IntroductionEpithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.MethodsBasal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.ResultsHere we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.ConclusionsTaken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

Most (80%) of the triple-negative breast cancers (TNBCs) express mutant p53 proteins that acquire oncogenic activities including promoting metastasis. We previously showed that wild-type ERβ (ERβ1) impedes epithelial to mesenchymal transition (EMT) and decreases the invasiveness of TNBC cells. In the present study we searched for signaling pathways that ERβ1 uses to inhibit EMT and invasion in TNBC cells. We show that ERβ1 binds to and opposes the transcriptional activity of mutant p53 at the promoters of genes that regulate metastasis. p63 that transcriptionally cooperates with mutant p53 also binds to ERβ1. Downregulation of p63 represses the epithelial phenotype of ERβ1-expressing cells and alters the expression of mutant p53 target genes. These results describe a novel mechanism through which ERβ1 can disturb oncogenic signals to inhibit aggressiveness in TNBCs.

Unfolded protein response (UPR) is an adaptive reaction that allows cancer cells to survive endoplasmic reticulum (EnR) stress that is often induced in the tumor microenvironment because of inadequate vascularization. Previous studies report an association between activation of the UPR and reduced sensitivity to antiestrogens and chemotherapeutics in estrogen receptor α (ERα)-positive and triple-negative breast cancers, respectively. ERα has been shown to regulate the expression of a key mediator of the EnR stress response, the X-box-binding protein-1 (XBP-1). Although network prediction models have associated ERβ with the EnR stress response, its role as regulator of the UPR has not been experimentally tested. Here, upregulation of wild-type ERβ (ERβ1) or treatment with ERβ agonists enhanced apoptosis in breast cancer cells in the presence of pharmacological inducers of EnR stress. Targeting the BCL-2 to the EnR of the ERβ1-expressing cells prevented the apoptosis induced by EnR stress but not by non-EnR stress apoptotic stimuli indicating that ERβ1 promotes EnR stress-regulated apoptosis. Downregulation of inositol-requiring kinase 1α (IRE1α) and decreased splicing of XBP-1 were associated with the decreased survival of the EnR-stressed ERβ1-expressing cells. ERβ1 was found to repress the IRE1 pathway of the UPR by inducing degradation of IRE1α. These results suggest that the ability of ERβ1 to target the UPR may offer alternative treatment strategies for breast cancer.

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BackgroundUpregulation of estrogen receptor beta (ERβ) in breast cancer cells is associated with epithelial maintenance, decreased proliferation and invasion, and a reduction in the expression of the receptor has been observed in invasive breast tumors. However, proof of an association between loss of ERβ and breast carcinogenesis is still missing.MethodsTo study the role of ERβ in breast oncogenesis, we generated mouse conditional mutants with specific inactivation of ERβ and p53 in the mammary gland epithelium. For epithelium-specific knockout of ERβ and p53, ERβ F/F and p53 F/F mice were crossed to transgenic mice that express the Cre recombinase under the control of the human keratin 14 promoter.ResultsSomatic loss of ERβ significantly accelerated formation of p53-deficient mammary tumors. Loss of the receptor also resulted in the development of less differentiated carcinomas with stronger spindle cell morphology and decreased expression of luminal epithelial markers.ConclusionsOur results show that synergism between ERβ and p53 inactivation functions to determine important aspects of breast oncogenesis and cancer progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0872-z) contains supplementary material, which is available to authorized users.