TI - Results Ectopic sFRP1 expression in MDA-MB-231 cells blocks WNT signaling . AB - Interference with autocrine WNT signaling via sFRP1 has been shown to block in vitro proliferation of human breast cancer cell lines [6,7] . In the following experiments we examined the effects of blocking WNT signaling using the basal-like breast cancer model MDA-MB-231 [15] . Vectors encoding Myc-tagged sFRP1 and the empty control were transfected into MDA-MB-231 cells , which express no sFRP1 mRNA ( data not shown ) , and stable clones were selected in G418-containing medium . Three MDA-MB-231/sFRP1 clones expressing moderate to strong levels of the Myc-tagged sFRP1 , as well as control clones , were selected for further analyses ( Figure 1a ) . WNT pathway activity was examined in the cells using various markers . As a consequence of WNT binding to FZD , cytoplasmic scaffolding proteins of the Dishevelled family ( DVL1 , DVL2 and DVL3 ) become PHOSphorylated on serine and threonine residues . DVL PHOSphorylation , which is the most proximal signaling event downstream of FZD activation , can be monitored by a decrease in the electrophoretic mobility of p-DVL [16,17] . DVL1 , DVL2 and DVL3 , total beta-catenin and stabilized active beta-catenin were examined by western analysis in the individual clones . In the sFRP1-expressing cells , there was a decrease in the level of the three p-DVLs , compared with the vector control cells . Furthermore , the level of total beta-catenin and active beta-catenin was reduced in the two clones expressing the highest level of sFRP1 ( Figure 1a ) . Next we examined transcriptional activity of the canonical WNT signaling pathway following transient expression of the TOPFlash TCF reporter plasmid in the cells . TOPFlash luciferase reporter activity showed a significant 2.7-fold decrease in the sFRP1-expressing cells compared with controls ( Figure 1b ) , confirming that ectopic expression of sFRP1 reduces canonical pathway activity in MDA-MB-231 cells . The PHOSphorylation status of ERK , another signaling protein that is active in MDA-MB-231 cells , was not altered in the sFRP1-expressing cells ( Figure 1a ) , suggesting that the effects of sFRP1 are specific for the WNT pathway . For further studies , the three sFRP1-expressing clones were pooled ( P1 ) and a second pool of sFRP1-expressing MDA-MB-231 cells consisting of gt 100 clones was generated ( P2 ) . Corresponding control pools , control-P1 and control-P2 , were also generated . Quantification of a western analysis shows that MDA-MB-231/sFRP1-P1 has 2.8-fold higher levels of sFRP1 than does MDA-MB-231/sFRP1-P2 ( Figure 1c ) .