The
retinoic acid receptor beta (
RAR-beta) gene encodes one of the primary receptors for
retinoic acid, an important signaling molecule in lung growth, differentiation and
carcinogenesis.
RAR-beta has been shown to be down-regulated by methylation in human
lung cancer. We have used previously lung
tumors induced in mice to evaluate the timing and effect of specific
carcinogen exposures on targeting genes altered in human
lung cancer. These studies were extended to characterize the role of methylation of the
RAR-beta gene in murine
lung cancers.
After treatment with the demethylating agent
5-aza-2'-deoxycytidine (DAC),
RAR-beta was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism.
Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the
5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by
carcinogen exposures with the detection of methylated alleles in virtually all primary
tumors associated with cigarette
smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of
tumors induced by
methylene chloride or
vinyl carbamate.
RAR-beta methylation was also detected in 54% of preneoplastic
hyperplasias induced by treatment with NNK.
Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of
adenocarcinoma development. These studies demonstrate that aberrant methylation of
RAR-beta is an early and common alteration in murine lung
tumors induced by several environmentally relevant exposures.