Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR, and reduced estrogen receptor (ER) α mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERα-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays, and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex ™ (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERα that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

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.

Aromatase inhibitors (AI) are rapidly becoming the first choice for hormonal treatment of estrogen receptor-A (ERA)-positive breast cancer in postmenopausal women. However, de novo and acquired resistance frequently occurs. We have previously identified a lysine to arginine transition at residue 303 (K303R) in ERA in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to tamoxifen treatment. Thus, we questioned whether resistance to AIs could arise in breast cancer cells expressing the ERA mutation. As preclinical models to directly test this possibility, we generated K303R-overexpressing MCF-7 cells stably transfected with an aromatase expression vector. Cells were stimulated with the aromatase substrate, androstenedione, with or without the AI anastrozole (Ana). We found that Ana decreased androstenedione-stimulated growth of wildtype cells, whereas K303R-expressing cells were resistant to the inhibitory effect of Ana on growth. We propose that a mechanism of resistance involves an increased binding between the mutant receptor and the p85A regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K), leading to increased PI3K activity and activation of protein kinase B/Akt survival pathways. Inhibition of the selective ''addiction'' to the PI3K/Akt pathway reversed AI resistance associated with expression of the mutant receptor. Our findings suggest that the K303R ERA mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and that targeting the PI3K/Akt pathway may be a useful strategy for treating patients with tumors resistant to hormone therapy.

We previously identified a lysine to arginine transition at residue 303 (K303R) in ERα in invasive breast cancers, which confers resistance to the aromatase inhibitor (AI) anastrozole (Ana) when expressed in MCF-7 breast cancer cells. Here we show that AI resistance arises through an enhanced cross-talk of the IGF-1R/IRS-1/Akt pathway with ERα, and the serine (S) residue 305 adjacent to the K303R mutation plays a key role in mediating this cross-talk. The ERα S305 residue is an important site that modifies response to tamoxifen; thus, we questioned whether this site could also influence AI response. We generated stable transfectants expressing wild-type (WT), K303R ERα, or a double K303R/S305A mutant receptor, and found that the AI-resistant phenotype associated with expression of the K303R mutation was dependent on activation of S305 within the receptor. Ana significantly reduced growth in K303R/S305A-expressing cells. Preventing S305 phosphorylation with a blocking peptide inhibited IGF-1R/IRS-1/Akt activation, and also restored AI sensitivity. Our data suggest that the K303R mutation and the S305 ERα residue may be a novel determinant of aromatase inhibitor response in breast cancer, and blockade of S305 phosphorylation represents a new therapeutic strategy for treating tumors resistant to hormone therapy.