Abstract 2139: RhoC is a determinant of metastatic potential and affects the abundance of breast cancer stem cells
Authors: Lian Zhu, Devin T. Rosenthal, Jie Zhang, Liwei Bao, Zhifen Wu, Kathy Toy, Celina G. Kleer, Sofia D. Merajver
Journal: Cancer Research (2012)

Abstract Introduction: While the ALDH1 subpopulation of cancer stem cells have been identified as necessary for breast cancer metastasis, the molecular regulators of ALDH1 marked metastasis are less well known. We observed that expression of the metastatic oncogene RhoC GTPase is concurrently altered with ALDH1 activity. Based on this observation we asked whether expression of RhoC drives the aggressive metastatic nature of ALDH1 (+) breast cancer stem cells. Methods: The normal-like mammary epithelial cell line, MCF-10A, with endogenously low RhoC expression, was transfected with either a control plasmid (vec) or a constitutively active RhoC plasmid (G14V). The RhoC-overexpressing Inflammatory Breast Cancer (IBC) cell line SUM149 was transfected with either a scrambled control shRNA (scr) or a RhoC-targeting shRNA (shRhoC). The transfected cells were sorted for positive (+) and negative (-) ALDH1 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. The effect of RhoC expression on the cell phenotype in vitro was also studied by subjecting the same cell lines above to time lapse microscopy and 3D cell culture. Results: While we expectedly saw a lower overall number of lung metastases in the ALDH1(-) population when compared to the ALDH1(+), the number of lung metastases in the ALDH1(+) population varied depending on the level of RhoC expressed in the xenografted cells. Inhibiting RhoC in the SUM149 cells significantly decreased the number of mice presenting with lung metastases when compared with the SUM149 scr control group. Alternatively, overexpressing RhoC with the G14V vector in the 10A ALDH1(+) population significantly increased the number of metastases when compared with the 10A vec control ALDH1(+). Interestingly, 80% of the MCF-10A G14V ALDH1(+) lung metastases resulted without the formation of a primary tumor. Alternatively, the number of ALDH1(+) cells in each cell line was also counted via the ALDEFLOUR assay, and the number of ALDH1(+) cells in the high RhoC expressing cell lines (SUM149 scr, 10A G14V) was approximately twice the number in the corresponding low RhoC expressing cell lines (SUM149 shRhoC, 10A G14V). We also find that overexpressing RhoC in the slow moving 10A G14V line increases the ALDH1(+) population's cell speed significantly. We also find correspondingly that ALDH1(+) subpopulations that exhibit high RhoC expression forms invasive acinar-like structures in 3D cell culture. Conclusions: From these findings, we conclude that RhoC expression is an essential driver of the metastatic potential and abundance of the ALDH1(+) breast cancer stem cell phenotype. 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 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2139. doi:1538-7445.AM2012-2139