Loss of Rho GDIα and Resistance to Tamoxifen via Effects on Estrogen Receptor α

Background Estrogen receptor (ER) alpha is a successful therapeutic target in breast cancer, but patients eventually develop resistance to antiestrogens such as tamoxifen. Methods To identify genes whose expression was associated with the development of tamoxifen resistance and metastasis, we used microarrays to compare gene expression in four primary tumors from tamoxifen-treated patients whose breast cancers did not recur vs five metastatic tumors from patients whose cancers progressed during adjuvant tamoxifen treatment. Because Rho guanine dissociation inhibitor (GDI) alpha was underexpressed in the tamoxifen-resistant group, we stably transfected ER alpha-positive MCF-7 breast cancer cells with a plasmid encoding a short hairpin (sh) RNA to silence Rho GDI alpha expression. We used immunoblots and transcription assays to examine the role of Rho GDI alpha in ER-related signaling and growth of cells in vitro and as xenografts in treated nude mice (n = 8-9 per group) to examine the effects of Rho GDI alpha blockade on hormone responsiveness and metastatic behavior. The time to tumor tripling as the time in weeks from randomization to a threefold increase in total tumor volume over baseline was examined in treated mice. The associations of Rho GDI alpha and MTA2 levels with tamoxifen resistance were examined in microarray data from patients. All statistical tests were two-sided. Results Rho GDI alpha was expressed at lower levels in ERa-positive tumors that recurred during tamoxifen treatment than in ERa-positive tamoxifen-sensitive primary tumors. MCF-7 breast cancer cells in which Rho GDIa expression had been silenced were tamoxifen-resistant, had increased Rho GTPase and p21-activated kinase 1 activity, increased phosphorylation of ER alpha at serine 305, and enhanced tamoxifen-induced ER alpha transcriptional activity compared with control cells. MCF-7 cells in which Rho GDI alpha expression was silenced metastasized with high frequency when grown as tumor xenografts. When mice were treated with estrogen or estrogen withdrawal, tripling times for xenografts from cells with Rho GDI alpha silencing were similar to those from vector-containing control cells; however, tripling times were statistically significantly faster than control when mice were treated with tamoxifen (median tripling time for tumors with Rho GDI alpha small interfering RNA = 2.34 weeks; for control tumors = not reached, hazard ratio = 4.13, 95% confidence interval = 1.07 to 15.96, P = .040 [ adjusted for multiple comparisons, P = .119]). Levels of the metastasis-associated protein MTA2 were also increased upon Rho GDI alpha silencing, and combined Rho GDI alpha and MTA2 levels were associated with recurrence in 250 tamoxifen-treated patients. Conclusion Loss of Rho GDI alpha enhances metastasis and resistance to tamoxifen via effects on both ER alpha and MTA2 in models of ER alpha-positive breast cancer and in tumors of tamoxifen-treated patients.