Abstract 3336: Anti-angiogenic agents increase breast cancer stem cells via generation of tumor hypoxia
Authors: Santos Sarah J. , Kakarala Pramod , Heath Amber , Clouthier Shawn , Wicha Max S. 
Published: June 2012

Abstract
The cancer stem cell hypothesis posits that many cancers, including breast cancer, are driven by a subpopulation of cells displaying stem cell properties. These cancer stem cells may also be responsible for mediating tumor metastasis and therapeutic resistance. Recently, a number of therapeutic agents designed to target tumor angiogenesis have been developed and have entered clinical trials in breast and other malignancies. Although these agents may delay tumor progression, once these tumors progress, they display accelerated growth and, as a result, these agents have had only limited effects on prolonging patient survival. Since cancer stem cells may mediate tumor invasion and metastasis, we hypothesize that antiangiogenic agents might increase cancer stem cell populations through the generation of tumor hypoxia. We examined the effect of the multikinase VEGFR inhibitor sunitinib on mammary cancer stem cells in mouse xenografts. Sunitinib treatment increased the percentage of cells expressing the stem cell marker Aldehyde dehydrogenase as assessed by the Aldefluor assay, in SUM159 and MDA-MB-231 breast cancer xenografts in NOD/SCID mice. Tumor cells obtained from sunitinib treated mice exhibit enhanced tumor initiating capacity when transplanted in secondary NOD/SCID mice. In order to determine the relationship between generation of tumor hypoxia and increase cancer stem cell frequency, we utilized hypoxyprobe staining to identify areas of tumor hypoxia and ALDH-1 staining to identify cancer stem cells. Tumors from sunitinib-treated mice displayed regions of tumor hypoxia containing enriched populations of ALDH1-expressing cells which also displayed increased nuclear β-catenin staining suggesting activation of the Wnt pathway in these cells. The effect of hypoxia on the cancer stem cell population was recapitulated in vitro by the demonstration that culture of SUM159 and MDA-MB-231 cells under moderate hypoxia (1-2% oxygen) increased the Aldefluor population by over two-fold. Knockdown of HIF1α utilizing an siRNA completely inhibits the increase in cancer stem cells following hypoxia treatment while knockdown of HIF2α exhibited a significant but lesser effect. Cells grown under hypoxic conditions also exhibited increased expression of phospho-Akt and activated β-catenin. Together these studies suggest that antiangiogenic agents increase the cancer stem cell populations through generation of tissue hypoxia, which, in turn, activates Wnt signaling mediated by HIF1α and HIF2α, Akt and β-catenin. The increase in cancer stem cell populations may account for the limited effectiveness of angiogenesis inhibitors and suggests that the use of these agents should be combined with agents capable of targeting the cancer stem cell population.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2011-3336