Allelic methylation bias of the RARB2 tumor suppressor gene promoter in cancer

Abstract

Retinoic acid receptor B2 (RARB2) is frequently inactivated in cancer. Methylation in the 5'-untranslated region and first exon is known to play a role; however, few studies have analyzed the detailed methylation pattern of the promoter region. We show that hypo- and hypermethylated alleles coexist in 5/11 cell lines in which RARB2 is inactivated. We present evidence supporting the mitotic transmission of these divergent methylation patterns and find a correlation between methylation divergence and heterozygosity at the 3p24 loci, suggesting an allelic methylation bias in these lines. Using a newly devised strategy based on allelic identification via methylation-sensitive restriction enzyme digestion combined with the use of a single nucleotide polymorphism, rs755661, we demonstrate that such a bias exists in three cancer cell specimens heterozygous at rs755661 and therefore amenable to this study. This previously unreported phenomenon of allelic methylation bias suggests that a promoter methylation-independent mechanism may be responsible for inactivation at the hypomethylated allele and this inactivation is reminiscent of an aberrant form of de novo imprinting. Approaches to interpreting methylation data should incorporate the notion of allelic methylation bias.

The effect of modifiable potentials on hypermethylation status of retinoic acid receptor-beta2 and estrogen receptor-alpha genes in primary breast cancer

“…Aberrant hypermethylation of the RARbeta2 promoter has been principally reported in primary breast cancer and premalignant lesions [9,29]. In this study, we have shown that RARbeta2 promoter region undergoes hypermethylation in a 36.4% of primary breast tumors.…”

Knockdown of RARB2 identifies a dual role in cancer

“…We tested this hypothesis using antisense oligodeoxynucleotides (ASO) to knockdown RARB2 mRNA levels in several RARB2-expressing tumor-derived cell lines, including A-549, NCI-H23, and NCI-H125. These cell lines were previously found to carry only unmethylated RARB2 alleles (Pappas et al, 2008). We found a correlation between the extent of knockdown and the inhibition of cell growth.…”

“…First, although large deletions of the chromosomal region 3p(14-23) carrying RARB2 (located at 3p21) have long been known to occur in lung cancer (WhangPeng et al, 1982;Naylor et al, 1987), intragenic mutations involving the remaining allele [the ''second-hit'' in Knudson's two-hit hypothesis (Knudson, 1971)] have not been reported [Houle, B., Pappas, J.J. and Bradley W.E.C., unpublished observations, and (Widschwendter et al, 2000)]. While RARB2 methylation has been reported by several groups including ours (Cote and Momparler, 1997;Cote et al, 1998;Bovenzi et al, 1999;Sirchia et al, 2000Sirchia et al, , 2002Virmani et al, 2000;Bovenzi and Momparler, 2001;Pappas et al, 2008) and may inactivate the remaining allele, the complete lack of mutations remains unusual as well as unexplained, unless RARB2 methylation occurs at a higher rate than is usual for ratelimiting mutations (Knudson, 2001). Second, although expression of classical tumor suppressor genes is very frequently inactivated in tumor cells, RARB2 remains expressed in about half of all lung, breast, and colon cancers.…”

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

“…However, few studies have analyzed the detailed methylation pattern of the promoter region [33,40-42,42], and to our knowledge, studies have not analyzed isolated alleles by sequencing only one subclone per bisulfite conversion reaction ( see Section 2.5.1). The vast majority of studies have used methylation-specific PCR, pooling potentially mixed populations of alleles together, as previously described in ref.…”