The Src inhibitor AZD0530 cooperates with antiestrogens to inhibit human breast cancer growth in vitro and in vivo

A41

Estrogen plays an important role in human breast cancer development and progression. It is a mitogen for estrogen receptor (ER) positive breast cancers. Upon estrogen binding, estrogen receptor- alpha (ER) recruits and activates Src leading to activation of MEK/MAPK and PI3K/Akt. Activation of these signaling kinases modulates cell cycle regulators to stimulate cell cycle progression. In this study, we investigated whether a novel Src inhibitor drug could synergize with antiestrogens (feslodex, tamoxifen and anastrozole) to inhibit cell proliferation in cell culture and in xenograft tumors in vivo. Aromatase inhibitors (AI) are used to block estrogen synthesis as an important treatment for postmenopausal women with estrogen receptor positive breast cancer. The AI anastrozole alone caused an incomplete cell cycle arrest in ER positive human breast cancer cells stably transfected with the aromatase gene (MCF-Arom5). Although the Src inhibitor, AZD0530 alone had no effect on cell cycle, it enhanced the antiproliferative effect of anastrozole on cultured MCF-AROM5. Using a dose of AZD0530 that on its own did not affect cell proliferation, it took 10 fold less anastrozole to arrest these cells when the anastrozole was used together with AZD0530. Treatment with anastrozole alone stimulated Src and MAPK activity, whereas treatment with anastrozole and AZD0530 together inhibited their activities. The two drugs together caused a greater p27 increase and cyclin E-Cdk2 inhibition than observed with either drug alone reflecting a more profound cell cycle arrest with the combination therapy. We also provide evidence for synergy between anastrozole and AZD0530 in vivo in MCF-AROM5 xenograft tumors in athymic mice. Without androstenedione, tumor growth was minimal through the whole experiment. In contrast, tumor volume increased rapidly with androstenedione treatment, reflecting the mitogenic effect of intracellular aromatase producing estrogen in this model. Daily oral AZD0530 (50mg/kg) had no effect on tumor growth compared with the androstenedione. Anastrozole alone suppressed tumor growth, with 0.25mg/kg anastrozole yielding a treated/control (%) of 62.1%. The combination therapy significantly delayed tumor growth, yielding a treated/control (%) of 46.7%. Our calculations indicate synergism between AZD0530 and anastrozole on xenograft tumors in vivo. All animals survived and showed no signs of toxicity or weight loss throughout the whole experiment. We also found that faslodex and tamoxifen, these two important estrogen blockers, could synergize with AZD0530 to inhibit the cell cycle by causing an upregulation of p27 levels, bingding of p27 to cyclinE-Cdk2 and kinase inhibition. These data show that antiestrogens cooperates with AZD0530 to inhibit ER positive breast cancer growth and support further clinical investigation of this combination for women with ER-positive breast cancer.