Abstract
Abstract Background: Many cancers, including breast cancer, are hierarchically organized and contain a subpopulation of self-renewing cells that display stem cell properties. Breast cancer stem cells (BCSCs) drive tumor growth and metastasis and contribute to therapeutic resistance. Recent studies suggest that BCSCs exist in alternative mesenchymal-like (EMT) and epithelial-like (MET) states which are characterized by expression of the BCSC markers CD44+/CD24- and aldehyde dehydrogenase (ALDH), respectively. BCSCs maintain the plasticity to transition between EMT and MET-like states in a process regulated by the tumor microenvironment. However, the pathways involved in regulation of these stem cell states remain undefined. Approach and Results: In order to elucidate the mechanisms which regulate EMT and MET BCSCs, we performed gene expression and proteomic analysis of BCSC populations enriched for EMT (CD44+/CD24-) or MET, ALDH (Aldefluor-positive). Among the genes enriched in the MET BCSC populations were Annexin A3 (ANXA3). Validation by qPCR and western blotting confirmed increased ANXA3 mRNA and protein expression in the ALDH+ fractions of MCF7, SUM 159, T47D and SUM 149 breast cancer cells, as well as normal mammary epithelial cells. Immunohistochemical analysis revealed that ANXA3 expression was significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating significant ANXA3 expression. To determine the functional role of ANXA3 in regulating BCSCs we established breast cancer cell lines with inducible knockdown of ANXA3 utilizing a doxycycline (DOX) inducible shRNA. ANXA3 knockdown reduced cell proliferation and decreased colony formation in soft agar. In addition, ANXA3 knockdown significantly decreased the proportion of ALDH+ BCSCs. In contrast, ANXA3 knockdown increased the relative proportion of EMT-like (i.e. CD44+/CD24-) cells as well as expression of EMT regulatory genes including Twist1 and Snail1. In addition, ANXA3 knocked down significantly decreased tumorsphere formation in vitro and tumor initiating capacity of breast cancer cells in NOD/SCID mice. Conclusions: These results suggest that ANXA3 may play a significant role in regulating the self-renewal of the MET-like ALDH+ BCSC. Studies are underway to determine the mechanisms of this regulation and to assess the potential of ANXA3 as a cancer stem cell therapeutic target. Citation Format: Yadwinder S. Deol, Sean P. McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Tahra K. Luther, Yang Cong, Ebrahim Azizi, Justin Colacino, Shawn G. Clouthier, Max Wicha. Annexin A3 regulates MET-like aldehyde dehydrogenase positive breast cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2015-2220
Published Version
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