Long chain alkylphenol mixture promotes breast cancer initiation and progression through an ERα36-mediated mechanism
Résumé
Growing source of evidence suggests that exposure to estrogen mimicking agents is a risk factor for breast cancer onset and progression. Long chain alkylphenols are man-made compounds still present in household products, industrial and agricultural processes, leading to a global environmental and human contamination. These molecules are known to exert estrogen-like activities through binding to classical estrogen receptors. Recently, we have demonstrated that a realistic mixture of 4-tert-octylphenol and 4-nonylphenol can stimulate proliferation and modulate epigenetic status of testicular cancer germ cells through a rapid, Estrogen Receptor alpha 36 (ERα36)- dependent non genomic pathway (Ajj et al, 2013; doi: 10.1371/journal.pone.0061758). In a retrospective study of breast tumor samples, we also validated ERα36 expression as a reliable prognostic factor for cancer progression from an estrogen dependent proliferative tumor toward an estrogen dispensable metastatic disease (Chamard-Jovenin et al, 2015; doi: 10.1186/s12918-015-0178-7).
Since high ERα36 expression enhances expression of migration/invasion markers in breast tumors, we addressed the question of its involvement in response to alkylphenol exposure in vitro (MCF-10A mammary epithelial cell line and MCF-7 estrogen-sensitive cancer cells) and in vivo (C57/Bl6 mice).
Methods
In order to characterize the molecular events in alkylphenol exposed cells, ERα36 overexpression (knock in) or gene-silencing (knock down) strategies combined to microarray analyses of the mixture target genes were used in MCF-10A cells. Molecular and cellular biology experiments confirmed the predicted phenotypes. A customized database was designed to analyze comprehensive gene expression results, nonlinear correlation analyses, and mutual information computations helpful for the modeling of alkylphenol/ERα36-dependent pathways.
In vivo, alkylphenol mixture doses, representative of human exposure, were orally given to C57/Bl6 pregnant females and histological analyses were then performed on F1 mammary glands.
Key Results
Our results highlight a key role for ERa36 in alkylphenol non genomic src protein kinase /PI3-kinase/serine-threonine kinase Akt/ nuclear factor-kappa B signaling in non cancerous epithelial breast cells. Flow cytometry analyses, scratch-wound assays and caspase clivage measurements indicate that the alkylphenol mixture may promote a neoplastic like phenotype, i. e. proliferation, apoptosis escape and migration in MCF-10A epithelial cells through an ERα36 dependent pathway. These results are currently used to build a model of alkylphenol-directed breast cancer induction and progression.
In vivo, mammary gland hyperplasia is observed following alkylphenol exposure during embryonic life.
Conclusions
Hence, alkylphenol and/or ERa36-dependent control of the proliferation, adhesion and survival pathways opens the way to a better understanding of the link between endocrine disruptor exposure and the burden of hormone sensitive cancers.
This work is supported by ANSES (n°2012-2-014).