WFDC2 suppresses prostate cancer metastasis by modulating EGFR signaling inactivation

Abstract

WAP four-disulfide core domain 2 (WFDC2) is a small secretory protein that has been widely studied in ovarian cancer. It has been proven that WFDC2 promotes proliferation and metastasis in ovarian cancer, and serves as a diagnostic biomarker. However, the specific function of WFDC2 in prostate cancer has not been reported. Here, we first screened the diagnostic marker and favorable prognostic factor WFDC2 in prostate cancer by bioinformatics. WFDC2 expression was negatively correlated with Gleason score and metastasis in prostate cancer. Then, we revealed that overexpression of WFDC2, and addition of recombinant protein HE4 can significantly inhibit prostate cancer metastasis in vivo and in vitro. By co-immunoprecipitation and co-localization assays, we proved that WFDC2 binds to the extracellular domain of epidermal growth factor receptor (EGFR). Immunoblot showed that WFDC2 overexpression and recombinant protein HE4 addition inactivated the EGFR/AKT/GSK3B/Snail signaling pathway, and then restrained the progression of epithelial-mesenchymal transition. In conclusion, our study identified that the tumor suppressor WFDC2 can suppress prostate cancer metastasis by inactivating EGFR signaling.

Fig. 1. High expression of WFDC2 has tropism toward a non-prostate cancer phenotype.

a ROC analysis for distinguishing non-tumor tissues from prostate cancer tissues based on tissue microarray. b, c Barplot of GSEA analysis and separated gene sets for the diagnostic role of WFDC2. d The expression of WFDC2 by different Gleason score based on GSE70770, GSE116918, and TCGA-PRAD. e Dotplot showing the overview of the tissue microarray. The color of the bar represents the percentage of tissue samples in each sample type and the size of the dot represents the sample number. f HE staining (top panel) and IHC staining (middle and bottom panel) of the tissue microarray. The scale bar of HE staining and IHC staining (middle) is 400 μm and the scale bar of IHC staining (bottom) is 100 μm.

Fig. 2. Overexpression of WFDC2 suppressed prostate cancer metastasis in vivo and in vitro.

a The expression of WFDC2 in GSE3325 and GSE8511. b The relationship between WFDC2 and EMT analyzed by GSEA. c, d Transwell assay in DU-145 and PC-3 after WFDC2 upregulation. The scale bar is 150 μm. e, f Wound healing assay in DU-145 and PC-3 after WFDC2 upregulation. The scale bar is 150 μm. g Immunoblot assay of EMT-related proteins in DU-145 and PC-3 after WFDC2 upregulation. h Immunofluorescence staining of WFDC2, p-EGFR, E-cadherin, and Vimentin in DU-145 and PC-3 after WFDC2 upregulation. i Diagram of mouse tail vein injection with PC-3-GFP vector or PC-3-GFP WFDC2 overexpressed. j Representative animal imaging and matching X-rays 6 weeks after tail vein injection of PC-3-GFP vector or PC-3-GFP WFDC2 overexpressed. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test. The scale bars are 10 mm and 4 mm, respectively.

Fig. 3. Overexpression of WFDC2 and addition of recombinant protein HE4 restrain the progression of EMT by inactivating the EGFR/AKT/GSK3B/Snail signaling pathway.

a, b The 12 positively correlated pathways and 14 negatively correlated pathways after overlapping GSE70770 and TCGA. c Immunoblot assay of EGFR/AKT/GSK3B/Snail signaling protein in DU-145 and PC-3 after WFDC2 upregulation. d, e The relative mRNA expression of EGFR, AKT, GSK3β, Snail, CDH1, and CDH2 in DU-145 and PC-3 after WFDC2 upregulation. f, g Wound healing assay in DU-145 and PC-3 after adding recombinant protein HE4. The scale bar is 150 μm. h, i Transwell assay in DU-145 and PC-3 after adding recombinant protein HE4. The scale bar is 150 μm. j Immunoblot assay of p-EGFR, t-EGFR, p-GSK3β, t-GSK3β, Snail, and E-cadherin in DU-145 and PC-3 after adding recombinant protein HE4. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test.

Fig. 4. WFDC2 binds to the EGFR extracellular domain.

a Co-IP assay of WFDC2 and EGFR in 293-T cell line. b Co-localization assay of WFDC2 and EGFR in DU-145 and PC-3 cell lines. The scale bar is 15 μm. c Diagram of EGFR-FL, EGFR-NT, and EGFR-CT. d Co-IP assay between WFDC2 and EGFR-FL, EGFR-NT, and EGFR-CT in 293-T cell line. e Graphic model of WFDC2 suppressed prostate cancer metastasis by inactivating EGFR signaling.

Fig. 5. EGFR and EGF can rescue the metastatic ability repressed by WFDC2.

a–c Transwell assay in DU-145 and PC-3 after overexpressing EGFR and WFDC2. The scale bar is 150 μm. d Immunoblot assay of EGFR-related and EMT-related proteins in DU-145 and PC-3 after overexpressing EGFR and WFDC2. e Immunoblot assay of EGFR-related and EMT-related proteins in DU-145 and PC-3 after adding EGF and HE4. f–h Transwell assay in DU-145 and PC-3 after adding EGF and HE4. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test. The scale bar is 150 μm.

Fig. 6. Prognostic value validation via different datasets.

a Forest plot summary of the log-rank analysis of WFDC2 in different datasets. The blue diamond squares on the transverse lines represent the HR, and the black transverse lines represent the 95% CI. The p value and 95% CI for each dataset are displayed in detail. b Recurrence-free survival (RFS) of WFDC2 in GSE70770. c RFS of WFDC2 in MSKCC cohort. d RFS of WFDC2 in GSE116918. e Metastasis-free survival of WFDC2 in GSE116918. f Disease-free survival of WFDC2 in TCGA-PRAD.