N 6 -methyladenosine (m 6 A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m 6 A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxiainducible factor (HIF)-1α-and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m 6 A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m 6 A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF-and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O 2 , HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.hypoxia-inducible factors | metastasis | pluripotency factors | self-renewal | tumorigenesis B reast cancer has the highest incidence of all cancers affecting women (1). It is also the leading cause of cancer-related death for women worldwide (2). Although progress has been made in treating early-stage breast cancer, there is no effective strategy to prevent or treat metastasis, which is the major cause of breast cancer-related mortality (3). Within breast cancers, a small subpopulation of cells, which are designated as tumor-initiating cells or breast cancer stem cells (BCSCs), have the unique property of generating daughter BCSCs, which are capable of infinite proliferation through self-renewal, and transient amplifying cells, which are capable of a limited number of cell divisions and give rise to differentiated breast cancer cells (4, 5). Only BCSCs are capable of forming a secondary (recurrent or metastatic) tumor (5, 6). As in the case of ES cells (ESCs) (7), the BCSC phenotype is specified by the expression of core pluripotency factors, including Kruppel-like factor 4 (KLF4), Octamer-binding transcription factor 4 (OCT4), SRY-box 2 (SOX2), and NANOG (8-12). Delineation of the molecular mechanisms that regulate the BCSC phenotype is needed to better understand metastasis and design more effective therapies.Intratumoral hypoxia is a common finding in advanced cancers. The median partial pressure of oxygen (pO 2 ) within primary breast cancers is 10 mm Hg [1.4% (vol/vol) O 2 ], compared with 65 mm Hg [9.3% (vol/vol) O 2 ] in normal breast tissue (13). Hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 are heterodimeric transcriptional activators, consisting of an O 2 -regu...

Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER + /PR + breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.digoxin | mammary fat pad implantation | tumor relapse

Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.