Abstract
Abstract Women who receive adjuvant tamoxifen for estrogen receptor (ER) positive breast cancer may develop drug resistant tumors through several pathways. DNA methylation of CpG islands in gene promoters is a mechanism of gene silencing, which has been implicated in acquired tamoxifen resistance, but remains poorly understand. Accordingly, we evaluated DNA methylation and mRNA expression in tamoxifen resistant cell lines and clinical specimens to elucidate markers and mechanisms that may be associated with treatment failures. DNA methylation was assessed in tamoxifen-sensitive MCF-7 cells and two derived cell lines (TMX2-11 and TMX2-28) rendered tamoxifen-resistant by treatment for 6 months with 10-6 M tamoxifen. Methylation profiling was performed using the Illumina HM450 BeadChip. We identified 3,000 CpG sites in which methylation levels of both ERα-positive TMX2-11 and ERα-negative TMX2-28 cells were significantly increased compared with the parental MCF-7 cell line. This analysis identified methylation of MAGED1, a tumor antigen and putative regulator of p53 transcription, as a candidate marker of acquired tamoxifen resistance. Using the HM450 BeadChip (which includes 5 CpG promoter sites for MAGED1), DNA hypermethylation was found in all 5 CpGs in TMX2-28 cells and 4 CpGs in TMX2-11; which was confirmed by pyrosequencing. Treatment with 2.5 μM AZA for 96 hours decreased methylation levels by 10% in TMX2-11 and 31% in TMX2-28 and increased gene expression in TMX2-28 (>400-fold). To assess whether DNA methylation of MAGED1 is associated with decreased expression in human tumors, we analyzed Illumina HM27 BeadChip methylation data and paired expression array data for 208 frozen primary breast cancers collected in the Polish Breast Cancer Study. Analysis of MAGED1 showed a significant negative correlation between expression and methylation in one of the two promoter CpG sites present on the BeadChip (cg17991347: rho = -0.199, p = 0.004). This correlation suggests the potential that promoter methylation may influence MAGED1 expression in breast cancers. To determine whether treatment with tamoxifen results in increased methylation of MAGED1 in recurrent tumors, we prepared DNA from 33 primary and 36 recurrent paired FFPE breast tumors from patients at Baystate Medical Center (Springfield, MA) for HM450 BeadChip methylation analysis. Results from the primary and recurrent tumors from women who did and did not receive tamoxifen treatment is ongoing to assess the role of MAGED1 in tamoxifen-resistance. Functional studies in which MAGED1 is selectively silenced in MCF-7 cells and overexpressed in the tamoxifen-resistant lines are planned to evaluate the potential role of MAGED1 in tamoxifen acquired resistance. Citation Format: Rahul M. Jawale, Krisitin Williams, Maxwell Lee, Howard H. Yang, Jonine Figueroa, Mark Sherman, Christopher N. Otis, Kathleen Arcaro. Tamoxifen-resistant breast cancer: DNA methylation and expression of MAGED1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2014-385
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