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Increased estrogen receptor expression leads to a novel DNA binding signature which differentiates luminal A and luminal B breast cancers

  • Author / Creator
    Haddon, Lacey
  • Eighty percent of all breast cancers are estrogen receptor positive (ER+) and molecular profiling has identified two ER+ subtypes: luminal A and luminal B. The accurate diagnosis of these luminal subtypes is one of the greatest clinical challenges with current gene assays obtaining only 75% accuracy at best. The need for an improved diagnostic arises from clinical evidence that luminal subtypes have a differential response to the anti-estrogen tamoxifen, which has been the standard of care for all ER+ patients. While luminal A patients have an excellent response to tamoxifen alone, luminal B patients often respond poorly and require additional chemotherapy. Luminal A tumors have the highest levels of ER expression and clinical studies have shown the response to tamoxifen increases as the level of ER expression increases. These tumors occur predominantly in postmenopausal women after the levels of serum estrogen (E2) decreases suggesting that premenopausal levels of E2 may help prevent luminal A cancers. Tamoxifen has well-documented estrogenic properties and there is strong clinical evidence that E2 is growth suppressive for some ER+ patients. This would suggest that the anti-proliferative effect of tamoxifen in tumors with high levels of ER expression is a response to E2. All in vitro ER+ cell lines have the luminal B molecular profile and a marked proliferative response to E2 thereby impeding the study of a potential growth suppressive effect of E2 in luminal A tumors. This has led to the current dogma that E2 is growth promoting for all ER+ tumors. However, in vitro studies have shown that cells with increased ER expression become growth suppressed by E2. These findings led to the hypothesis that the two biological subtypes of ER+ breast cancers represent a differential response to E2 that is regulated by the level of ER. A stable MCF-7 transfectant with an inducible ER plasmid (MCF7-ER) was generated to study the effect of increased ER expression on proliferation, gene regulation, ER-DNA binding and chromatin reconfiguration in the absence and presence of E2. Increased ER expression led to an anti-proliferative response to E2 which was mediated through inhibition of cell cycle progression. The cell cycle block detected after E2 treatment correlated with an increase in p21 expression that may be directly regulated through ER binding at the p21 (CDKN1A) gene. A significant decrease in E2F1 expression was also detected and correlated with the down-regulation of several E2F regulated cell cycle genes in MCF7-ER cells after E2 treatment. These results suggest that increased ER expression mediates an anti-proliferative response to E2 through regulation of the E2F1 pathway. Investigation of the differential response to E2 using full transcriptome analysis (RNA-Seq) was done to determine the effect of increased ER expression on gene regulation. There were 72 basally up-regulated genes in the MCF7-ER cells in the absence of E2 which became down-regulated after E2 treatment. Chromatin immunoprecipitation followed by full genome sequencing (ChIP-Seq) experiments in chapter 4 showed ER binding at previously mapped anchor regions for long-range loops for five of the differentially expressed genes in the presence and absence of E2. Fluorescence in situ hybridization (FISH) experiments confirmed the presence of a long-range loop near the TFF1 gene in the MCF7-ER cells in the absence of E2 which was maintained after E2 treatment. These results suggest that ER-mediated DNA reconfiguration may serve as a biological mechanism that regulates the differential response to E2. Further investigation of E2-regulated genes with a unique DNA loop formation may enable the development of a new clinical assay that can predict an ER+ patient’s response to E2.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R30K26T16
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.