Breast cancer is the leading cause of deaths from cancer in women. Cancer recurrence is the most common cause of mortality in breast cancer patients. The cancer stem cell (CSC) hypothesis proposes that CSCs are the center of cancer development and recurrence. Targeting CSCs, in combination with standard chemotherapy, may prevent cancer recurrence and improve long-term survival. Stem cells can be enriched in non-adherent sphere cultures. To identify molecular targets in breast CSCs, we evaluated the transcription levels of stem cell-related genes in 4T1 mouse mammary cancer cells grown as spheres or in a monolayer culture. The most differentially expressed gene was found to be wingless-type MMTV integration site family member 1 (Wnt1) in the 4T1 sphere culture. Functionally, knockdown of Wnt1 in breast cancer cell lines suppressed the in vitro properties of the stem-like cells, including their sphere-forming ability and ALDH activity, whereas the addition of recombinant Wnt1 to breast cancer cell lines enhanced the in vitro properties of these stem-like cells. In addition, knockdown of Wnt1 in 4T1 cells affected the properties of the stem-like cells in vivo, including their tumorigenic potential and tumor initiation ability. Collectively, these results suggest that Wnt1 expression may give rise to the properties of CSCs in breast tumors. Therefore, targeting Wnt1-associated signaling proteins may provide an effective therapeutic approach for the treatment of advanced breast cancer. (c) 2012 Elsevier Inc. All rights reserved.