Zeb2 drives invasive and microbiota-dependent colon carcinoma

Abstract

Colorectal cancer (CRC) is highly prevalent in Western society, and increasing evidence indicates strong contributions of environmental factors and the intestinal microbiota to CRC initiation, progression and even metastasis. We have identified a synergistic inflammatory tumor-promoting mechanism through which the resident intestinal microbiota boosts invasive CRC development in an epithelial-to-mesenchymal transition-prone tissue environment. Intestinal epithelial cell (IEC)-specific transgenic expression of the epithelial-to-mesenchymal transition regulator Zeb2 in mice (Zeb2IEC-Tg/+) leads to increased intestinal permeability, myeloid cell-driven inflammation and spontaneous invasive CRC development. Zeb2IEC-Tg/+ mice develop a dysplastic colonic epithelium, which progresses to severely inflamed neoplastic lesions while the small intestinal epithelium remains normal. Zeb2IEC-Tg/+ mice are characterized by intestinal dysbiosis, and microbiota depletion with broad-spectrum antibiotics or germ-free rederivation completely prevents cancer development. Zeb2IEC-Tg/+ mice represent the first mouse model of spontaneous microbiota-dependent invasive CRC and will help us to better understand host–microbiome interactions driving CRC development in humans.

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Fig. 1: Mono-allelic expression of Zeb2 in the mouse intestinal epithelium induces CRC.
Fig. 2: Zeb2IEC-Tg/+ mice develop severe pathology in the colon but not in the small intestine.
Fig. 3: Zeb2 transgenic expression in IECs induces intestinal barrier disruption, bacterial infiltration and colon inflammation.
Fig. 4: Myeloid cell infiltrates contribute to CRC in Zeb2 IEC transgenic mice.
Fig. 5: Microbiota dysbiosis in Zeb2 IEC transgenic mice.
Fig. 6: Microbiota dependency for CRC development in Zeb2 IEC transgenic mice.
Fig. 7: Axenic Zeb2IEC-Tg/+ mice are protected from CRC development.
Fig. 8: ZEB2 expression in human CRC.

Data availability

Statistical source data for Figs. 17 and Extended Data Figs. 1, 3 and 510 are provided with the paper. RNA sequencing data on ZEB2-overexpressing DLD-1 cells have been deposited in the Gene Expression Omnibus under accession code GSE148823. Sequencing data from the shallow whole-genome sequencing on Zeb2IEC-Tg/+ and control colon tumor tissue have been deposited in the Sequence Read Archive database under accession code PRJNA626401. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Change history

  • 10 July 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank M. Gomez, S. Ganal, J. Kirundi, K. McCoy and A. MacPherson for generating axenic Zeb2IEC-Tg/+ mice at the University of Bern, Switzerland. We thank L. Bellen and K. Barbry for animal care and the VIB Flow Core for training, support and access to the instrument park. K.S. was a predoctoral fellow with the Institute for the Promotion of Innovation by Science and Technology (IWT) and was supported by a ‘Kom op tegen Kanker’ (Stand Up To Cancer) grant from the Flemish Cancer Society. Work in the G.v.L. laboratory is supported by research grants from the FWO, Strategic Basic Research (SBO) program, Geneeskundige Stichting Koningin Elisabeth (GSKE), CBC Banque Prize, Charcot Foundation, Stichting Tegen Kanker, Cancer Research Institute Ghent and Concerted Research Actions (GOA) of Ghent University. The laboratory of G.B. is supported by grants from the FWO, SBO, GOA, Vlaamse Liga Tegen Kanker and Stichting Tegen Kanker. The laboratory of L.V. is supported by grants from the FWO, SBO and Cancer Research Institute Ghent.

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Contributions

K.S., I.P., G. Blancke, E.H., E.D., M.S., H.V., S.J., J.T., N.V. and D.N. performed the experiments. K.S., I.P., E.H., E.D., E.R., J.J.H., A.W., D.N., P.V.V., G.D.H., P.B., E.E., P.W., B.N.L., C.C., S.T., S.G., G. Berx, L.V. and G.v.L. analyzed the data. E.T. and L.B. provided reagents. G. Berx, L.V. and G.v.L provided ideas and coordinated the project. K.S., G. Berx, L.V. and G.v.L. wrote the manuscript.

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Correspondence to Geert Berx or Lars Vereecke or Geert van Loo.

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Extended data

Extended Data Fig. 1 Expression of Zeb2 in the mouse intestinal epithelium.

a, Schematic representation of the conditional ROSA26-based Zeb2 overexpression mouse model. The mouse Zeb2 open reading frame was targeted to the ROSA26 locus preceded by a floxed (fl) transcriptional stop (PGK-Neo-3XpA) cassette and followed by IRES-EGFP reporter sequence. b, R26-Zeb2Tg/Tg mice were crossed with a Cre transgenic line under the control of Villin promoter, generating R26-Zeb2Tg/+ villin-creTg/+ transgenic (Tg/+) mice and R26-Zeb2Tg/+ villin-cre+/+ (WT) controls. c-d, Kaplan-Meier survival curve (c) and clinical stool score (d) of male (n=12 mice) and female (n=10 mice) Zeb2IEC-Tg/+ mice based on Hemoccult assay. Data are presented as mean ± SEM. Source data

Extended Data Fig. 2 Histopathology of Zeb2IEC-Tg/+ mice.

a, Representative H&E stained colon section from wild-type control mouse showing normal proximal colon (representative for n=10 mice). Left panel, magnification of boxed area. b, Representative H&E stained sections of Zeb2IEC-Tg/+ mouse (representative for n=7 mice) demonstrating multicentric invasive adenocarcinoma in a background of atypical mucosal hyperplasia with multifocal intraepithelial neoplasia and intramucosal carcinoma. Also sparse inflammatory cell infiltrates, predominantly lymphocytes and plasma cells, can be observed in the lamina propria and submucosa. Left panel, higher magnification showing severe atypical hyperplasia/intramucosal carcinoma characterized by tumultuous proliferation of irregular glandular units (upper), and of the invading adenocarcinomatous component characterized by irregular glandular profiles with abundant mucus collection, transmural infiltration of the intestinal wall and spread along the mesenteric ligament (lower). c, PAS staining of section of distal colon and rectum of Zeb2IEC-Tg/+ mouse (representative for n=7 mice). Abundant collection of PAS-positive mucus is evident in the irregular neoplastic glandular units transmurally invading the intestinal wall and infiltrating along the mesenteric ligament. d, Macroscopic view on proximal colon tissue dissected in 44-week old WT and Zeb2IEC-Tg/+ mice. e, Macroscopic dissected proximal colon in 31-week old Zeb2IEC-Tg/+ mouse displaying transmural cancerous outgrowth.

Extended Data Fig. 3 Zeb2IEC-Tg/+ mice develop severe pathology in colon but not in small intestine.

a, Quantification of Ki67-, CD44-, P-Stat3- and P-Smad3-positive cells in different regions of the colon (mucosa, submucosa, muscle or serosa) of 25-30-week old wild-type (WT) and Zeb2IEC-Tg/+ mice (Tg) (Ki67 Tg/+, n=9 mice and WT, n=6 mice; CD44 Tg/+, n=5 mice and WT, n=5 mice; P-Stat3 Tg/+, n=7 mice and WT, n=5 mice; P-Smad3 Tg/+, n=6 mice and WT, n=3 mice). For each tissue region and mouse, 3 separate areas were analyzed and averages per mouse per region were plotted in GraphPad Prism. Data are presented as mean ± SEM. ** p<0.01, *** p<0.001, **** p<0.0001. Two way ANOVA, corrected for multiple comparisons using Sidak test in GraphPad Prism. b, Representative FACS plots of Ki67+EPCAM+ cells in colon (left panel) and small intestine (right panel) in 5 week old wild-type (WT) and Zeb2IEC-Tg/+ mice. The experiment was performed twice with similar results. c, Quantitative PCR analysis for Zeb2 and eGFP in lysates from the small intestine of Zeb2IEC-Tg/+ (Tg/+, n=6 mice) and wild-type littermate controls (WT, n=5 mice). Data are presented as mean ± SEM and were analyzed with two-tailed Mann Whitney test, p-value = 0.0043 for both. d, Quantification of Ki67-positive cells in different regions of the small intestine of 25-30-week old wild-type (WT, n=4 mice) and Zeb2IEC-Tg/+ mice (Tg, n=5 mice). For each tissue region and mouse, 3 separate areas were analyzed and averages per mouse per region were plotted in GraphPad Prism. Data are presented as mean ± SEM and were analyzed with two-way ANOVA with Sidak correction. e, Quantitative PCR analysis for inflammatory markers (TNF, IL-1β, Cox2, Cxcl5 and A20), CD44, and Paneth cell markers (Crypt-1 and LysP) in lysates from the small intestine of Zeb2IEC-Tg/+ (Tg/+, n=6 mice) and wild-type littermate controls (WT, n=5 mice). Data are presented as mean ± SEM and were analyzed with two-tailed Mann Whitney test, p-value = 0.0087 for IL-1β and 0.0043 for Cox2; ns, non-significant. Source data

Extended Data Fig. 4 Tumorigenic potential and copy number profiling of Zeb2 transgenic tissue.

a, Orthotopic transplantation of intestinal tumor cells from Zeb2 transgenic mice into the cecum of NSG mice. Zeb2 transgenic cells could be identified by immunohistochemical staining for the GFP transgene. Scale bar: 200µm; boxed area Scale bar: 100µm. Data representative for one experiment on 5 NSG mice. b, Copy number analysis by shallow whole genome sequencing of Zeb2IEC-Tg/+ colon tumor tissue (n=4 mice, lanes 3-6), normal lung tissue (n=2 mice, lanes 1-2) and normal colon tissue from a wild-type littermate control mouse (lane 7). Genomic aberrations are positioned along the murine chromosomes and visualized in red for deletions and blue for amplifications.

Extended Data Fig. 5 Induction of ZEB2 expression leads to a loss of cell adhesion complexes such as tight and gap junctions.

RNA-Seq analysis on Doxycycline (Dox)-inducible ZEB2-overexpressing DLD-1 colorectal cancer cell line clones. Data represents the mean centered rlog-transformed counts (n=3 biological repeats). Source data

Extended Data Fig. 6 Zeb2 expression and intestinal barrier integrity in the small intestine.

Quantitative PCR analysis for expression of tight junction proteins (Claudin-4, -6 and -7, Occludin, Tjp-1, F11R/JAM-1) and desmosomes (Desmocollin-1 and -3, Desmoglein-2, Plakophillin-1) in lysates from the small intestine of Zeb2IEC-Tg/+ (Tg/+, n=6 mice) and wild-type littermate controls (WT, n=5 mice). Data are presented as mean ± SEM and were analyzed with two-tailed Mann Whitney test. *, p-value = 0.0173; ** p-value = 0.0043; ns, non-significant. Source data

Extended Data Fig. 7 Microbiota dysbiosis in Zeb2IEC-Tg/+ mice.

