Tumor initiating cells (TICs) serve as the root of tumor growth. After identifying TICs in spontaneous breast tumors of the MMTV-Wnt1 mouse model, we confirmed the specific expression and activation of Yes-associated protein 1 (Yap1) within TICs. To investigate the role of Yap1 in the self-renewal of breast TICs and the underlying mechanism, we sorted CD49fhighEpCAMlow cells as breast TICs. Active Yap1 with ectopic expression in breast TICs promoted their colony formation in vitro (p< 0.01) and self-renewal in vivo (p< 0.01), and led to a 4-fold increase in TIC frequency (p< 0.05).A conditional knock-out mouse was reconstructed to generate Yap1 knock-out breast tumors. The loss of Yap1 led to a dramatic growth disadvantage of breast TICs in vitro (p< 0.01) and in vivo (p< 0.01), and it also led to an over 200-fold decrease in TIC frequency (p< 0.01). The expression of active Yap1 was negatively correlated with that of phosphorylated Smad3 (p-Smad3).Transforming growth factor β (TGF-β) served as a strong enhancer of Smad3 and an inhibitor of clonogenesis of TICs. The presence of SIS3, a specific inhibitor of Smad3, could rescue the TGF-β -induced growth inhibition and reverse the Smad3 inhibition by Yap1. Analysis of a database containing 2,072 human breast cancer samples showed that higher expressions of Yap1 correlated with a poorer outcome of a 15-year survival rate and median overall survival (mOS)in patients, especially in those with basal breast tumors without estrogen receptor 1 (ER) expression. The findings indicate that active Yap1 promotes the self-renewal of breast TICs by inhibiting Smad3 signaling.
Keywords: Smad3; Yap1; breast cancer; self-renewal; tumor initialing cells.