Abstract 2280: Increased activity of the Rho family of proteins results in a tamoxifen-resistant phenotype in ERα-positive breast cancer cells

Abstract

Abstract Background: We discovered that shRNA knockdown of Rho GDIα can render estrogen receptor (ER)-positive breast cancer cells more aggressive by increasing their metastatic ability and altering their tamoxifen (tam) sensitivity. Rho GDIα is a negative regulator of the Rho family of proteins (Rho, Rac, and Cdc42), which play an important role in the regulation of the actin cytoskeleton. While the Rho GDIα pathway is known to influence metastasis in breast cancer, it is unclear how this pathway causes ERα-positive breast cancer cells to become tam-resistant. Since loss of Rho GDIα causes an increased activity of the Rho proteins it is possible that increased activity of downstream effectors may lead to tam resistance through crosstalk with the ERα signaling pathway. We have shown that increased phosphorylation of the S305 site in ERα leads to tam resistance and this site is a target for PAK1 phosphorylation which lies downstream of several growth factor signaling pathways. Results: Stable knockdown of Rho GDIα in ER-positive breast cancer cells, using shRNA, resulted in tam resistance in vivo. Tam stimulated tumor growth in athymic nude mice injected with Rho GDIα knockdown cells. These mice exhibted metastatic lung lesions when treated with either estrogen or tam but not in untreated mice. Tam was unable to reduce in vitro growth of Rho GDIα knockdown cells in soft agar assays. Rhotekin pulldown assays revealed higher activity of Rho proteins in Rho GDIα knockdown cells compared to vector control cells. PAK1 kinase assays revealed that PAK1, a downstream effector of the Rho pathway, had increased activity in Rho GDIα knockdown cells. This increased activity led to increased phosphorylation of ERα on S305. As expected, acetylation of ERα was decreased and this decrease in acetylation was dependent on phosphorylation of S305. ERE-luciferase assays showed that Rho GDIα knockdown cells had higher levels of ERE luciferase activity in these cells, tam significantly stimulated ERE-luciferase activity, suggesting agonist activity as a mechanism of resistance. Further possible effectors of Rho GDIα are being investigated as possible candidates playing a role in the crosstalk between these two pathways, as well as growth factor signaling pathways which may signal to ER and play a role in the tam-resistant phenotype. Interestingly, cells which became tam resistant due to long term culture in the presence of tam showed a decrease in endogenous Rho GDIα levels. Discussion: Loss of Rho GDIα in ERα-positive breast cancer cells conferred a tam-resistant phenotype in vitro and in vivo. Cells with decreased Rho GDIα levels had increased phosphorylation of ERα, suggesting crosstalk between the Rho family of signaling pathways and the ERα signaling pathways. Disrupting this crosstalk may be a mechanism of preventing or reversing tam resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2280. doi:10.1158/1538-7445.AM2011-2280