Gujral TS et al. (2014), A noncanonical Frizzled2 pathway regulates epit...

XB-ART-51542

Cell 2014 Nov 06;1594:844-56.

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A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis.

Gujral TS , Chan M , Peshkin L , Sorger PK , Kirschner MW , MacBeath G .

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Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. We find that the Wnt receptor Frizzled2 (Fzd2) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.

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Species referenced: Xenopus
Genes referenced: dvl2 foxc1 fyn fzd2 itk map2k1 mapk1 ocln snai2 stat3.2 vim wnt5a wnt5b

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	Figure 1. Fzd2 and Its Cognate Ligands Wnt5a/b Are Overexpressed in Late-Stage Cancers, and Their Expression Correlates with Mesenchymal Markers(A) Heatmaps showing correlation of Fzd2 and its ligands Wnt5a/b with mesenchymal markers in 59 breast, 62 colon, 28 liver, and 186 lung cancer cell lines.(B) Bar graph showing that Fzd2 mRNA expression is significantly increased in late stages (stages III and IV) of primary liver and lung cancers compared with normal tissue (p < 0.05).(C) Fzd2 regulates cell migration. Top, relative wound density (RWD) of Fzd2-shRNA- or control-shRNA- (sh-Ctl) expressing FOCUS and BT549 mesenchymal cells. Bottom, RWD of Fzd2-expressing or control vector-expressing Huh7 and Dld1 epithelial cells. RWD is a measure of the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at every time point.(D) Fzd2 signaling regulates EMT program. Representative images showing that expression of Fzd2 in Huh7 cells decreased levels of the epithelial markers E-cadherin and Occludin and increased levels of the mesenchymal markers Foxc1 and Vimentin. Blue, nucleus stain.(E) Volume plot of 75 EMT genes measured by qPCR in FOCUS cells expressing Ctl-shRNA or Fzd2-shRNA (left) or Huh7 cells expressing vector only or Fzd2 expression vector (right). A set of genes that were significantly downregulated (p < 0.05) upon knockdown of Fzd2 are shown in red, whereas significantly upregulated (p < 0.05) genes are shown in green.Error bars indicate SEM. See also Figures S1 and S2.
	Figure 2. Stat3 Is a Key Mediator of Fzd2-Mediated Downstream Signaling, EMT Program, and Cellular Migration(A) Comparison of 45 different signal transduction pathways in FOCUS cells transfected with Fzd2 or control shRNA using a 45-transcription factor reporter array. Signaling pathways that showed significant change in Fzd2 knockdown samples are indicated. Neg and Pos denote negative and positive luciferase controls.(B) Bar graph showing increase in transcription activity of Stat3 upon Wnt5a stimulation in Fzd2-expressing Huh7 cells.(C) Bar graph showing decrease in phosphorylation of Stat3, Erk1, and Mek1 upon Fzd2 knockdown in FOCUS cells. The relative phosphorylation of Akt (Ser473) is unchanged in Fzd2-shRNA-expressing cells.(D) Wnt5a stimulation increases phosphorylation of Stat3, Erk, and Mek in a Fzd2-dependent manner.(E) Treatment with Stat3 inhibitor reduces FOCUS cell migration. Dose response curves showing EC50 (50% reduction in cell migration compared with DMSO control) in FOCUS and SNU449 liver cancer cell lines treated with Stat3 or Mek inhibitors.(F) Western blots showing that Fzd2 and Stat3 associate in a coimmunoprecipitation assay. Lysates immunoblotted with anti-Stat3 and anti-Fzd2 are also shown.(G) Perturbing Stat3 expression reverses EMT in FOCUS cells. Bar graph shows expression of epithelial and mesenchymal marker genes in FOCUS cells with knockdown of Stat3.(H) Stat3 activity regulates cell migration. Knocking down expression of Stat3 decreases Fzd2-mediated cell migration in FOCUS cells (left), whereas expression of constitutively active Stat3 (Stat3C) increased migration of Dld1 epithelial cells (middle). Treatment with Stattic (Stat3 inhibitor) decreased migration of Fzd2-overexpressing Huh7 cells (right) in a dose-dependent manner.Error bars indicate SEM. See also Figures S3, S4, and S5 and Table S1.
