ARTICLE ABSTRACTPurpose: The use of aromatase inhibitors (AI) in the treatment of estrogen receptor (ER)-positive, postmenopausal breast cancer has proven efficacy. However, inappropriate activation of ER target genes has been implicated in the development of resistant tumors. The ER coactivator protein AIB1 has previously been associated with initiation of breast cancer and resistance to endocrine therapy.Experimental Design: Here, we investigated the role of AIB1 in the deregulation of ER target genes occurring as a consequence of AI resistance using tissue microarrays of patients with breast cancer and cell line models of resistance to the AI letrozole.Results: Expression of AIB1 associated with disease recurrence (P = 0.025) and reduced disease-free survival time (P = 0.0471) in patients treated with an AI as first-line therapy. In a cell line model of resistance to letrozole (LetR), we found ERα/AIB1 promoter recruitment and subsequent expression of the classic ER target genes pS2 and Myc to be constitutively upregulated in the presence of both androstenedione and letrozole. In contrast, the recruitment of the ERα/AIB1 transcriptional complex to the nonclassic ER target cyclin D1 and its subsequent expression remained sensitive to steroid treatment and could be inhibited by treatment with letrozole. Molecular studies revealed that this may be due in part to direct steroid regulation of c-jun-NH2-kinase (JNK), signaling to Jun and Fos at the cyclin D1 promoter.Conclusion: This study establishes a role for AIB1 in AI-resistant breast cancer and describes a new mechanism of ERα/AIB1 gene regulation which could contribute to the development of an aggressive tumor phenotype. Clin Cancer Res; 18(12); 3305–15. ©2012 AACR.