Abstract P5-03-09: Receptor Activator of Nuclear Factor Kappa B (RANK) as a potential therapeutic target in triple-negative breast cancer

Abstract

Abstract Background: Triple-negative breast cancer (TNBC) has a poorer prognosis in comparison to other breast cancer subtypes because of its high metastatic rate. There is evidence that cancer stem cells (CSCs), known to have an epithelial-to-mesenchymal (EMT) phenotype, may contribute to the metastasis of breast cancer. Denosumab (an anti-RANK ligand antibody) is currently being explored as a treatment for breast cancer in phase II and III clinical trials. RANK is thought to regulate mammary stem cells. Therefore, we hypothesized that RANK promotes tumorigenicity and metastasis of TNBC via the regulation of EMT and enrichment of CSCs. We sought to determine the effects of RANK expression on the tumorigenic and metastatic activities of TNBC cells and whether RANK is a potential therapeutic target in TNBC. Methods: Primary breast tumor specimens from the MD Anderson Cancer Center Tumor Bank were stratified by ER/HER2 status. We analyzed RANK mRNA expression levels, obtained by Affymetrix gene chip array, in ER+/HER2-negative breast tumors (n = 22) and ER−/HER2-negative breast tumors including TNBC tumors (n = 18). To study the effects of RANK suppression in TNBC cells, we generated two stable RANK shRNA MDA-MB-231 TNBC cell lines and a negative shRNA cell line as a control and examined whether knockdown of RANK expression correlated with a decrease in mesenchymal markers, vimentin and snail, and stem cell marker, ALDH1, invasion, migration, and tumorigenicity in TNBC cells. Results: An analysis of RANK mRNA expression levels among human primary breast cancer specimens revealed increased RANK expression in ER−/HER2-negative primary breast tumors including TNBC tumors, compared with ER+/HER2-negative primary breast tumors (p = 0.034). Knocking down RANK in MDA-MB-231 cells significantly inhibited migration (p = 0.0005) and invasion (p = 0.0006) and reduced the stem cell subpopulation, as shown by a decrease in mammospheres (p = 0.0188) and ALDH1 activity. Further, in a three-dimensional (3D) cell culture model, we observed significant inhibition of projections in MDA-MB-231 cells with knockdown of RANK (p < 0.05), which correlated with downregulation of the mesenchymal markers vimentin and snail shown by qRT PCR analysis. Conclusion: Our data demonstrate that RANK may promote the tumorigenesis of TNBC by enriching cancer stem cells. In future studies, we will investigate the effect of RANK inhibition on metastasis in vivo using a TNBC xenograft model. Our long-term goal is to develop RANK-targeted therapy for TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-09.