A novel retinoic acid receptor beta isoform and retinoid resistance in lung carcinogenesis

J Natl Cancer Inst. 2005 Nov 16;97(22):1645-51. doi: 10.1093/jnci/dji371.

Abstract

Background: We previously reported that all-trans-retinoic acid (RA) treatment can prevent in vitro transformation of immortalized human bronchial epithelial (HBE) cells.

Methods: To determine whether methylation inhibits RARbeta expression in HBE cells, we used sodium bisulfite sequencing to compare RARbeta P2 promoter methylation patterns in RA-sensitive (BEAS-2B) and RA-resistant (BEAS-2B-R1) HBE cells. Immunoblotting was used to assess induction of the RARbeta, placental transforming growth factor beta (PTGF-beta), Fos-related antigen 1 (Fra-1), and transglutaminase II (TGase II) proteins by RA following treatment with azacitidine, a DNA demethylating agent. The expression, transcriptional activity, and growth suppressive activity of RARbeta1', a novel RAR isoform, were evaluated in lung cancer cells transfected with RARbeta1', and expression was also studied in paired normal lung tissues and lung tumors. All statistical tests were two-sided.

Results: Hypermethylation was observed in the 3' region of the RARbeta P2 promoter of BEAS-2B-R1 but not BEAS-2B cells. Azacitidine treatment of BEAS-2B-R1 cells restored RA-inducible RARbeta2 and PTGF-beta expression but not that of RARbeta1', Fra-1, or TGase II. RARbeta1' expression was repressed in RA-resistant BEAS-2B-R1 cells and in lung cancers, compared with adjacent normal lung tissues. BEAS-2B-R1 cells transiently transfected with RARbeta1' had increased RA-dependent activation of a retinoic acid receptor element (RARE)-containing reporter plasmid compared with vector control (mean = 3.2, 95% confidence interval [CI] = 3.1 to 3.3 versus mean = 1.4, 95% CI = 1.3 to 1.5; P<.001). In H358 lung cancer cells transiently transfected with RARbeta1', RA treatment restored target gene expression compared with that in vector-transfected cells and suppressed cell growth compared with that in untreated cells (4 microM; treated mean = 0.49 versus untreated mean = 1.0, difference = 0.51, 95% CI = 0.35 to 0.67, P = .003; 8 microM: treated mean = 0.50 versus untreated mean = 1.0, difference = 0.50, 95% CI = 0.26 to 0.74, P = .015).

Conclusion: Restoration of RARbeta1' expression may overcome retinoid resistance in lung carcinogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimetabolites, Antineoplastic / pharmacology*
  • Azacitidine / pharmacology*
  • Bone Morphogenetic Proteins / metabolism
  • Bronchial Neoplasms / prevention & control*
  • Cell Line, Tumor
  • DNA Methylation / drug effects
  • GTP-Binding Proteins / metabolism
  • Gene Expression Regulation, Neoplastic / drug effects
  • Growth Differentiation Factor 15
  • Humans
  • Immunoblotting
  • Luciferases / analysis
  • Lung Neoplasms / drug therapy*
  • Lung Neoplasms / metabolism
  • Membrane Proteins / metabolism
  • Promoter Regions, Genetic / drug effects
  • Protein Isoforms
  • Proto-Oncogene Proteins c-fos / metabolism
  • Receptors, Retinoic Acid / genetics
  • Receptors, Retinoic Acid / metabolism*
  • Respiratory Mucosa / metabolism
  • Respiratory Mucosa / pathology*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription, Genetic / drug effects
  • Transfection
  • Transglutaminases / metabolism
  • Tretinoin / pharmacology

Substances

  • Antimetabolites, Antineoplastic
  • Bone Morphogenetic Proteins
  • GDF15 protein, human
  • Growth Differentiation Factor 15
  • Membrane Proteins
  • Protein Isoforms
  • Proto-Oncogene Proteins c-fos
  • Receptors, Retinoic Acid
  • fos-related antigen 1
  • retinoic acid receptor beta
  • Tretinoin
  • Luciferases
  • transglutaminase 2
  • Transglutaminases
  • GTP-Binding Proteins
  • Azacitidine