Abstract
Recent researches suggest the Wnt signaling pathway is involved in maintaining the characteristics and population of cancer stem cells. Secreted frizzled-related protein (SFRP) family is composed of five secreted glycoproteins (SFRP1 to SFRP5) which play modulators in the Wnt signaling pathway. SFRPs contain the Frizzled-like cysteine-rich domain (CRD) at the N-terminus which interacts with Wnt ligands, resulting in the prevention of Wnt ligand-Frizzled receptor interaction and the Wnt signaling pathway activation. However, little is known about whether SFRPs only act as extracellular modulators or could be through another mechanism to regulate the Wnt signaling pathway. In our study, we found that SFRPs could translocate into the nucleus and associate with β-catenin in SFRPs-expressing cells. The interaction domain mapping indicated that the binding region of β-catenin for SFRPs was similar to that for T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF). Thus, we further investigated the interaction of TCF4 and β-catenin in the nucleus by co-immunoprecipitation. Interestingly, although β-catenin accumulated in the nucleus of SFRPs-expressing cells, SFRP1, SFRP2, SFRP4 and SFRP5 could significantly suppress TCF4 binding β-catenin compared with Mock cells, whereas SFRP3 could promote the recruitment of β-catenin to TCF4. After that, a glycogen synthase kinase 3 beta (GSK3β) specific inhibitor was used to activate the intracellular Wnt signaling cascade and test whether SFRPs could regulate the transcriptional activity of β-catenin/TCF and consequent biological functions beyond acting as extracellular modulators. We found that SFRP1, SFRP2, SFRP4 and SFRP5 significantly inhibited the transcriptional activity of β-catenin/TCF and sphere formation ability after GSK3β specific inhibitor treatment, whereas SFRP3 obviously enhanced the β-catenin/TCF-mediated transcriptional activity and sphere formation ability. Taken together, in addition to the role of extracellular modulators, SFRPs can also regulate the Wnt signaling via associating with β-catenin to modulate the interaction of TCF4 and β-catenin in the nucleus, which impacts on Wnt/β-catenin-elicited the tumor-initiating ability. To all of above, we provide a novel mechanism of SFRPs regulating the Wnt signaling at the intracellular level, which challenges the rule of SFRPs acting as extracellular modulators in the Wnt signaling pathway.