Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation.
BACKGROUND
De-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target beta-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are 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 protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation 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/beta-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation.


METHODS
Using the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; beta-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages 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 beta-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active beta-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 mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) 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.


CONCLUSIONS
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.
Schlange
Thomas
T
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Matsuda
Yutaka
Y
Lienhard
Susanne
S
Huber
Alexandre
A
Hynes
Nancy E
NE
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D019898:Autocrine Communication; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation; D024161:Collagen Type XI / Q000378:metabolism; D017077:Culture Media, Conditioned; D000072261:Dishevelled Proteins; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D004965:Estrogen Antagonists / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D015533:Transcriptional Activation; D051155:Wnt1 Protein / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism; D000094742:Secreted Frizzled-Related Proteins
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D024161:Collagen Type XI; D017077:Culture Media, Conditioned; D000072261:Dishevelled Proteins; D066246:ErbB Receptors; D004965:Estrogen Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010750:Phosphoproteins; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D013629:Tamoxifen; D051155:Wnt1 Protein; D051176:beta Catenin; D019061:src-Family Kinases; C499319:COL11A2 protein, human; C489427:MYC protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C495247:WNT1 protein, human; C016601:afimoxifene