Wisp3/Ccn6 Regulates Epithelial-Mesenchymal Transition And Tumor Initiating Cell Properties In Triple Negative Breast Cancer Cells Through Slug

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Although triple-negative breast cancers (TNBCs) constitute 10%-20% of all breast cancers, they are aggressive tumors with higher recurrence and poorer prognosis than other breast cancer subtypes. TNBCs frequently exhibit features of epithelial to mesenchymal transition (EMT) and are enriched in breast tumor initiating cells (BTICs). Recent studies have demonstrated that aberrant activation of the EMT program induces stem cell features in breast cells, suggesting that blocking the cross-talk between EMT and stemness may be an effective treatment for TNBCs. WISP3/CCN6, a secreted matrix associated protein, is expressed in normal breast tissues and is decreased in aggressive breast cancer samples. We have shown that CCN6 knockdown in nontumorigenic breast cells triggers EMT and invasion. We hypothesize that CCN6 regulates the cross-talk between EMT and BTICs pathways in TNBCs. Methods: We overexpressed CCN6 in the TNBC cell lines, MDA-MB-231, SUM-159 and MDA-MB-436, which are aggressive and exhibit a mesenchymal-like phenotype and low endogenous CCN6 expression. We tested the effect of CCN6 overexpression in these cell lines using motility and invasion assays in vitro. Stem cell features were investigated by mammosphere assays, and the ALDEFLUOR assay by flow cytometry. We developed mammary xenografts in nude mice using serial dilutions to investigate the role of CCN6 in tumor initiation and metastasis. Results: Overexpression of CCN6 inhibits cell motility and invasion, and leads to phenotypic changes of mesenchymal-epithelial transition (MET), with downregulation of the mesenchymal cell marker vimentin, increased epithelial marker cytokeratin, and decreased protein levels of the EMT transcription factor Slug. CCN6 overexpression significantly decreased the ALDEFLUOR-positive population and mammosphere formation of TNBC cell lines. In vivo, CCN6 overexpression delayed tumor initiation and reduced metastasis in human breast cancer xenografts. Slug reconstitution was sufficient to rescue the effects of CCN6 on MET and stem cell properties of TNBCs. Conclusions: These data demonstrate that CCN6 regulates EMT and tumor initiating cell features in TNBC at least in part through Slug. Further, our study suggests that CCN6 may be a novel regulator of the EMT-stem cell cross talk in TNBC, with possible clinical application. Citation Format: Wei Huang, Kathy A. Toy, Celina G. Kleer. WISP3/CCN6 regulates epithelial-mesenchymal transition and tumor initiating cell properties in triple negative breast cancer cells through Slug. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2014-1037