SmgGDS splice variants: Potential novel targets for breast cancer therapeutics.

Abstract

225 Background: Breast cancer malignancy is promoted by the small GTPases K-Ras, Rap1, RhoA, and Rac1. We recently reported that the activity of these GTPases is regulated by two splice variants of SmgGDS, named SmgGDS-558 and SmgGDS-607. SmgGDS is overexpressed in lung and prostate cancer, but its role in breast cancer is unknown. Here we examine whether SmgGDS splice variants are expressed in breast cancer and promote breast cancer malignancy. Methods: Immunohistochemical staining for SmgGDS was performed on archival human breast tumors (N=183) and normal breast tissue (N=22). Immunoblotting was used to detect SmgGDS splice variants in human breast cancer cell lines. siRNAs that silence different SmgGDS splice variants were used to examine the effects of SmgGDS knockdown on breast cancer cell spreading, cell proliferation via [3H]thymidine uptake, anchorage-independent cell growth using soft agar colony assays, and NF-kB activation using luciferase reporter assays. Results: SmgGDS expression was detected in >65% of DCIS and invasive lobular and ductal carcinoma, and in 100% of metastatic tumors, but was detected in only 18% of normal breast tissue specimens (p<0.01). All tested cell lines expressed both SmgGDS splice variants. Silencing SmgGDS-558 or SmgGDS-607 with siRNA significantly diminished cell proliferation (100±2.0 vs. 11.3±1.0 vs. 50.5±2.2, p<0.05) in MCF-7 cells, with similar results in MDA-MB-231 and T47D cells. MCF-7 soft agar colony formation was significantly decreased 71% and 49% when SmgGDS-558 or SmgGDS-607 was silenced. MCF-7 cell spreading was promoted by silencing SmgGDS-558, but not SmgGDS-607. NF-kB activation was also decreased by 30% in resting and TNF-α-treated MCF-7 and MDA-MB-231 cells by silencing either splice variant. Conclusions: SmgGDS is disproportionally expressed in human breast cancers. Reduction of SmgGDS-558 and SmgGDS-607 levels dramatically reduced breast cancer cell proliferation, soft agar colony formation, and NF-kB activation. Our results indicate that SmgGDS splice variants regulate breast cancer malignancy, most likely through their regulation of small GTPases. This study identifies SmgGDS splice variants as novel therapeutic targets in breast cancer.