Nithya Ramnath1, Ernest Nadal, Chae Kyung Jeon, Juan Sandoval, Justin Colacino, Laura S Rozek, Paul J Christensen, Manel Esteller, David G Beer, So Hee Kim. 1. *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.
Abstract
INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
INTRODUCTION: 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 humanlung adenocarcinoma (AC) by unknown mechanisms. METHODS: 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. RESULTS: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. CONCLUSIONS: 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.
Authors: P L Jones; G J Veenstra; P A Wade; D Vermaak; S U Kass; N Landsberger; J Strouboulis; A P Wolffe Journal: Nat Genet Date: 1998-06 Impact factor: 38.330
Authors: Y Higashimoto; M Ohata; K Nishio; Y Iwamoto; H Fujimoto; K Uetani; T Suruda; Y Nakamura; M Funasako; N Saijo Journal: Anticancer Res Date: 1996 Sep-Oct Impact factor: 2.480
Authors: Majda Haznadar; Kristopher W Krausz; Ezra Margono; Christopher M Diehl; Elise D Bowman; Soumen Kanti Manna; Ana I Robles; Bríd M Ryan; Frank J Gonzalez; Curtis C Harris Journal: Cancer Med Date: 2018-05-03 Impact factor: 4.452