Phthalates stimulate the epithelial to mesenchymal transition through an HDAC6-dependent mechanism in human breast epithelial stem cells.

Phthalates are environmental hormone-like molecules that are associated with breast cancer risk and are involved in metastasis, a process that requires the epithelial-mesenchymal transition (EMT). However, few studies have addressed the potential effects of phthalates on stem cells. Here we tested the hypothesis that phthalates such as butyl benzyl phthalate and di-n-butyl phthalate induce EMT in R2d cells, a stem cell-derived human breast epithelial cell line that is responsive to estradiol for tumor development. We observed that phthalates induced EMT as evidenced by morphological changes concomitant with increased expression of mesenchymal markers and decreased expression of epithelial markers. Molecular mechanism studies revealed that histone deacetylase 6 (HDAC6) is required for phthalate-induced cell migration and invasion during EMT in vitro and metastasis into the lungs of nude mice. We also constructed a series of mutant HDAC6 promoter fragments and found that the transcription factor AP-2a plays a novel role in regulating the HDAC6 promoter. Furthermore, phthalates stimulated estrogen receptors and triggered the downstream EGFR-PKA signaling cascade, leading to increased expression of AP-2a in the nucleus. We also observed that phthalates increased expression of the PP1/HDAC6 complex and caused Akt activation and GSK3β inactivation, leading to transcriptional activation of vimentin through the β-catenin-TCF-4/LEF1 pathway. Understanding the signaling cascades of phthalates that activate EMT through HDAC6 in breast epithelial stem cells provides the identification of novel therapeutic target for human breast cancer.