SMAD4 Suppresses Colitis-associated Carcinoma Through Inhibition of CCL20/CCR6-mediated Inflammation

Abstract

Background & aims: We previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss.

Methods: In silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining.

Results: In human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, T_h17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone.

Conclusions: Regulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.

Keywords: Colorectal Cancer; Inflammation; Inflammatory Bowel Disease; SMAD4.

Figure 1.. Lack of colon epithelial SMAD4 expression is associated with increased epithelial CCL20 expression and CCR6+ cells.

(A) Representative IHC images of human UCACs (n=34), UC-dysplasia (n=18), UC (n=34), and healthy colon control (n=5) samples for the indicated markers (brown; scale bars: 100μm). (B-D) Quantification according to SMAD4 status indicated on X axis. (B) Quantification of stromal CCR6+ cells/mm², (C) epithelial CCL20 staining score, and (D) CCL20 epithelial percent positivity (data points represent mean per sample if multiple cores exist; Mann-Whitney U test; *P < .05, ** P < .001).

Figure 2.. SMAD4 loss is associated with increased immune cell recruitment in human ulcerative colitis-associated cancer specimens.

(A) MxIF of human UCACs (SMAD4+ n=4, SMAD4- n=3) stained for indicated markers (CD3 = yellow, CD11b = blue, CD68 = red, DAPI = gray; scale bars: 100μm). (B) Quantification of immune cell types relative to total tissue area. (C) Quantification of stromal and (D) epithelial-associated immune cells relative to their respective tissue area (data points represent mean per tumor sample if multiple cores exist; Mann-Whitney U test; *P < .05, ** P < .001).

Figure 3.. Smad4 loss results in increased GALT area only when Ccr6 is expressed.

(A) Quantification of GALT number and cross-sectional area from 5 Smad4^ΔLrig1 and 5 SMAD4+ control mice under homeostatic conditions, (B) and in 4 SMAD4+ Ccr6^GFP/+, 6 SMAD4+ Ccr6^GFP/GFP, 8 Smad4^∆Lrig1 Ccr6^GFP/+, and 11 Smad4^∆Lrig1 Ccr6^GFP/GFP mice, 3 months after completion of DSS-induced colitis. (C) Immunofluorescent staining and quantification for GFP (red) in GALTs from indicated genotypes (Data points represent mean quantification per mouse; DAPI: blue, scale bars: 100μm; Mann-Whitney U test; *P < .05, ** P < .001).

Figure 4.. Loss of epithelial Smad4 results in increased sub-epithelial immune cells in mouse colon in a CCR6-dependent manner.

Colonic stroma from 5 SMAD4+ Ccr6^GFP/+, 6 SMAD4+ Ccr6^GFP/GFP, 5 Smad4^∆Lrig1 Ccr6^GFP/+, and 4 Smad4^∆Lrig1 Ccr6^GFP/GFP mice was analyzed by flow cytometry 1 month after completion of DSS-induced colitis. (A) Representative flow cytometry scatter plots showing surface staining of colon stromal CD3+ or CD11c+ in CD45+ cells in a Smad4^ΔLrig1 Ccr6 ^GFP/+ mouse and a Smad4^ΔLrig1 Ccr6 ^GFP/GFP mouse. Numbers in plot indicate percentage of cells in each gate. (B) Proportion of live stromal cells that were CD45+. (C) Proportion of CD45+ cells that were CD3+. (D) Proportion of CD3+ cells stained for CD8, CD4, (E) CD4 FoxP3, or CD4 IL17a. (F) Proportion of CD45+ cells that were CD11c+. (G) Proportion of CD11c+ cell stained for CD11b or CX3CR1 (Welch’s t test; *P < .05, ** P < .001).

Figure 5.. Loss of epithelial Smad4 results in increased colon epithelial-associated immune cells in a CCR6-dependent manner.

Cells isolated from colonic epithelium of 5 SMAD4+ Ccr6^GFP/+, 6 SMAD4+ Ccr6^GFP/GFP, 5 Smad4^∆Lrig1 Ccr6^GFP/+, and 4 Smad4^∆Lrig1 Ccr6^GFP/GFP mice were analyzed by flow cytometry 1 month after completion of DSS-induced colitis. (A) Representative flow cytometry scatter plots showing surface staining for epithelial-associated CD3+ or CD11c+ in CD45+ cells of a Smad4^ΔLrig1 Ccr6 ^GFP/+ mouse and a Smad4^ΔLrig1 Ccr6 ^GFP/GFP mouse. Numbers in plot indicate percentage of cells in each gate. (B) Proportion of live stromal cells that were CD45+. (C) Proportion of CD45+ cells that were CD3+. (D) Proportion of CD3+ cells stained for CD8, CD4, (E) CD4 FoxP3, or CD4 IL17a. (F) Proportion of CD45+ cells that were CD11c+. (G) Proportion of CD11c+ cell stained for CD11b or CX3CR1 (Welch’s t test; *P < .05, ** P < .001).

Figure 6:. Loss of Ccr6 prevents tumorigenesis after DSS-induced colitis.

2 SMAD4+ Ccr6^GFP/+, 6 SMAD4+ Ccr6^GFP/GFP, 8 Smad4^∆Lrig1 Ccr6^GFP/+, and 11 Smad4^∆Lrig1 Ccr6^GFP/GFP mice were analyzed for invasive tumors 3 months after the last cycle of DSS. (A) Number of mice that developed CAC s in each group (Fisher’s exact test). (B) Number of invasive tumors noted per mouse in each genotype (Mann-Whitney U test). (C) Representative images of invasive adenocarcinoma of the distal colon of a Smad4^∆Lrig1 Ccr6^GFP/+ mouse as well as the single invasive crypt seen in a Smad4^∆Lrig1 Ccr6^GFP/EGFP and a Smad4+ Ccr6^GFP/GFP mouse (MM: muscularis mucosa; SIC: single invasive crypt; CAC: colitis associated cancer; Scale bars: 100μm; *P < .05, ** P < .001).

Figure 7.. Loss of epithelial Smad4 resulted in significantly increased FoxP3+ cells in GALTs only in the presence of CCR6.

Staining for FoxP3 (brown) in representative samples for (A) GALTs or (B) crypt-associated regions comparing 4 SMAD4+ Ccr6^GFP/+, 6 SMAD4+ Ccr6^GFP/GFP, 8 Smad4^∆Lrig1 Ccr6^GFP/+, and 11 Smad4^∆Lrig1 Ccr6^GFP/GFP mice. (scale bars: 100μm; graphs represent quantification of staining; data points represent mean quantification per mouse Mann-Whitney U test; *P < .05).