ELF5 and DOK7 regulation in anti-estrogen treated cells and tumors

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.

WITHDRAWN: Assessment of DOK7 and SPDEF as Potential Epigenetic Biomarkers in Whole Blood of Patients with Gastric Adenocarcinoma and Intestinal Metaplasia

“…It is a cytoplasmic activator of the muscle-speci c kinase (MuSK), involved in neuromuscular junction formation and maintenance [11]. CpG site hypermethylation was observed within the DOK7 promoter region in cancers with epithelial origins, such as breast [12] and lung [13] cancers.…”

The ELF3 transcription factor is associated with an epithelial phenotype and represses epithelial-mesenchymal transition

“…Conversely, downregulating ELF3 showed opposite trends ( Fig 4C , iii). While additional experimental data supporting this hypothesis is needed to validate the importance of these relationships in tamoxifen resistance, the observed upregulation of another ETS family member, ELF5, in tamoxifen-resistant MCF7 cells (Kalyuga et al ., 2012; Fitzgerald et al ., 2016) and tamoxifen-resistant brain metastases (Piggin et al ., 2020), as well as differential expression of ELF3 in tamoxifen-treated vs. control groups (Gielen et al ., 2005), lends credence to this hypothesis.…”

“…Conversely, downregulating ELF3 showed opposite trends (Fig 4C,iii). While additional experimental data supporting this hypothesis is needed to validate the importance of these relationships in tamoxifen resistance, the observed upregulation of another ETS family member, ELF5, in tamoxifen-resistant MCF7 cells (Kalyuga et al, 2012;Fitzgerald et al, 2016) and tamoxifen-resistant brain metastases (Piggin et al, 2020), as well as differential expression of ELF3 in tamoxifen-treated vs. control groups (Gielen et al, 2005), lends credence to this hypothesis. Epithelial and Sensitive (Epi-Sen), Hybrid and Resistant (Hyb-Res), Hybrid and Sensitive (Hyb-Sen), and Mesenchymal and Resistant (Mes-Res) in control, 20-fold up or downregulation of ELF3; * represents a statistically significant difference in the fraction of cases that end up in the epithelial phenotype .…”