ARTICLE ABSTRACTCancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f+/EpCAM−/Lin−) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f+/EpCAM−, CD44+/EpCAM−, CD44+/CD24−, or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44+/CD24−/ANTXR1+ cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44+/CD24−/ANTXR1− cells. Activation of ANTXR1 by its natural ligand C5A, a fragment of collagen VI α3, increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor–lipoprotein receptor-related protein 6 (LRP6), phosphorylation of GSK3α/β, and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression, self-renewal, invasion, tumorigenicity, and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A, which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore, ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus, ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment, linking it to a stemness signaling network that drives metastatic progression. Cancer Res; 73(18); 5821–33. ©2013 AACR.