Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer

Abstract

Background and aims: Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.

Methods: We generated VCMsh2/Tgfbr2 mice in which Msh2^loxP and Tgfbr2^loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis.

Results: VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.

Conclusions: Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

Keywords: Colon Cancer; DNA Mismatch Repair; Gut Microbiota; Inflammation.

Graphical abstract

Figure 1

Inactivation of Msh2 and Tgfbr2 in the intestinal epithelium induces colon tumorigenesis. (A) Kaplan-Meier analysis. VCMsh2/Tgfbr2 (n = 89) vs VCMsh2 (n = 28) P < .0001. (B) Comparison of tumor occurrence. VCMsh2/Tgfbr2 CRCs (n = 42; 7 months) vs SI tumors (n = 18; 9.5 months), ∗∗∗∗P < .0001; VCMsh2/Tgfbr2 SI tumors (n = 18; 9.5 months) vs VCMsh2 SI tumors (n = 28, 12 months) ∗∗P = .0019. (C) I, Macroscopic image of VCMsh2/Tgfbr2 CRC. Representative images: II, hematoxylin and eosin staining of VCMsh2/Tgfbr2 mucinous CRC (scale bar = 200 μm); III, dysplastic VCMsh2/Tgfbr2 colon (scale bar = 50 μm); IV, hypertrophy or hyperplasia in goblet cells of VCMsh2/Tgfbr2 colon mucosa (scale bar = 20 μm); V, colitis in VCMsh2/Tgfbr2 (scale bar = 20 μm); VI, VCTgfbr2 mucinous CRC (scale bars = 50 μm, 200 μm). (D) Kaplan-Meier analysis. FCMsh2/Tgfbr2 (n = 38) vs FCMsh2 (n = 31), ∗∗∗P < .0001. (E) FCMsh2/Tgfbr2 mucinous CRC (scale bars = 50 μm, 200 μm).

Figure 2

Histologic and molecular features of VCMsh2/Tgfbr2 intestinal tumors. (A) Representative images of Alcian blue staining identifying the blue mucinous lakes: I, VCMsh2/Tgfbr2 CRC (scale bar = 100 μm); II, VCMsh2/Tgfbr2 SI tumor (scale bar = 20 μm); III, VCMsh2 SI tumor (scale bar = 20 μm). (B) Inflammation score comparison. VCMsh2/Tgfbr2 SI (n = 35) vs VCMsh2/Tgfbr2 colon (n = 37), ∗P = .04; VCMsh2/Tgfbr2 colon vs VCMsh2 colon (n = 9), ∗P = .04. (C) 8-oxoG ELISA. VCMsh2/Tgfbr2 CRCs (n = 6) vs VCMsh2/Tgfbr2 SI tumors (n = 5), ∗P = .03; VCMsh2/Tgfbr2 CRCs (n = 6) vs VCMsh2 SI tumors (n = 6), ∗P = .015. (D) 8-oxoG staining. Increased 8-oxoG nuclear accumulation in VCMsh2/Tgfbr2 CRCs compared with VCMsh2/Tgfbr2 SI tumors (scale bar = 50 μm). (E) Membrane bound beta-catenin in VCMsh2/Tgfbr2 CRC and beta-catenin nuclear accumulation in VCMsh2/Tgfbr2 SI tumor (scale bar = 50 μm).

Figure 3

Mutational analysis of VCMsh2/Tgfbr2 intestinal tumors. (A) Number of mutations detected and predicted effect classification in VCMsh2/Tgfbr2 CRCs (n = 5) and SI tumors (n = 5). (B) Distribution of base substitution mutations; frequencies of transversions (Tv) and transitions (Ti). (C, D) Top 25 genes with a mutation frequency of 40% or higher. (E) Venn diagram showing common and unique high-impact-effect mutations between VCMsh2/Tgfbr2 SI tumors and CRCs. (F) IPA analysis of the 27 common high-impact mutations from E. (G) Mutation frequencies of selected genes; human datasets are originated from cBioPortal datasets.

Figure 4

Impact analysis of VCMsh2/Tgfbr2 mutations in intestinal tumors. (A) IPA-generated scheme of mutated genes in VCMsh2/Tgfbr2 CRCs and their involvement in IBD-associated diseases. (B) Venn diagram between predicted high-impact mutations from VCMsh2/Tgfbr2 CRCs and human IBD-MSI or MSS CRCs. (C) List of genes previously found mutated in human IBD-CRCs and colitis that are frequently mutated in both VCMsh2/Tgfbr2 CRCs and human MSI IBD-CRCs but only rarely in MSS IBD-CRCs and VCMsh2/Tgfbr2 SI tumors. Each column represents 1 sample: VCMsh2/Tgfbr2 CRCs/SI tumors, n = 5; human MSI IBD-CRCs, n = 7; human MSS IBD-CRCs, n = 24. The box colors indicate the predicted mutational effect as in the Figure 3C and D color legend. (D) Mutations distribution among human and mouse CRCs datasets among the 3 major top canonical pathways highlighted by IPA analysis.

Figure 5

The tumor microenvironment in VCMsh2/Tgfbr2 CRCs is characterized by an inflammation-specific gene expression signature. (A) Multidimensional scaling plot showing clear separation between VCMsh2/Tgfbr2 CRCs (green) and matched tumor-free mucosa (red). (B) GSEA of VCMsh2/Tgfbr2 CRCs vs matched tumor-free colon mucosa, comparison with multiple signatures. (C) Ratio of CD45+/Epcam+ cells in VCMsh2/Tgfbr2 CRCs (n = 11) vs colon mucosa (n = 11), ∗∗∗P < .0001. (D) Representative images of staining for different immune cells: macrophages (F4/80 antibody), T cells (CD3 antibody), and B cells (B220 antibody) in VCMsh2/Tgfbr2 colon tumor-free mucosa and CRC (scale bars = 200 μm, 50 μm, 20 μm). FDR, false discovery rate; NES, normalized enrichment score.

Figure 6

Characterization of inflammatory factors in the VCMsh2/Tgfbr2 CRCs tumor microenvironment. (A) qPCR. TNF-α: wild-type (WT) vs VCMsh2/Tgfbr2 CRCs colon (n = 7), ∗P = .01. IL-6: WT (n = 3) vs VCMsh2/Tgfbr2 CRCs (n = 5), ∗P = .036; VCMsh2/Tgfbr2 CRCs vs VCMsh2/Tgfbr2 colon (n = 5), ∗∗P = .0079. IL-17A WT (n = 5) vs VCMsh2/Tgfbr2 CRCs (n = 4), ∗P = .015; VCMsh2/Tgfbr2 CRCs vs VCMsh2/Tgfbr2 colon (n = 4), ∗P = .028. (B) Representative images of TNF-α staining: I, VCMsh2/Tgfbr2 colon tumor-free mucosa; II, VCMsh2/Tgfbr2 CRC (scale bar = 20 μm); III, VCMsh2/Tgfbr2 SI tumor-free mucosa; IV, SI tumor (scale bar = 50 μm). (C) Representative images of staining for IL-6 and IL-17 comparing VCMsh2/Tgfbr2 colon tumor-free mucosa and tumor (scale bars = 200 μm, 50 μm). (D) GSEA of VCMsh2/Tgfbr2 CRCs vs matched colon mucosa with a signature indicative of TGFB1-treated fibroblast. (E) qPCR. Tgfb1, CRCs fold change relative to tumor-free colon (n = 5), ∗∗P = .0079. (F) iNOS staining comparing VCMsh2/Tgfbr2 colon mucosa and CRC in VCMsh2/Tgfbr2 CRCs (scale bar = 200 μm).

Figure 7

Epithelial tumor cells expression profile analysis of VCMsh2/Tgfbr2 CRCs. (A) GSEA of sorted epithelial cells from VCMsh2/Tgfbr2 CRCs and matched tumor-free colon mucosa. (B) qPCR. Nlrp6: WT (n = 3) vs VCMsh2/Tgfbr2 CRCs (n = 5), ∗P = .035; VCMsh2/Tgfbr2 colon (n = 5) vs VCMsh2/Tgfbr2 CRCs (n = 5), ∗∗P = .0079.

Figure 8

Microbiota composition modulates the susceptibility of VCMsh2/Tgfbr2 mice to IBD-CRC. (A) Kaplan-Meier analysis. VCMsh2/Tgfbr2 mice (n = 89) vs VCMsh2/Tgfbr2-SPF mice aged in the SPF barrier (n = 50), P < .0001. (B) Inflammation score. VCMsh2/Tgfbr2 mice with CRC (n = 24), VCMsh2/Tgfbr2-SPF mice with CRC (n = 15), VCMsh2/Tgfbr2-SPF mice with SI tumors (n = 7); no significant differences. (C) qPCR. TNF-α: VCMsh2/Tgfbr2 (n = 7) vs VCMsh2/Tgfbr2-SPF colon (n = 6), ∗∗P = .008. (D) Relatedness (β-diversity) of fecal microbiota composition between the 2 different cohorts of young VCMsh2/Tgfbr2 mice (n = 3 conventional mice and n = 6 SPF mice). Principal coordinate (PC) analysis plot on Bray-Curtis distance matrix shows a clear separation between the 2 barriers (analyses of similarities test, R=1, P = .014).

