Mechanisms of acquired resistance to endocrine therapy in breast cancer, a major clinical challenge, are poorly understood. We have used a mass spectrometry-based screen to identify proteins that are associated with the endocrine-resistant phenotype. In this study, we report the identification of a novel pathway of resistance to endocrine therapy involving interactions of the developmental transcription HOXC11 with the steroid receptor coactivator protein SRC-1, which is a strong predictor of reduced disease-free survival in breast cancer patients. HOXC11 and SRC-1 cooperate to regulate expression of the calcium-binding protein S100β in resistant breast cancer cells. Nuclear HOXC11 and S100β were found to strongly predict poor disease-free survival in breast cancer patients (n = 560; hazard ratios: 5.79 and 5.82, respectively; P < 0.0001). Elevated serum levels of S100β detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio: 5.3; P = 0.004). Our findings define a biomolecular interaction network that drives an adaptive response to endocrine therapy with negative consequences for survival in breast cancer.

Purpose:This study investigates the role of the p160 coactivators AIB1and SRC-1independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments. Experimental Design: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients. Results: Coassociation of the p160s and estrogen receptor a was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrinesensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor a following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1and SRC-1with estrogen receptor a in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively). Conclusions: SRC-1is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor a can predict the response of patients to endocrine treatment.Adjuvant endocrine therapy offers substantial benefit in terms of reduction in risk of tumor recurrence in women with estrogen receptor -positive tumors. However, although most patients initially respond to tamoxifen, in 30% to 40% of cases these tumors recur within 5 years. This precipitates cessation of the regime and the initiation of second-line therapy. However, despite new targeted treatments for breast cancer, the vast majority of patients still depend on endocrine manipulation for management of their breast cancer.The magnitude of estrogen receptor gene regulation is influenced not only by the ligand but also by the presence of specific coregulatory proteins, present at rate-limiting levels, which modulate transcription. Over the past 10 years a number of nuclear receptor -interacting proteins have been isolated using various screening strategies. These include the p160 family coactivator proteins -steroid receptor coactivator-1 (SRC-1/NCoA-1), SRC-2 (TIF2/GRIP1), and SRC-3 (AIB1/ pCIP/RAC3/ACTR). The coactivator proteins drive nuclear receptor transcriptional activity by doing the significant reactions required for control of enhancer-d...

Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor positive breast cancer. In two breast cancer cell line models of AI resistance we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors which recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.

The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other non-steroidal transcription factors, drives this tumor adaptability. In this discovery study we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct ER-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular and in vivo studies. ADAM22 functioned in cellular migration and differentiation and its levels were increased endocrine resistant tumors compared to endocrine sensitive tumors in a mouse xenograft models of human breast cancer. Clinically ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer.