FIGURE 6.

Wnt signaling (A–C) drives the alternative promoter usage of SRI (with preference to the promoter proximal to exon 2), (D–F) suppresses the inclusion of CD46 exon 8, and (G–I) inhibits the alternative polyadenylation of RECQL5 with transcription terminating at exon 8.3, in the two independent in vivo tumor models descibed in Figure 1A. (A,D,G) Each of the plots includes a schematic illustration of the exon structure of genes with Wnt regulated differential exon usage, together with the heatmaps indicating the magnitude of variable exon usage in both the HPAF-II orthotopic and ColoPDX tumor models. A positive value (in red) indicates that Wnt signaling promotes the inclusion of the exon, whereas a negative value (in blue) indicates that Wnt signaling suppresses the inclusion of the exon. (B,E,H) In vitro qPCR validation is also shown for the Wnt regulated (B) change in promoter preference of SRI in favor of the promoter proximal to exon 2, (E) skipping of CD46 exon 8, and (H) inhibition of the gene expression of the truncated RECQL5α/γ isoforms. ETC-159 was administered to Wnt-addicted HPAF-II cells with or without the stabilized β-catenin. The percentage change in expression upon ETC-159 treatment was calculated. The boxplot shows the average relative gene expression (center line) and the 95% confidence interval (length of the box). A two-tailed t-test was used to determine significance between comparisons. For statistical tests, (ns) P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001. (C,F,I) The differential inclusion of Wnt regulated exons (measured as ΔPSI) in multiple tumors from the TCGA SpliceSeq database were assessed for the Wnt regulated (C) alternative promoter usage of SRI (with preference for the promoter proximal to exon 2), (F) skipping of exon 8 of CD46, and (I) inhibition of the expression of RECQL5γ isoform with transcription terminating at exon 8.3. A positive ΔPSI value indicates that the variable exon is included more in the tumors relative to the matched normals. The tumor types are sorted by the Wnt score (see Materials and Methods) with colorectal cancer (COAD) having the highest Wnt score. The higher the Wnt score, the stronger the relative influence of Wnt/β-catenin signaling in the cancer. A two-tailed t-test was used with H₀: average ΔPSI value = 0, for each cancer type. For statistical tests, (ns) P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001.