Androgen receptor (AR) is an attractive target in breast cancer because of its frequent expression in all the molecular subtypes, especially in estrogen receptor (ER)-positive luminal breast cancers. We have previously shown a role for AR overexpression in tamoxifen resistance. We engineered ER-positive MCF-7 cells to overexpress aromatase and AR (MCF-7 AR Arom cells) to explore the role of AR in aromatase inhibitor (AI) resistance. Androstendione (AD) was used as a substrate for aromatization to estrogen. The nonsteroidal AI anastrazole (Ana) inhibited AD-stimulated growth and ER transcriptional activity in MCF-7 Arom cells, but not in MCF-7 AR Arom cells. Enhanced activation of pIGF-1R and pAKT was found in AR-overexpressing cells, and their inhibitors restored sensitivity to Ana, suggesting that these pathways represent escape survival mechanisms. Sensitivity to Ana was restored with AR antagonists, or the antiestrogen fulvestrant. These results suggest that both AR and ERα must be blocked to restore sensitivity to hormonal therapies in AR-overexpressing ERα-positive breast cancers. AR contributed to ERα transcriptional activity in MCF-7 AR Arom cells, and AR and ERα co-localized in AD + Ana-treated cells, suggesting cooperation between the two receptors. AR-mediated resistance was associated with a failure to block ER transcriptional activity and enhanced up-regulation of AR and ER-responsive gene expression. Clinically, it may be necessary to block both AR and ERα in patients whose tumors express elevated levels of AR. In addition, inhibitors to the AKT/IGF-1R signaling pathways may provide alternative approaches to block escape pathways and restore hormone sensitivity in resistant breast tumors.

It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers, however we do not yet know how to best treat these patients. We have modeled the three most frequent hormone binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR we identified mutations at high frequencies ranging from 12% for Y537N, 5% for Y537S, and 2% for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort, and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.

The idea that somatic ER mutations could play an important role in the evolution of hormone dependent breast cancers was proposed some years ago [1,2], but has remained controversial until recently. A significant amount of new data has confirmed these initial observations and shown their significance, along with much additional work relevant to the treatment of breast cancer. Thus, it is the purpose of this review to summarize the research to date on the existence and clinical consequences of ER mutations in primary and metastatic breast cancer.

Purpose Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) -positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI), a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDI and seek to determine AR’s contribution to resistance. Methods We engineered ER -positive cell lines with stable knock-down (KD) of Rho GDI (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Results Tam-resistant tumors and cell lines with low Rho GDI levels exhibited upregulated AR expression. Microarray of Rho GDI KD cells indicated that activation of EGFR and ER was associated with Tam treatment. When AR levels were elevated interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ER. Tam exhibited agonist activity in AR over-expressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. Conclusions We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ER -positive cells with low expression of Rho GDI.

The androgen receptor (AR) is a proven clinical target in prostate cancer. Recent research indicates that it is an emerging hormonal target in breast cancer as well, with potential clinical benefit in both estrogen receptor(ER) positive and negative tumors. Compared to the ER, AR contains unique functional domains with relevance to its altered role in human breast cancer. The majority of ER-positive tumors express AR, and a significant percentage of ER-negative tumors might benefit from combined targeting of AR and the ErbB2/HER2 oncogene. Signaling downstream of AR might also affect many clinically important pathways which are also emerging clinical targets in breast cancer. AR expression might also play a role during tumor progression to metastatic disease. The role of AR as a new important biomarker in breast cancer will be reviewed herein.