Abstract P6-05-05: The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen
Authors: Lacey Haddon, Xiuying Hu, Hosna Jabbari, Judith Hugh
Journal: Cancer Research (2020)

Abstract Background: The premise that estrogen promotes the growth of all estrogen receptor positive (ER) + breast cancers has led to anti-estrogen therapies as the standard of care for all ER+ tumors. Paradoxically, estrogen (E2) has demonstrated a therapeutic effect in the adjuvant, palliative and prevention of breast cancers in some women suggesting that there is a subset of ER+ breast cancers that may be growth-suppressed by E2. Our companion window-of-opportunity trial (abstract submitted) found E2 is growth suppressive for some post-menopausal women with high ER expressing tumors. We utilized an in vitro cell model to investigate whether differential growth responses to E2 could be due to transcriptional differences mediated by the level of the ER. Method: We generated a stable MCF-7 transfectant (MCF7-ER) using a fluorescently labelled lentiviral plasmid with a doxycycline inducible ER and compared the response to E2 against an MCF-7 mock transfectant (MCF7-EM). We assessed changes in proliferation (S-phase fraction), gene regulation (RNA-Seq) and differential ER-DNA binding (chromatin immunoprecipitation (ChIP-Seq)). To determine if high levels of ER were causing changes in the underlying chromatin configuration, we compared our ChIP-Seq profiles against previously mapped DNA loop anchor regions obtained by chromatin interaction analysis by paired end-tag sequencing (ChIA-PET) on the MCF-7 parental cell line treated with E2 (Fullwood M et al. Nature 2009). Changes in DNA loop formation were assessed by fluorescence in situ hybridization (FISH) and chromatin conformation capture (3C). Results: We confirmed previously published findings that increased ER expression in the presence of E2 leads to significant decreases in S-phase fraction (P = 0.003) with impaired transition at the G1/S and G2/M checkpoints. RNA-Seq studies defined 342 basally-upregulated genes in the MCF7-ER cells that became down-regulated after E2 treatment indicative of inverse regulation compared to the effect of E2 in the MCF7-EM mock transfectants. In E2-deprived conditions, there was an increased number of ER-DNA binding peaks in the high ER expressing cells (2,776 in MCF7-ER vs. 1,906 in MCF7-EM), suggesting unliganded ER may mediate basal gene transcription. Only 17 (5%) of the basally upregulated genes had ER binding at a proximal or distant anchor associated with previously mapped ER-DNA loops. This suggests that high ER expressing cells may develop a novel chromatin configuration that is distinct from those of parental MCF-7 cells. FISH analysis confirmed a significant increase in loop formation at the promoter region of the TFF1 gene in the MCF7-ER cells compared to MCF7-EM mock transfectants (P = 0) in the absence of E2. This DNA loop was maintained, though slightly decreased, after E2 treatment. Conclusions: These results support a theoretical model where increased ER expression may enable a novel chromatin configuration which mediates a basal level of gene expression that switches to a growth-suppressive transcriptional response upon E2 treatment. Further elucidation of an anti-proliferative DNA configuration signature using MCF7-ER cells may generate a novel set of interactions that could serve as a strategy to predict a patient’s response to estrogens and endocrine therapies. Citation Format: Lacey Haddon, Xiuying Hu, Hosna Jabbari, Judith Hugh. The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-05.