Fig 2: Activation of the WNT signaling pathway strengthens the TGFß-induced migratory phenotype. (A) ChIP-qPCR showing time-dependent recruitment of SMAD2/3 binding to the WNT7B gene locus in MCF10A MII before (–) or after TGFß (5 ng/ml) treatment (1.5 and 16 h). Results of four independent experiments are shown by dot plot chart; *P < 0.05. (B) qRT-PCR analysis showing time-dependent WNT7B mRNA expression in MCF10A MII before (–) or after TGFß (5 ng/ml) treatment (1.5 or 16 h). Results of six independent experiments are shown by dot plot chart; **P < 0.01. (C) Left: qRT-PCR analysis of WNT7B mRNA expression in MCF10A MII cells transfected with the indicated control (siNTC) or JUNB and SMAD4 specific siRNAs, and stimulated for 16 h with TGFß (5 ng/ml). Results of four independent experiments are shown by dot plot chart; **P < 0.01 versus siNTC TGFß 16 h. Right: Western blot control of knock-down efficiency. (D) ChIP-qPCR showing time-dependent recruitment of JUNB to the WNT7B gene locus in MCF10A MII before (–) or after TGFß (5 ng/ml) treatment (1.5 and 16 h). (E) Collagen invasion assay of MCF10A MII spheroids stably expressing control GFP or ectopic WNT7B-MYC. Spheroids were embedded in collagen in the absence or presence of TGFß, the TGFßRI inhibitor (ALK5i) SB505124 (2.5 µM) or the WNT inhibitor (WNTi) IWP-2 (5 µM), as indicated. Left: representative pictures of spheroids taken 36 h after being embedded in collagen. Right: relative invasion was quantified as the mean area that the spheroids occupied 36 h after being embedded in collagen. Data represent means ± SD (n = 6 spheroids per condition) and are representative of three independent experiments; ***P < 0.001. (F) Western blot analysis of the MCF10A MII cells stably expressing control GFP or ectopic WNT7B-MYC. Cells were treated for 12 h with TGFß (5 ng/ml) in the absence or presence of DMSO control, the TGFßRI inhibitor (ALK5i) SB505124 (2.5 µM) or the WNT inhibitor (WNTi) IWP-2 (5 µM), as indicated. (G) qRT-PCR target gene analysis of the cells shown in E and F, treated for 16 h with TGFß (5 ng/ml) as indicated. A representative results of three independent experiments is shown.

Fig 3: WNT7B promotes breast cancer cell invasion. (A) MCF10A MII mCherry stably expressing control GFP (MII GFP) or ectopic WNT7B-MYC (MII WNT7B) were injected into the DoC of 48-hpf zebrafish embryos. Left: representative images of zebrafish at 6 days post-injection (dpi). Right: quantification of invasive cell cluster numbers in GFP or WNT7B-MYC expressing MCF10A MII cells injected zebrafish larvae. (B) Kaplan-Meyer analysis of overall survival of breast cancer datasets from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (40); all subtypes, n = 1904; ER+ subjects, n = 1445; ER- subjects, n = 429; luminal A subtype, n = 679; luminal B subtype, n = 461; HER2- subtype, n = 220; basal-like subtype, n = 199). Survival analysis was performed using a log-rank test. (C) Z-scored expression values of mRNA were obtained with cBioPortal (42,43). (***P < 0.001; n.s. not significant, Welch's t-test).

Fig 4: WNT7b activation was reversely associated with prognosis of the CRC cohort. Post-surgical survival rates analysis exhibited significantly differentiated prognosis between subgroups. Log-rank test, P = 0.0396

Fig 5: After silencing Fzd7/Wnt7b expression using lentiviral shRNA targeting Fzd7/Wnt7b, spheroid formation per 1000 cells decreased from 22.0 ± 2.6/20.0 ± 3.6 (con) to 15.0 ± 1.0/14.3 ± 3.1 in the Capan-2 cells, and from 22.0 ± 2.6/18.7 ± 1.5 (con) to 16.0 ± 4.4/13.7 ± 2.1 in the Panc-1 cells (shFzd7/Wnt7b), the difference was statistically significant. The diameter of the cell spheres decreased from 190.74 ± 79.74/165 ± 71.7 µm (con) to 135 ± 62.06/121 ± 51.58 µm in Capan-2 cells, and from 177 ± 68.4/191 ± 68.61 µm (con) to 123 ± 52.47/130 ± 49.82 µm in the Panc-1 cells (shFzd7/Wnt7b), the difference was statistically significant (A). In Capan-2 and Panc-1, S cells expressed higher levels of Fzd7, Wnt7b, and ABCG2, the difference was statistically significant (B), and the proportion of CD24+CD44+ cells in the Capan-2 and Panc-1 cells increased from 15.9 ± 3.4% and 12.6 ± 3.2% in P cells to 52.9 ± 5.3% and 51.0 ± 5.7% in S cells, respectively, the difference was statistically significant (C). *p < 0.05, **p < 0.01, ***p < 0.001. shRNA, short hairpin