Background:
De-regulation of the WNT signaling pathway via mutations in APC and Axin, proteins that target b-catenin for destruction, have been linked to various types of human cancers. These genetic alterations are rarely if ever observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-Related Protein1 (sFRP1), a negative Wnt pathway regulator, leading to down-regulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that in addition to the canonical WNT/b-catenin pathway, WNT signaling activates the ERK1/2 pathway in mouse mammary epithelial cells via EGFR transactivation.
Methods:
Using the WNT modulator sFRP1 and siRNA mediated Dishevelled (DVL) knock-down we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay; b-catenin, EGFR, ERK1/2 activation and PARP cleavage were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors or blocking antibodies.
Results:
Phospho-DVL and stabilized b-catenin are present in many breast tumor cell lines indicating autocrine WNT signaling activity. Interfering with this loop decreases active b-catenin levels, lowers ERK1/2 activity, blocks proliferation and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1 and MCF-7 cells. The effects of WNT signaling are partly mediated by EGFR transactivation in human breast cancer cells in a metalloprotease and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor positive breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor.
Conclusion:
Our data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments.