Frizzled-8 integrates Wnt-11 and transforming growth factor-β signaling in prostate cancer

Wnt-11 promotes cancer cell migration and invasion independently of β-catenin but the receptors involved remain unknown. Here, we provide evidence that FZD8 is a major Wnt-11 receptor in prostate cancer that integrates Wnt-11 and TGF-β signals to promote EMT. FZD8 mRNA is upregulated in multiple prostate cancer datasets and in metastatic cancer cell lines in vitro and in vivo. Analysis of patient samples reveals increased levels of FZD8 in cancer, correlating with Wnt-11. FZD8 co-localizes and co-immunoprecipitates with Wnt-11 and potentiates Wnt-11 activation of ATF2-dependent transcription. FZD8 silencing reduces prostate cancer cell migration, invasion, three-dimensional (3D) organotypic cell growth, expression of EMT-related genes, and TGF-β/Smad-dependent signaling. Mechanistically, FZD8 forms a TGF-β-regulated complex with TGF-β receptors that is mediated by the extracellular domains of FZD8 and TGFBR1. Targeting FZD8 may therefore inhibit aberrant activation of both Wnt and TGF-β signals in prostate cancer.

List of primers used in this study, with their sequences and concentration. List of antibodies used in this study, with their companies, catalog numbers, species and concentrations for each application; WB western blotting, IP immunoprecipitation, IF immunofluorescence, IHC immunohistochemistry. Table 3 Parameter Explanation Unit

Area
Area of the segmented structure pixels

Roundness
Roundness of the segmented structure %

Roughness
Roughness of the surface of the segmented structure %

AppIndex
Indexes the severity of the invasiveness by utilising Roundness and Roughness parameters ratio Definitions of parameters evaluated with AMIDA analysis of 3D culture assays. Clinical and pathological characteristics of the prostate cancer patients used in the TMAs. Comparison of FZD8 and Wnt-11 protein expression in benign and malignant prostate tissue. FZD8 and Wnt-11 protein expression levels were compared in tumor and benign epithelium and cancer stroma and benign stroma. Both were significantly higher in cancer and significantly lower in cancer stroma. Pearson Chi-square test with correction and Fisher´s exact test, two-sided are shown Statistical analysis of FZD 8 and Wnt-11 staining intensities in TMAs. Adjacent sections of tumor and benign prostate from 99 patients were analyzed; 9 tumor sections were subsequently found to contain no cancer and were removed from the analysis, as were samples stained for Wnt-11 and FZD 8 that were missing/damaged. Staining was classified as low (score 0 or 1) or high (score 2 or 3). Entries are absolute numbers with % in parentheses. Statistical significance was determined using Chi-squared test, Pearson correction and Fisher´s exact test, two-sided; p < 0.05 was considered to be statistically significant.       Fig.  5c and d, respectively); error bars show SD for three or four independent experiments, respectively (*P<0.05 by Student´s t test). Representative images are on the right, scale bar 100 µm. (c) Cell number of PC-3M and DU145 cells transfected with control and FZD8 siRNAs; error bars show SD of at least three independent experiments (*P<0.05 by Student´s t test). These data were used to normalize numbers of migrating cells in Fig. 2a and Supplementary Fig. 5a. (d) Cell number of PC-3M and DU145 cells transfected with control and FZD8 siRNAs; error bars show SD of at least three independent experiments (*P<0.05 by Student´s t test). These data were used to normalize numbers of invading cells in Fig. 2b and Supplementary Fig. 5b. (e) Invasion assays using PC-3M cells stably silenced for FZD8 using lentiviruses expressing two different FZD8 shRNAs (shFZD8.1 and shFZD8.2), values are relative to pLKO and normalized to numbers of viable cells plated in parallel ( Supplementary Fig. 5g Figure 9. FZD8 regulates TGF-β signaling (a) Relative CAGA12luciferase/renilla activity in DU145 cells transfected with control or FZD8 siRNAs and treated -/+ 0.1 ng ml -1 TGF-β for 24 h; error bars show SD from three independent experiments (*P<0.05 by ANOVA with Tukey post-hoc test). (b) Relative CAGA12luciferase/renilla activity in PC-3M cells transfected with control or dsiFZD8 and treated -/+ 1 ng ml -1 TGF-β for 24 h; error bars show SD from three independent experiments (*P<0.05 by ANOVA with Tukey post-hoc test). (c) Q-PCR analysis of the indicated genes in DU145 cells transfected with control or FZD8 siRNAs; error bars show SD for four independent experiment (*P<0.05, **P<0,001 by Student´s t-test). (d) Western blots of extracts from DU145 cells transfected with control or FZD8 siRNAs and treated with 1 ng ml -1 TGF-β for 30 min were probed for pSMAD2, pSMAD3, SMAD2/3 and b-tubulin as a loading control; graph on the right shows relative levels of expression, as determined by densitometry analysis of bands from TGF-β-treated extracts, normalized to b-tubulin and relative to control siRNA, from three independent experiments (*P<0.05 by Student´s t-test). (e) Invasion assays of control and FZD8-silenced DU145 cells treated with 5 ng ml -1 of TGF-β for 48h; error bars represent SD of three independent experiments Left: relative cell invasion normalized to siCtrl in the presence of TGF-β, taking into account effects of silencing on cell number ( Supplementary Fig. 9g). Middle: representative pictures of invaded cells, scale bar 100 µm. Right: representative experiment. (f) and (g) Cell number of PC-3M and DU145 cells, respectively transfected with control and FZD8 siRNAs and treated with 5 ng ml -1 of TGF-β for 48h; error bars show SD of three independent experiments (*P<0.05 by ANOVA with Tukey post-hoc test). These data were used to normalize numbers of invading cells in Fig. 6d and Supplementary Fig. 9e, respectively.  Figure 11. Contribution of ATF-2 to TGFb signaling (a) Relative CAGA12-luciferase/renilla activity in PC-3M cells transfected with empty vector (V) or dominant-negative ATF2 (∆-ATF2) and treated with 1 ng ml -1 TGF-β for 24 h; error bars show SD for three independent experiments (*P<0.05, **P<0,001 by ANOVA with Tukey post-hoc test). (b) Relative 3TPlux luciferase/renilla activity in PC-3M cells transfected with empty vector (V) or dominant-negative ATF2 (∆-ATF2) and treated -/+ 1 ng ml -1 TGF-β for 24 h; error bars show SD from three independent experiments (*P<0.05, **P<0,001 by ANOVA with Tukey post-hoc test). (c) Q-PCR analysis showing mRNA expression of the indicated genes, relative to 36B4, in DU145 cells transfected with empty vector (V) or ∆-ATF2 and treated with 1 ng ml -1 TGF-β for 24 h; error bars show SD for four independent experiments (*P<0.05 by Student´s t-test). (d) Western blots of anti-HA immunoprecipitates (IP) and extracts (inputs) from PC-3M cells transfected for 24h with HA-tagged ATF2 and c-JUN, and SMAD3 plasmids were probed for ATF2 and c-JUN (HA) and SMAD3 (Flag); blots are representative of two independent experiments.