Mortality from breast cancer results from the ability of some tumors to metastasize to distant sites. Unfortunately, there are only a few preclinical breast cancer models that metastasize to distant sites to study the complex process of metastasis, and almost all of these are estrogen receptor (ER)-negative. We have discovered that a cytoskeletal regulatory gene called Rho guanine disassociation inhibitor alpha (Rho GDI alpha) is expressed at lower levels in metastatic, hormone-resistant ER-positive human breast tumors, suggesting that it potentially might be a biomarker of resistance and/or metastasis. We have data suggesting that Rho GDI alpha may indeed be a metastasis suppressor, because when we knockdown its expression in ER-positive breast cancer cells to simulate our clinical tumor data, we generated a phenotype that was resistant to the antiestrogen tamoxifen (Tam), and which metastasized to lungs with a high frequency when grown as xenografts in athymic nude mice. We have also discovered that Rho GDI alpha knockdown activates the Rho signaling pathway, increasing downstream p21-activating kinase (PAK1) activity, which then increased phosphorylation of ER alpha at serine 305. Concomitantly, levels of the metastasis associated (MTA2) protein and the androgen receptor (AR) were enhanced with Rho GDI alpha knockdown. MTA2 overexpression in ER-positive breast cancer cells rendered them hormone dependent, and in silico analysis of published datasets show that high MTA2 levels are associated with poor clinical outcome in patients treated with Tam. Treatment of Rho GDIα knockdown cells with the AR antagonist bicalutamide blocked tamoxifen’s agonist effects, suggesting that AR overerxpression may play a role in the Tam-resistant phenotype. The role of Rho GDIα, MTA2, and AR in metastasis of Rho knockdown cells is being explored and will be presented.