Suleiman Massarweh

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 cross-talk 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.

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)-positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifen's antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2.

Introduced more than 100 years ago, endocrine therapy is still the most important systemic therapy for all stages of estrogen receptor (ER) -positive breast tumors. A major clinical problem limiting the usefulness of this therapy is tumor resistance, either de novo or acquired during the course of the treatment. Relatively new discoveries emphasize the complexity of ER signaling and its multiple regulatory interactions with growth factor and other kinase signaling pathways. Both genomic (nuclear) and nongenomic (membrane and cytoplasmic) ER activities contribute to this intimate cross-talk, which is probably a fundamental factor in endocrine resistance. New targeted therapies, especially against the epidermal growth factor receptor/HER-2 pathway, should be carefully evaluated in more (bio)logical strategies to enable them to be exploited appropriately. A strategy of combining endocrine therapy (particularly tamoxifen) with these inhibitors, to circumvent de novo and acquired resistance, will be discussed. We will also emphasize open questions and future challenges in the dynamic research field of molecular ER biology from the endocrine therapy perspective.

Data suggest that breast cancer growth is regulated by coordinated actions of the estrogen receptor (ER) and various growth factor receptor signaling pathways. In tumors with active growth factor receptor signaling (e.g., HER2 amplification), tamoxifen may lose its estrogen antagonist activity and may acquire more agonist-like activity, resulting in tumor growth stimulation. Because treatments designed to deprive the ER of its ligand estrogen will reduce signaling from both nuclear and membrane ER, aromatase inhibitors might be expected to be superior to tamoxifen in tumors with high growth factor receptor content, such as those overexpressing HER2. Recent clinical studies suggest that this is the case in humans, as trials of aromatase inhibitors show superior results compared with tamoxifen, especially in tumors overexpressing HER2. Although estrogen deprivation therapy is often effective in ER-positive breast cancer, de novo and acquired resistance are still problematic. Experimental models suggest that in one form of resistance to estrogen deprivation therapy, the tumor becomes supersensitive to low residual estrogen concentrations perhaps because of activation of mitogen-activated protein kinase. Such tumors respond to additional treatment with fulvestrant or even tamoxifen. On the other hand, in tumors overexpressing HER2, acquired resistance to estrogen deprivation therapy involves the loss of ER and ER-regulated genes and further up-regulation of growth factor signaling rendering the tumor hormonal therapy resistant. This process can be delayed or reversed by simultaneous treatment with growth factor pathway inhibitors. This strategy is now being tested in clinical trials.

Endocrine therapy, and especially tamoxifen, is the most important systemic treatment of estrogen receptor (ER)-positive breast cancer at all stages. A serious obstacle, however, is intrinsic or acquired resistance to these therapies, which in the case of selective ER modulators, such as tamoxifen, involves some imbalance of their agonist versus antagonist actions. Recent data suggest that levels of both ER coregulatory proteins and extra and intracellular signaling from growth factor-related pathways may be important in adjusting this mixed agonist/antagonist activity of selective ER modulators in resistant breast tumors. We suggest that ER coregulators' mediation of growth factor and other cellular signaling to the ER pathway is an important feature in endocrine response and resistance in breast cancer. Indeed, we find that failure of the antitumor activity of tamoxifen in patients with breast cancer is actually determined by both the levels of and the interaction between the ER coactivator amplified in breast cancer-1 (AIB1) and the epidermal growth factor-related protein HER. Thus, the interactions of these diverse elements are essential considerations in defining new predictive and therapeutic tools.