Transforming Growth Factor Beta Promotes Inflammation and Tumorigenesis in Smad4‐Deficient Intestinal Epithelium in a YAP‐Dependent Manner

Abstract Transforming growth factor beta (TGF‐β), a multifunctional cytokine, plays critical roles in immune responses. However, the precise role of TGF‐β in colitis and colitis‐associated cancer remains poorly defined. Here, it is demonstrated that TGF‐β promotes the colonic inflammation and related tumorigenesis in the absence of Smad family member 4 (Smad4). Smad4 loss in intestinal epithelium aggravates colitis and colitis‐associated neoplasia induced by dextran sulfate sodium (DSS) and azoxymethane/dextran sulfate sodium (AOM/DSS), leading to over‐activated immune responses and increased TGF‐β1 levels. In Smad4‐deficient organoids, TGF‐β1 stimulates spheroid formation and impairs intestinal stem cell proliferation and lineage specification. YAP, whose expression is directly upregulated by TGF‐β1 after Smad4 deletion, mediates the effect of TGF‐β1 by interacting with Smad2/3. Attenuation of YAP/TAZ prevents TGF‐β1‐induced spheroid formation in Smad4 − / – organoids and alleviates colitis and colitis‐associated cancer in Smad4‐deficient mice. Collectively, these results highlight an integral role of the TGF‐β/Smad4 axis in restraining intestinal inflammation and tumorigenesis and suggest TGF‐β or YAP signaling as therapeutic targets for these gastrointestinal diseases intervention.

(a) Anti-p65 immunostaining of colon sections from the indicated mice treated with DSS for five days. Scale bar: 50 μm (b) Immunoblotting of colonic epithelium lysates derived from indicated mice after 3.5% DSS treatment for 5 days. (c) Immunoblotting of proliferation-and apoptosis-related proteins in the colonic epithelium from the indicated mice after DSS treatment. n=2 mice for each genotype. (a) GSEA analysis of the genes related to immune cell proliferation and epithelial cell apoptosis in littermate control Smad4 fl/fl and Villin CreER ;Smad4 fl/fl mice upon DSS-induced colitis. (b) Immunofluorescence staining of IL-7R and IL-17R in the distal colon derived from DSS-treated mice and statistical analysis of the cell percentage shown in the right (n=3). Scale bar: 50 μm. (c) Immunofluorescence and quantification analysis of F4/80 in the colon sections derived from Smad4 fl/fl and Villin CreER ;Smad4 fl/fl mice following DSS treatment for 5 days. n=3. Scale bar: 50 μm. (d) RT-qPCR analysis of the markers of M1-and M2-macrophages in colonic tissue derived from Smad4 fl/fl and Villin CreER ;Smad4 fl/fl mice following DSS treatment for 5 days. n=3 mice for each genotype.
(e) RT-qPCR analysis of the indicated inflammasome genes in the colonic epithelium from DSS-treated mice. n=3 mice for each genotype. (f) Immunoblotting of colonic epithelium lysates from the indicated mice with or without DSS treatment. (n=4 mice for each group). (g) Immunoblotting of colonic tumor lysates from the indicated mice challenged with AOM/DSS. (n=2 mice for each group). Data are presented as means ± SD. Statistical significance is determined by unpaired, two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.  (c) Immunofluorescence co-staining of colon sections with the antibodies against TGF-β1 or tight junction protein ZO-1, and statistics is shown on the right. (d) Heatmap of TGF-β target genes in the indicated mice after DSS treatment. n=2 mice per group.
(e) RT-qPCR analysis of TGF-β1 expression in the indicated mice after DSS treatment. n=3 biological replicates. (f) Box plots for SMAD4 and TGFB1 expression from the datasets of intestinal biopsies from UC and non-inflammatory individuals (GSE38713). Data are presented as means ± SD. Statistical significance is determined by unpaired, two-tailed Student's t-test. *p < 0.05, **p < 0.01,

Figure S5. TGF-β induces spheroids and evokes an epithelial-intrinsic immune response in Smad4 -/organoids.
(a) Morphology of Smad4-deficient organoids with or without 0.5 nM TGF-β1 treatment. Quantification of spheroids and budding organoids is evaluated (lower panel). Scale bar: 100 μm. (b) Morphology of WT and Smad4-deficient organoids after stimulating with different concentrations of BMP4. Quantification of the ratio of spheroids is shown on the right. Scale bar: 100 μm. (c) RT-qPCR analysis showing gene expression of proinflammatory cytokines and chemokines in the indicated intestinal organoids with TGF-β1 treatment for 8 hours. Data are presented as means ± SD. Statistical significance is determined by unpaired, two-tailed Student's t-test. **p < 0.01, ***p < 0.001, n.s, no significance.  (e) Immunostaining of YAP (red) indicating its subcellular distribution in indicated organoids with quantification shown on the right. Scale bars: 50 μm. (f) RT-qPCR analysis of the expression of YAP target genes in indicated organoids with or without TGF-β1 and VP treatment. (g) Bright field images of WT and Smad4-deficient organoids with/without YAP(S94A) overexpression after TGF-β1 treatment. Quantification is shown on the right. n=3 biological replicates for each timepoint. Scale bar: 50 μm. Data are presented as means ± S.D. Statistical significance is determined by unpaired, two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s, no significance.

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Data files S1. Gene expression of colonic epithelial cells upon colitis by RNA-Seq analysis. Data files S2. Gene expression by RNA-seq in small intestinal organoids.