	Figure 3. Fyn Kinase Is a Critical Regulator of Fzd2-Mediated Stat3 Activity(A) Identification of informative kinases in Fzd2-mediated cell migration using kinome regularization. Plot shows LOOCV error using elastic net regularization fit. The error bars represent cross-validation error plus 1 SD. The kinases identified at absolute minima (blue dashed line) were termed the most informative kinases.(B) Evolution of regression coefficients. Plot showing regression coefficients for Fyn kinase against value of elastic-net penalty α. Nonzero regression coefficients for kinases picked at α > 0.5 (gray region) are considered significantly informative.(C) Src family kinase inhibitor reduces Fzd2-mediated cellular migration. Relative wound density of cancer cells treated with varying concentration of Dasatinib was monitored for 96 hr. Dose-response curves of Dasatinib treatment in seven cancer cell lines and respective EC50 are shown.(D) Knockdown of Fzd2 expression reduces phosphorylation of SFKs in FOCUS cells, whereas overexpression of Fzd2 increases Src phosphorylation in Huh7 cells.(E) Fyn kinase phosphorylates Stat3. Western blots showing phosphorylation of Stat3 upon knockdown of Fyn in FOCUS cells.(F) Wnt5-Fzd2-dependent Stat3 transcription activity can be rescued by overexpression of active Src in Fzd2 or Fyn knockdown cells.(G) Overexpression of active SFK (SrcY527F) in Huh7 cells increased transcriptional activity of Stat3.Error bars indicate SEM. See also Figure S6.
	Figure 4. Fyn Regulates Fzd2-Mediated EMT Program and Cellular Migration(A and B) (A) Perturbing Fzd2-dependent Fyn activity reverses EMT. Plots showing mRNA expression of selected EMT genes measured by qPCR in FOCUS cells expressing shRNA against Fyn or (B) Huh7 cells expressing active Src (Src Y527F).(C) Representative images showing expression of active Src in Huh7 cells decreased levels of epithelial markers, E-cadherin, and Occludin and increased levels of mesenchymal markers, Foxc1, Slug, and Vimentin. Blue nucleus stain. Scale bar, 100 pixels.(D) Heat map showing effect of Fyn inhibitor (Dasatinib) on expression of EMT-associated genes.(E and F) Fyn-shRNA showed significant decrease in Fzd2-mediated cell migration in FOCUS cells, whereas expression of SrcY527F increases cell migration in Huh7 cells. Treatment with Stattic (Stat3 inhibitor) decreased migration of Huh7 cells expressing SrcY527F to the wild-type Huh7 levels.Error bars indicate SEM. See also Figure S6.
	Figure 5. Fzd2 Is Tyrosine Phosphorylated and Is Directly Associated with the Fyn-SH2 Domain(A) Schematics showing domain structures of Fyn-kinase and Fzd2 proteins. Fyn contains an SH3 domain, SH2 domain, and a kinase domain. Fzd2 is a seven transmembrane domain-containing protein. Tyrosine residues in the first cytosolic loop (Y275) and in the C-terminal tail (Y552) are highlighted. Dvl-binding sequence in the C-terminal domain of Fzd2 is also shown.(B) Fzd2 is tyrosine phosphorylated in three HCC cell lines. Western blots showing tyrosine phosphorylation of Fzd2 detected by immunoblotting with anti-phosphotyrosine antibody (pY100) in immunoprecipitates. Total protein levels of Fzd2 and β-actin in whole-cell lysates are also shown.