Figure 9

Microbiota changes associated with VCMsh2/Tgfbr2 IBD-CRC. (A) Phyla relative abundance of young mice from the conventional and SPF barriers at 3 months of age. (B) Ratio of Bacteroides/Firmicutes phyla between young conventional and SPF mice: P = .048 (Mann-Whitney U test). (C, D) Box plots of relative abundance, young age comparison between barriers: Prevotella, P = 7.02E-16; Helicobacter, P = 2.22E-12; Desulfovibrio, P = .006; Peptococcus, P = 7.29E-08; Clostridium XVIII, P = 0.006. (E) Bar plots of the genera significantly increased in VCMsh2/Tgfbr2 mice with CRCs: Akkermansia, P = .0036; Acetatifactor, P = .0017; Coprobacillus, P = .0036. (F) Taxonomic distribution at the genus level from conventional VCMsh2/Tgfbr2 mice with either SI tumors or CRCs (n = 3); Akkermansia, P = .05. (G) Relative abundance of A. muciniphila in conventional VCMsh2/Tgfbr2 mice with CRCs (n = 4) vs VCMsh2/Tgfbr2 mice with SI tumors (n = 3). Each column represents a mouse with replicates, the normalized A. muciniphila 16S rRNA level is expressed as relative ratio to the sentinel mice, ∗∗∗∗P < 001.

Figure 10

Tgfbr2 status and microbiota composition modulate DSS-induced colonic inflammation and dMMR colorectal tumorigenesis. (A) Kaplan-Meier analysis. VCMsh2/Tgfbr2 + DSS (n = 20) vs VCMsh2 +DSS (n = 16), P < .0001; VCMsh2/Tgfbr2 +DSS vs VCMsh2/Tgfbr2-SPF +DSS (n = 15), P < .0001. (B) Inflammation score. VCMsh2/Tgfbr2 +DSS (n = 15) vs VCMsh2/Tgfbr2-SPF +DSS (n = 12), ∗∗P = .005. (C) Long-term body weight recovery during or after DSS treatment recorded every 7 days. VCMsh2 mice (n = 6), VCMsh2/Tgfbr2 mice (n = 13). Weight is shown as relative to the initial body weight, ∗∗P = .007.

Figure 11

The effect of Tgfbr2 loss of function in DSS-induced dMMR CRCs. (A) CRCs in VCMsh2/Tgfbr2 mice treated with DSS (objectives magnification): I, macroscopic mucinous CRCs. II, Alcian blue staining showing blue mucin lakes (scale bar = 200 μm). III, Beta-catenin membrane-bound staining (scale bar = 50 μm). (B) CRCs in VCMsh2 mice treated with DSS. I, CRC with a polyp-like appearance. II, Alcian blue staining showing prominent villous or tubulovillous histology and absence of mucin lakes (scale bar = 200 μm). III, Beta-catenin nuclear accumulation (scale bar = 50 μm).

Figure 12

Tgfbr2 loss and the microbial ecosystem cause distinct dysbiotic shifts during DSS-induced CRC development. (A) Principal coordinate (PC) analysis plots of β-diversity analysis based on Bray-Curtis metrics comparing VCMsh2/Tgfbr2 and VCMsh2 mice before and after DSS treatment. Significances are shown in Table 18. (B) α-Diversity measured before and after DSS treatment: VCMsh2/Tgfbr2 mice: Chao1 index, P = .002; Shannon index, P = .014; VCMsh2 mice P > .05 (ns). (C, D) β-diversity by PC analysis of Bray-Curtis dissimilarity distances showing a significant separation before and after DSS treatment in VCMsh2/Tgfbr2 and in VCMsh2. Analyses of similarities test statistics in Table 18.

Figure 13

Dysbiotic shifts during DSS-induced VCMsh2/Tgfbr2 tumorigenesis. (A, B) Box plots showing the relative abundance of genera significantly different after DSS treatment in VCMsh2/Tgfbr2 mice and VCMsh2 mice. (C) No significant differences in α-diversity in VCMsh2/Tgfbr2-SPF mice before and after DSS treatment. (D) Principal coordinate (PC) analysis plots of β-diversity analysis based on Bray-Curtis metrics in VCMsh2/Tgfbr2-SPF before and after DSS treatment, analyses of similarities R = 0.26, P = .033. (E) Bar plots showing the relative abundance of genera significantly different after DSS treatment in VCMsh2/Tgfbr2-SPF mice. (F, G) Box plots of relative abundance comparing DSS-treated VCMsh2/Tgfbr2 mice with either DSS-treated VCMsh2 or VCMsh2/Tgfbr2-SPF mice, indicating common genera shifts signature (Supplementary Excel File S12).