Epigenetic Repression of RARRES1 Is Mediated by Methylation of a Proximal Promoter and a Loss of CTCF Binding

Abstract

BackgroundThe cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown.Methodology/Principal FindingsWe first compared methylation occurring at the sequences (−664∼+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (−664∼−86), while the downstream element (−85∼+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores.Conclusion/SignificanceThis report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.

Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1

“…In validation of the importance of methylation of the promoter region of RARRES1, we performed 5-methylcytosine (5-mC) ChIP on RARRES1-silenced Hs578T cells using 4 locations ranging from ~1000bp upstream of the transcription start site (TSS) to within 100bp of the TSS (as described in Peng, 2012 [17], indicated in Figure 6B as A-D). We observed a decrease in 5-mC following decitabine treatment which was most pronounced in Region C and D (Figure 7B), which is located nearest to site 1.…”

“…Our own work identified the cancer stem cell marker and retinoic-acid (RA) producing enzyme, aldehyde dehydrogenase 1A3 (ALDH1A3), as promoting or suppressing tumor growth in a context-dependent manner in TNBC [15]. The RA receptor responder protein 1 (RARRES1) has also been identified as either suppressing or promoting tumor growth, depending on the study [16,17]. …”

Promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5, and ELP3 in invasive ductal breast carcinoma

“…Previous candidate gene studies investigated promoter hypermethylation of breast cancer lesions and identified aberrant methylation at the promoters of RASSF1A, Hin-1, RAR-b, Cyclin D2, twist, GSTP1, ABCB1, FOXC1, MGMT, MLH1, PPP2R2B, PTEN, BRCA2, CDH13, MSH6 and RARRES1, and others [8][9][10][11][12]. Identification of these genes has helped begin to elucidate the molecular pathogenesis of breast carcinoma, as it reveals intracellular pathways that are altered that likely contribute to oncogenesis.…”

The Promoter Methylation Status and mRNA Expression Levels ofCTCFandSIRT6in Sporadic Breast Cancer

“…CTCF was also recognized as a potential tumor suppressor gene in breast cancer (Filippova et al, 1998(Filippova et al, , 2002Rasko et al, 2001;Fiorentino and Giordano, 2012), and CTCF suppresses breast cancer growth (Tiffen et al, 2013). To our knowledge, all publications currently available mainly describe that loss of CTCF binding due to hypermethylation leads to subsequent silencing of tumor suppressor gene loci, including BRCA1 (Butcher et al, 2004), retinoblastoma (Rb) (De la Rosa-Velazquez et al, 2007), p16(INK4a) (Witcher andEmerson, 2009), p53 (Soto-Reyes andRecillas-Targa, 2010), and retinoic acid receptor responder 1 (RARRES1) (Peng et al, 2012). In our study, none of the invasive breast cancer and DCIS samples was methylated at the CTCF promoters examined, although we have used the commonly studied promoter/upstream by 500 bases and 5¢ UTR exons region with CpG island(s).…”

“…Previous candidate gene studies investigated promoter hypermethylation of breast cancer lesions and identified aberrant methylation at the promoters of RASSF1A, Hin-1, RAR-b, Cyclin D2, twist, GSTP1, ABCB1, FOXC1, MGMT, MLH1, PPP2R2B, PTEN, BRCA2, CDH13, MSH6, RARRES1, SLIT2, and WIF1 and others (Fackler et al, 2003;Lee, 2007;Muggerud et al, 2010;Moelans et al, 2011;Peng et al, 2012;Alvarez et al, 2013). Identification of these genes has helped begin to elucidate the molecular pathogenesis of breast carcinoma, as it reveals intracellular pathways that are altered that likely contribute to oncogenesis.…”