Abstract 3015: Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells
Authors: Yadwinder Deol S. , McDermott Sean , Lubman David M. , Chang Jenny C. , Nie Song , Cong Yang , Turdo Alice , Azizi Ebrahim , Luther Tahra K. , Clouthier Shawn G. , Wicha Max 
Published: May 2015

Abstract
Increasing evidence has demonstrated that breast cancers are driven by a cellular subset that displays stem cell properties. These cells mediate tumor metastasis and contribute to therapeutic resistance. We recently reported that breast cancer stem cells (BCSCs) are capable of inter-converting between two cellular states in a process regulated by the tumor microenvironment. CD44+/CD24- CSCs are characterized by expression of epithelial-mesenchymal transition (EMT) genes, invasive and largely quiescent, whereas mesenchymal-epithelial-like (MET) stem cells are characterized by expression of Aldehyde dehydrogenase (ALDH), epithelial morphology and self-renewal. In order to further characterize these two alternative populations of BCSCs, we carried out gene expression analysis of sorted ALDH+ and CD44+/24- populations from a series of primary human breast cancers and established cell lines. We used the CSC markers to fractionate MCF7 cells and performed proteomic analysis utilizing liquid chromatography-tandem mass spectrometry. Differentially expressed proteins were identified using t-test and ANOVA based on spectra counts. Both microarray and proteomic analysis identified Annexin A3 (ANXA3) to be consistently upregulated in ALDH/MET stem cell populations compared to matched CD44+/24-/EMT-like CSCs. These results were validated through qPCR and western blotting in utilizing the luminal cell line MCF7 as well as the claudinlow cell line SUM 159. In the latter, ANXA3 was upregulated 2-5 fold at both the mRNA and protein levels in ALDH+ compared to ALDH- populations. Further, immunofluorescence studies using confocal microscopy demonstrated co-expression of ANXA3 and ALDH in MCF7 and SUM 159 cells. To determine the clinical significance of ANXA3 in breast cancer, we performed immunohistochemical analyses on tissue microarrays of breast cancer patients. ANXA3 expression was found to be significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating ANXA3 staining. ANXA3 was expressed at significantly higher levels in luminal compared to basal and claudinlow tumors. Furthermore, 42% of HER2+ tumors also showed moderate to high expression of ANXA3. These results suggest that ANXA3 is predominantly expressed in luminal breast cancers, where it marks the ALDH+ epithelial-like CSC populations. The functional role of ANXA3 in breast CSCs is currently under investigation.
Citation Format: Deol S. Yadwinder, Sean McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Yang Cong, Alice Turdo, Ebrahim Azizi, Tahra K. Luther, Shawn G. Clouthier, Max Wicha. Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells. [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 3015. doi:10.1158/1538-7445.AM2014-3015