Abundance of significantly different taxa between wild-type (WT, n=21 mice) and Zeb2IEC-Tg/+ (Tg/+, n=34 mice) fecal samples based on MaAslin and t-test FDR correction analysis. The horizontal straight line indicates the group means and the dotted line indicates the group medians. Source data

Extended Data Fig. 8 Microbiota dysbiosis in Zeb2IEC-Tg/+ mice.

a, b, Altered bacterial diversity/community structure in mucosal samples from 25 week-old wild-type (WT, in green, n=28 mice) and Zeb2IEC-Tg/+ (Tg/+, in red, n=17 mice) mice. α-diversity measured by the Shannon Diversity Index at the genus level (p= 0.1439, Welch two Sample t-test) (a). β-diversity represented by a PCoA of Bray-Curtis dissimilarity distances of bacterial composition (b). c, d, Differentially abundant taxa between wild-type (WT) and Zeb2IEC-Tg/+ (Tg/+). Linear Discriminant Analysis Effect Size (LEfSe) cladogram of differentially abundant taxa in mucosal samples from WT (in green, n=21 mice) and Tg/+ (in red, n=34 mice) mice (c). Linear Discriminant Analyis (LDA) scores (d) of differentially abundant taxa combined with multiple testing corrections (analysis associated with Extended Data Fig. 8c): t-test with FDR correction (*: padj <0.05) (no significant differences were identified based on MaAslin analysis). [phylum (p), class (c), order (o), family (f), genera (g), species (s)]. e, Abundance of significantly different taxa between wild-type (WT) and Zeb2IEC-Tg/+ (Tg/+) mucosal samples based on t-test FDR correction analysis. The horizontal straight line indicates the group means and the dotted line indicates the group medians. f, g, Mycobiota profiling in Zeb2IEC-Tg/+ mice. Fungal diversity/community (WT n=33 mice, in green, and Tg/+ n=22 mice, in red). α-diversity measured by the Shannon Diversity Index at the genus level (p= 0.0717, Welch two Sample t-test) (f). β-diversity represented by a PCoA of Bray-Curtis dissimilarity distances of fungal composition (g). Each box represents the third (upper) and first (lower) quartiles. Lines inside the boxes represent the median. Source data

Extended Data Fig. 9 ABX treated Zeb2IEC-Tg/+ mice are protected from CRC development.

a, Clinical stool score of untreated (n=6 mice), vancomycin-treated (n=6 mice) and aztreonam-treated (n=4 mice) Zeb2IEC-Tg/+ mice based on Hemoccult assay. Data are presented as mean ± SEM. b, Colon pathology detected by endoscopy and represented as MEICS (murine endoscopic index of colitis severity) score. Each symbol represents one mouse. Data are presented as mean ± SEM. c, Colon histology of Aztreonam (left panel) and Vancomycin (right panel) treated Zeb2IEC-Tg/+ mice by H&E staining. Scale bar: 200µm. Representative images from one experiment with n=4 mice. Source data

Extended Data Fig. 10 Axenic Zeb2IEC-Tg/+ mice are protected from CRC development.

a, Quantification of Ki67-, CD44-, P-Stat3- and P-Smad3-positive cells in different regions of the colon (mucosa, submucosa, muscle or serosa) of 20 week-old germfree wild-type (WT) and Zeb2IEC-Tg/+ mice (Tg). For each tissue region and mouse, 3 separate areas were analyzed and averages per mouse per region were plotted in GraphPad Prism. Data are presented as mean ± SEM and were analyzed with two-way ANOVA with Sidak correction (Ki67 WT, n=4 mice and Tg/+, n=5 mice; CD44 WT, n=4 mice and Tg/+, n=4 mice; P-Smad3 WT, n=3 mice and Tg/+, n=6 mice). b, Flow cytometry analysis of colonic lamina propria immune cells in 12 week-old germfree WT (n=6 mice) and Zeb2IEC-Tg/+ (n=6 mice). Data are presented as mean ± SEM, two-sided student’s t test. Each symbol represents one mouse. Source data

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Slowicka, K., Petta, I., Blancke, G. et al. Zeb2 drives invasive and microbiota-dependent colon carcinoma. Nat Cancer 1, 620–634 (2020). https://doi.org/10.1038/s43018-020-0070-2

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