title: 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.
  
  
  METHODS
  Twenty-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.
  
  
  RESULTS
  All 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Environmental Science, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Lily M
authors.initials: LM
authors.lastname: Fitzgerald
authors.affiliation: Department of Veterinary and Animal Science, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Eva P
authors.initials: EP
authors.lastname: Browne
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Christie
authors.affiliation: Department of Veterinary and Animal Science, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Punska
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Leo O
authors.initials: LO
authors.lastname: Simmons
authors.affiliation: Department of Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Williams
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Pentecost
authors.affiliation: Pathology Department, Baystate Medical Center, Springfield, MA USA.
authors.forename: Rahul
authors.initials: R
authors.lastname: M Jawale
authors.affiliation: Pathology Department, Baystate Medical Center, Springfield, MA USA.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Otis
authors.affiliation: Department of Veterinary and Animal Science, University of Massachusetts Amherst, Amherst, MA USA.
authors.forename: Kathleen F
authors.initials: KF
authors.lastname: Arcaro
keywords: Breast cancer; DOK7; ELF5; Estrogen receptor; Progesterone receptor; Tamoxifen resistance