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Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR., (©2016 American Association for Cancer Research.)

Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.