(C) Fzd2-pY552 binds directly to the SH2 domain of Fyn. A peptide array consisting of phosphorylated tyrosine 275 (pY275), pY552, nonphosphorylated tyrosine 275 (Y275), Y552 and a peptide containing proline-rich region from the first cytoplasmic loop of Fzd2 (P276) were incubated with purified SH2 domain of Fyn, SH3 domain of Fyn, and purified Stat3 of GST control proteins. Protein-peptide interaction was measured by probing arrays with anti-GST antibody. A plot of relative fluorescence intensity measured on the array is shown.(D) Western blots showing GST pull-down of Fzd2 and Stat3 using purified SH2 domain of Fyn. The pull-downs were subjected to western blotting and immunoblotted with anti-Fzd2, Stat3, and GST antibodies. Total protein levels of Fzd2 and β-actin in whole-cell lysates are also shown.(E) SH2 domain of Fyn is critical for Wnt5/Fzd2-mediated Stat3 transcriptional activity. Stat3 transcriptional activity was measured in FOCUS cells transfected with the indicated constructs.(F) A bar graph showing Fzd2 tyrosine phosphorylation in FOCUS cells treated with DMSO, Dasatinib (1 μM), or Staurosporine (100 nM) for 30 min. Fzd2 phosphorylation was detected by immunoblotting with anti-phosphotyrosine antibody (pY100) in Fzd2 immunoprecipitates. Data are the mean of at least two independent samples, and error bars indicate SEM.Error bars indicate SEM. See also Figure S6.
	Figure 6. Fzd2 Knockdown or Treatment with an Anti-Fzd2 Antibody Reduces Tumor Growth and Metastasis in Mouse Xenograft(A) Knockdown of Fzd2 expression reduces tumor growth in nude mice. FOCUS cells were injected s.c. into athymic mice and the ability of cells to form tumor outgrowths was monitored in the presence (red shade) or absence (green shade) of siRNA against Fzd2.(B) Treatment with two different clones of anti-Fzd2 antibodies reduced tumor growth in nude mice in a dose-dependent manner. Athymic mice were subcutaneously injected with FOCUS-Luc cells. When the outgrowths were ∼200 mm3, mice were divided at random into three groups (vehicle control, mAb-Fzd2 10 mg/kg, and mAb-Fzd2 30 mg/kg) for clone1 and into two groups (vehicle control and mAb-Fzd2 30 mg/kg) for clone 2 treatment. The treated group received mAb-Fzd2 injection twice a week for 2 weeks, whereas the control group received s.c injection of vehicle.(C) Ex vivo detection of metastasis after subcutaneous injection of FOCUS-luc or Huh7-Luc cells in nude mice. Liver and lungs were dissected from mice treated with 30 mg/kg antibody clone 1 or 28 mg/kg antibody clone 2 as well as the vehicle-treated control group to examine metastasis. Liver and lungs were dissected from mice injected with Huh7 cells expressing Fzd2 or vector only controls.(D) Overexpression of Fzd2 expression in Huh7 cells does not affect tumor growth in nude mice. Huh7 cells transfected with either empty vector or vector encoding Fzd2 gene were injected s.c. into athymic mice, and the ability of cells to form tumor outgrowths was monitored.(E) A Fzd2-gene signature (55 genes), 3-gene signature (Fzd2, E-cadherin, and MMP9), and Fzd2-only correctly predicted metastasis in 46 cases of HCC. AUC represent area under the curve.(F) Kaplan-Meier survival curves for 46 HCC patients. The statistical p value was generated by the Cox-Mantel log-rank test.Error bars indicate SEM. See also Figure S7 and Table S2.
	Figure 7. A Schematic of a Noncanonical Fzd2 PathwayWnt5-Fzd2-Fyn-Stat3 axis contributes to EMT program, cellular migration, and tumor metastasis. Dashed line indicates the provisional nature of this pathway.

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Balanis, Epithelial to mesenchymal transition promotes breast cancer progression via a fibronectin-dependent STAT3 signaling pathway. 2013, Pubmed