Mammary epithelial cell phenotype disruption in vitro and in vivo through ERalpha36 overexpression

Abstract

Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), widely expressed in hormone sensitive cancer cells and whose high expression level correlates with a poor survival prognosis for breast cancer patients. While ERα36 activity have been related to breast cancer progression or acquired resistance to treatment, expression level and location of ERα36 are poorly documented in the normal mammary gland. Therefore, we explored the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein ERα36 mRNA was specifically expressed in the mammary gland. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36 and modulated by the JAK2/STAT3 signaling pathway. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration potential and resistance to staurosporin-induced apoptosis of the MCF-10A cell line. In vivo, ERα36 expression led to duct epithelium thinning and disruption in adult but not in prepubescent mouse mammary gland. These phenotypes correlated with a loss of E-cadherin expression. Here, we show that an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of adult endogenous steroid levels, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells

“…Pathogen-free C57BL/6J mice (here considered as wt, Charles River Laboratories) or ERα36 Tg C57BL/6J mice ( 16 ) were maintained in temperature-controlled and light-controlled (10 h light, 14 h dark cycle) conditions in the Nancy Faculty of Pharmacy animal facility. All experimental procedures were approved by the French Minister of Research Committee for animal experiment (Protocol no.…”

“…Transgenic C57BL/6J mice expressing a single copy of a human ERα36 transgene in the mammary gland under the control of the MMTV promoter were obtained as described in Thiebaut et al ( 16 ). Tg males were mated with 7-week-old C57BL/6J wt females in order to obtain 50% hemizygotic Tg litters wherein the transgene is paternally inherited (Figure S1 in Supplementary Material).…”

“…Parternal inheritance of the transgene was preferred to the maternal transmission in order to avoid a potential effect of the transgene expression during lactation. Before weaning, litters from wt females and Tg males were genotyped as previously described ( 16 ). In our study, wt litters issued from wt males and wt litters issued from hemizygote Tg males were compared.…”

“…The non-injected contralateral gland was used as a control. The following three mammary cell lines were used: ERα36 expressing MDA-MB-231 breast cancer line as a positive control for tumor formation, MCF-10A/Zeo as a non-cancerous negative control, or the previously described MCF-10A/ERα36 line overexpressing the human ERα36 receptor ( 16 ). Animals were sacrificed 4 weeks after grafting by lethal intraperitoneal injection of pentobarbital (see the above text).…”

“…Estrogen receptor alpha 36 is a variant of the canonical human ERα lacking the helix 9–12 domain, likely to bind a great diversity of compounds ( 15 ). Recently, we showed that expression of human ERα36 transgene triggers neoplastic-like alteration of mice mammary gland ( 16 ). Since ERα36 can enhance expression of migration/invasion markers in normal epithelial cells, we wondered if its expression or activity could be stimulated under alkylphenol exposure, alter mammary epithelial cell differentiation, and consequently lead to developmental, structural, or functional disruption of the mammary gland.…”