title: Epigenetic regulation of vitamin D metabolism in human lung adenocarcinoma.
abstract: BACKGROUND
  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.
authors.affiliation: *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.
authors.forename: Nithya
authors.initials: N
authors.lastname: Ramnath
authors.forename: Ernest
authors.initials: E
authors.lastname: Nadal
authors.forename: Chae Kyung
authors.initials: CK
authors.lastname: Jeon
authors.forename: Juan
authors.initials: J
authors.lastname: Sandoval
authors.forename: Justin
authors.initials: J
authors.lastname: Colacino
authors.forename: Laura S
authors.initials: LS
authors.lastname: Rozek
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Christensen
authors.forename: Manel
authors.initials: M
authors.lastname: Esteller
authors.forename: David G
authors.initials: DG
authors.lastname: Beer
authors.forename: So Hee
authors.initials: SH
authors.lastname: Kim
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D000368:Aged; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D004797:Enzyme-Linked Immunosorbent Assay; D044127:Epigenesis, Genetic; D005260:Female; D005434:Flow Cytometry; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D008297:Male; D009364:Neoplasm Recurrence, Local / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D009367:Neoplasm Staging; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase
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