子DNA甲基化呈负相关。用5-氮杂-2'-脱氧胞苷（5-Aza）和曲古抑菌素A（TSA）处理可增加肺AC中CYP24A1的表达。我们观察到，CYP24A1启动子高甲基化在体外降低了CYP24A1酶活性，而用5-Aza和/或TSA处理增加了CYP24A1酶对其底物1,25-D3的亲和力。此外，ChIP-qPCR分析揭示了CYP24A1启动子区域内特定的组蛋白修饰。用TSA处理可增加CYP24A1启动子处的H3K4me2和H3K9ac，同时降低H3K9me2，用5-Aza和/或TSA处理可增加维生素D受体（VDR）与CYP24A1启动子的维生素D反应元件（VDRE）的结合。
人肺AC中CYP24A1基因的表达部分受启动子DNA甲基化和抑制性组蛋白修饰的表观遗传调控。在靶向CYP24A1以优化1,25-D3在肺AC中的抗增殖作用时应考虑这些发现。
