Mechanisms of Tamoxifen Resistance: Increased Estrogen Receptor-HER2/neu Cross-Talk in ER/HER2–Positive Breast Cancer

Jiang Shou 0 1 Suleiman Massarweh 0 1 C. Kent Osborne 0 1 Alan E. Wakeling 0 1 Simale Ali 0 1 Heidi Weiss 0 1 Rachel Schiff ) 0 1 0 Affiliations of authors: The Breast Center (JS , SM, CKO, HW, RS) and the Departments of Medicine (JS , SM, CKO, HW, RS) and Molecular and Cellular Biology (CKO), Baylor College of Medicine , Houston, TX ; Cancer and Infec- tion Bioscience , AstraZeneca, Macclesfield, Cheshire, UK (AEW); Department of Cancer Medicine, Imperial College of Science, Technology & Medicine , London , UK (SA). Medicine , One Baylor Plaza, MS 600, Houston, TX 77030 ( 1 Journal of the National Cancer Institute , Vol. 96, No. 12, June 16, 2004 Background: Patients receiving adjuvant tamoxifen whose tumors express high levels of both HER2/neu (HER2) and the estrogen receptor (ER) coactivator AIB1 often develop tamoxifen resistance. We used a breast cancer model system with high expression of AIB1 and HER2 to investigate the possible mechanisms underlying this resistance. Methods: MCF-7 breast cancer cells, which express high levels of AIB1, and a tamoxifen-resistant derivative cell line engineered to overexpress HER2 (MCF-7/HER2-18) were treated with estrogen, tamoxifen, epidermal growth factor (EGF), or heregulin in the absence or presence of the EGF receptor (EGFR) tyrosine kinase inhibitor gefitinib. We analyzed phosphorylation of signaling intermediates by immunoblotting, ER transcriptional activity with reporter gene constructs and immunoblot analysis of endogenous gene products, promoter assembly by chromatin immunoprecipitation (ChIP) assay, and tumor cell growth in vitro by anchorage-independent colony formation and in vivo using xenografts in nude mice. Results: MCF-7/HER2-18 tumors were completely growth inhibited by estrogen deprivation but were growth stimulated by tamoxifen. Molecular crosstalk between the ER and HER2 pathways was increased in the MCF-7/HER-2 cells compared with MCF-7 cells, with cross-phosphorylation and activation of both the ER and the EGFR/HER2 receptors, the signaling molecules AKT and ERK 1,2 mitogen-activated protein kinase (MAPK), and AIB1 itself with both estrogen and tamoxifen treatment. Tamoxifen recruited coactivator complexes (ER, AIB1, CBP, p300) to the ER-regulated pS2 gene promoter in MCF-7/ HER2-18 cells and corepressor complexes (NCoR, histone deacetylase 3) in MCF-7 cells. Gefitinib pretreatment blocked receptor cross-talk, reestablished corepressor complexes with tamoxifen-bound ER on target gene promoters, eliminated tamoxifen's agonist effects, and restored its antitumor activity both in vitro and in vivo in MCF-7/HER2-18 cells. Conclusions: Tamoxifen behaves as an estrogen agonist in breast cancer cells that express high levels of AIB1 and HER2, resulting in de novo resistance. Gefitinib's ability to eliminate this cross-talk and to restore tamoxifen's antitumor effects should be tested in the clinic. [J Natl Cancer Inst 2004; 96:926 -35] The binding of estrogen to the estrogen receptor (ER) induces receptor phosphorylation, alters its conformation, triggers receptor dimerization, and facilitates binding of the receptor complex to the promoter region of target genes to activate transcription (1). These transcriptional effects of the ER are modulated by interactions with coregulatory proteins that function as either coactivators or corepressors (2). The conformation of estrogenbound ER favors the recruitment of coactivators that augment - transcriptional activity. By contrast, the ER conformation induced by binding of the selective estrogen receptor modulator (SERM) tamoxifen favors the recruitment of corepressors that inhibit transcriptional activity. Tamoxifen has different partial agonistantagonist activities in different tissues, however, and the differences may be related in part to the milieu of ER coactivators and corepressors in these tissues (3). For example, increased levels of coactivators such as AIB1 (SRC3) or SRC1 enhance the estrogen agonist properties of tamoxifen in vitro (4). ER can generate multiple growth-promoting signals both inside and outside the nucleus. Estrogen-induced expression of genes encoding growth factors, their receptors, and other signaling molecules can provide cell proliferation and survival stimuli (5,6). ER can also complex with other transcription factors, such as Fos and Jun proteins on AP1 response elements, to alter the transcription of genes not normally thought to be classical estrogen targets, such as cyclin D1, insulin-like growth factor 1, and collagenase (7). Finally, new evidence also indicates that ER located in or near the cell membrane can activate growth factor receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and HER2/neu (HER2), providing another mechanism for the growth-promoting effects of estrogen (8). The receptor cross-talk between the ER and growth factor receptors travels in both directions. For example, ERK1,2 mitogen-activated protein kinase (MAPK) that has been activated by signaling from the EGFR or HER2 phosphorylates both ER and the ER coactivator AIB1 (9). The resulting ER phosphorylation in the N-terminal region, which can also be induced by estrogen binding, increases transcription arising from the AF-1 domain of the ER; the resulting phosphorylation of AIB1, which is not directly induced by estrogen, augments its coactivator activity (9,10). These data raise the possibility that high tumor levels of ER coactivators, such as AIB1, could cause tamoxifen resistance and that HER2 cross-talk with ER could enhance the estrogen agonist activity of tamoxifen-bound ER. Tamoxifens agonist properties might require both high AIB1 levels and growth factor receptor cross-talk, which phosphorylates and further activates both AIB1 and ER. In support of this hypothesis, we recently reported that tamoxifen-treated breast cancer patients whose ER-positive tumors express high levels of AIB1 and HER2 experience substantially more recurrences than those with ER-positive tumors that have lower expression of one or both proteins (11). Furthermore, there was a strong correlation between overexpression of AIB1 and overexpression of HER2 in these tumors, suggesting that high levels of the two proteins provide a strong selective growth advantage for tumor cells with this genotype. The goal of the present study was to identify the mechanism for the tamoxifen resistance displayed by ER-positive tumors that express high levels of both AIB1 and HER2. We studied as an experimental model MCF-7 breast cancer cells, which express high levels of AIB1, and a derivative line, MCF-7/HER218, which expresses high levels of both AIB1 and HER2. We compared the estrogen agonist activity of tamoxifen-bound ER on in vivo tumor growth induced by estrogen and tamoxifen in the two cell lines, and we examined cross-talk between the HER2 and ER signaling pathways. We also investigated the components of the coregulatory complexes recruited by tamoxifen-bound ER (...truncated)