Antiangiogenic therapy has been thought to hold significant potential for the treatment of cancer. However, the efficacy of such treatments, especially in breast cancer patients, has been called into question, as recent clinical trials reveal only limited effectiveness of antiangiogenic agents in prolonging patient survival. New research using preclinical models further suggests that antiangiogenic agents actually increase invasive and metastatic properties of breast cancer cells. We demonstrate that by generating intratumoral hypoxia in human breast cancer xenografts, the antiangiogenic agents sunitinib and bevacizumab increase the population of cancer stem cells. In vitro studies revealed that hypoxia-driven stem/progenitor cell enrichment is primarily mediated by hypoxia-inducible factor 1α. We further show that the Akt/β-catenin cancer stem cell regulatory pathway is activated in breast cancer cells under hypoxic conditions in vitro and in sunitinib-treated mouse xenografts. These studies demonstrate that hypoxia-driven cancer stem cell stimulation limits the effectiveness of antiangiogenic agents, and suggest that to improve patient outcome, these agents might have to be combined with cancer stem cell-targeting drugs.antiangiogenesis | HIF-1α

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