Abstract
Background Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. MethodsTwenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 μM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women.ResultsAll 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors.ConclusionsThe distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.
Highlights
Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops
We have previously shown that tamoxifen treatment alters DNA methylation of various genes involved in cell growth in breast cancer cell lines [12]
We sought to characterize tamoxifenselected MCF-7 derivative cell lines based on changes in the expression and methylation of docking protein 7 (DOK7), E74-like factor 5 (ELF5), and ERα, and the protein levels of ERα, progesterone receptor (PR), and human epidermal growth factor 2 (HER2). To extend these findings clinically, we examined the relationship between hormone receptor status, anti-estrogen treatment and promoter methylation of DOK7 and ELF5 in a set of matched primary and recurrent breast tumors from 24 women
Summary
Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. 80 % of primary breast cancers express estrogen receptor α (ER), a nuclear transcription factor that is the product of the ESR1 gene [1,2,3]. We have previously shown that tamoxifen treatment alters DNA methylation of various genes involved in cell growth in breast cancer cell lines [12]. Despite their insensitivity to tamoxifen, most resistant breast cancers still express ER [9]. Cancers with decreased levels of PR are more likely to become resistant to endocrine therapy [15], and HER2 overexpression is known to promote tamoxifen resistance via cross-talk with ER [16]
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