Abstract 3842: RhoC is an essential determinant of breast cancer stem cell abundance and metastatic potential

Abstract Introduction: While ALDH has been identified as a marker for breast cancer stem cells, it is still not clear which signaling molecules drive the breast cancer stem cell phenotype. We observed that expression of the metastatic oncogene RhoC GTPase is concurrently altered with ALDH activity. Based on this observation we asked whether expression of RhoC impacts the aggressive metastatic nature of ALDH-positive breast cancer stem cells. Methods: The effect of RhoC expression was studied by xenografting mice with ALDH-sorted cell lines with normal and modified levels of RhoC. The normal-like mammary epithelial cell line MCF-10A, which has endogenously low RhoC expression, was transfected with either a control plasmid (10A vec) or a plasmid for constitutively active RhoC (10A G14V). The RhoC-overexpressing Inflammatory Breast Cancer (IBC) cell line SUM149 was transfected with either scrambled control shRNA (149 scr) or shRNA targeting RhoC (149 shRhoC). The transfected cells were sorted for positive and negative ALDH activity via the ALDEFLOUR assay. The sorted cells were then orthotopically xenografted into the mammary fat pads of NOD/SCID mice, and the mice were monitored for tumor incidence and lung metastasis. Results: While we expectedly saw a lower overall number of lung metastases in the ALDH(−) population when compared to the ALDH(+), the number of lung metastases in the ALDH(+) population varied depending on the level of RhoC expressed in the xenografted cells. Inhibiting RhoC in the SUM149 shRhoC cells significantly decreased the number of mice presenting with lung metastases when compared with the SUM149 scr control group. Alternatively, overexpressing RhoC in the 10AG14V ALDH(+) population significantly increased the number of metastases when compared with the 10A vec control ALDH(+). Interestingly, 80% of the MCF-10A G14V ALDH(+) lung metastases resulted without the formation of a primary tumor. Alternatively, the number of ALDH positive cells in each cell line was also counted via the ALDEFLOUR assay, and the number of ALDH positive cells in the high RhoC expressing cell lines (SUM149 scr, 10A G14V) were approximately twice the number in the corresponding low RhoC expressing cell lines (SUM149 shRhoC, 10A G14V). This correlates with a western blot which illustrates that a ALDH(−) tumor had a significantly lower level of RhoC when compared to an ALDH(+) tumor. Conclusions: From these findings, we conclude that RhoC expression is an essential determinant of both the metastatic potential and abundance of ALDH(+) breast cancer stem cells. We further determine that RhoC overexpression is capable of driving breast cancer metastasis independent from primary tumor formation. This data has important clinical implications for targeting metastatic cancer stem cells. Currently our lab is testing the therapeutic effects of a novel small molecule RhoC inhibitor. 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 3842. doi:10.1158/1538-7445.AM2011-3842