Previously, we showed that the bioactive green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits growth in soft agar of breast cancer cells with Her-2/neu overexpression. Using gene expression profiling, here we show that EGCG treatment of Her-2/neu–driven mammary tumor cells alters the expression of key regulators in the epithelial to mesenchymal transition (EMT) pathway, reducing invasive phenotype. Specifically, the epithelial genes E-cadherin, γ-catenin, MTA3, and estrogen receptor α (ERα) were up-regulated by EGCG, whereas the proinvasive snail gene was down-regulated. Consistently, EGCG inhibited branching colony growth and invasion in Matrigel. EGCG treatment similarly inhibited invasive phenotype of mouse mammary tumor cells driven by Nuclear Factor-κB c-Rel and protein kinase CK2, frequently found overexpressed in human breast disease. Recently, we identified the Forkhead box O transcription factor FOXO3a as a major transcriptional regulator of ERα. Given the pivotal role of ERα in preventing EMT, we hypothesized that the activation of FOXO3a by EGCG plays an important role in the observed reversal of invasive phenotype in ERα-positive breast cancer cells. EGCG treatment activated FOXO3a. Ectopic expression of a constitutively active FOXO3a overrode transforming growth factor-β1–mediated invasive phenotype and induced a more epithelial phenotype, which was dependent on ERα expression and signaling. Conversely, a dominant negative FOXO3a reduced epithelial phenotype of ERα-low breast cancer cells. These results identify, for the first time, a role for FOXO3a in the inhibition of invasive phenotype in breast cancer cells with active ERα signaling and elucidate a novel mechanism whereby EGCG represses EMT of breast cancer cells. [Cancer Res 2007;67(12):5763–70]

Activated estrogen receptor α (ERα) modulates transcription triggered by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demonstrated that the interference occurs without binding of ERα to DNA but probably results from protein·protein interactions. However, involvement of a direct interaction between ERα and AP-1 is still debated. Using glutathioneS-transferase pull-down assays, we demonstrated that ERα bound directly to c-Jun and JunB but not to FOS family members, in a ligand-independent manner. The interaction could occur when c-Jun was bound onto DNA, as shown in a protein-protein-DNA assay. It implicated the C-terminal part of c-Jun and amino acids 259–302 present in the ERα hinge domain. ERα but not an ERα mutant deleted of amino acids 250–303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, as shown by two-hybrid and coimmunoprecipitation assays. We also show that ERα, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein complex in vitro and in intact cells and that the ERα·c-Jun interaction could be crucial for the stability of this complex. VP16-ERα and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induced transcription in the presence of estradiol, and this synergistic effect was not observed with the ERα mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ERα, was used instead of c-Jun. Finally, ER241G was inefficient for regulation of AP-1 activity, and an ERα truncation mutant encompassing the hinge domain had a dominant negative effect on ERα action. These results altogether demonstrate that ERα can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizing a multiprotein complex containing p160 coactivator, is likely to be involved in estradiol regulation of AP-1 responses.

Induction of epithelial-to-mesenchymal transition (EMT) by TGF-β1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-β1–mediated EMT. Here, TGF-β1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-β1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-β1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-β1–induced EMT via repression of BMP-5 in breast cancer. Cancer Res; 72(23); 6268–78. ©2012 AACR.

The aryl hydrocarbon receptor (AhR) is an evolutionarily conserved transcription factor bound and activated by ubiquitous environmental pollutants. Historically, the AhR has been studied for its transcriptional regulation of genes encoding cytochrome P450 enzymes, which metabolize many of these chemicals into mutagenic and toxic intermediates. However, recent studies demonstrate that the AhR plays an important role in the biology of several cell types in the absence of environmental chemicals. Here, this paradigm shift is discussed in the context of a putative role for the AhR in mammary gland tumorigenesis. Data demonstrating high levels of constitutively active AhR in mammary tumors are summarized. Particular focus is placed on the likelihood that the AhR contributes to ongoing mammary tumor cell growth and on the possibility that the AhR inhibits apoptosis while promoting transition to an invasive, metastatic phenotype. A working model is proposed that may help explain the sometimes contradictory outcomes observed after AhR manipulation and that serves as a blueprint for the design of therapeutics which target the AhR in breast cancer. The theme that malignant cells reveal the functions for which the AhR has been evolutionarily conserved is presented throughout this discussion.