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Investigation of the gut microbiome, bile acid composition and host immunoinflammatory response in a model of azoxymethane-induced colon cancer at discrete timepoints

Abstract

Background

Distinct sets of microbes contribute to colorectal cancer (CRC) initiation and progression. Some occur due to the evolving intestinal environment but may not contribute to disease. In contrast, others may play an important role at particular times during the tumorigenic process. Here, we describe changes in the microbiota and host over the course of azoxymethane (AOM)-induced tumorigenesis.

Methods

Mice were administered AOM or PBS and were euthanised 8, 12, 24 and 48 weeks later. Samples were analysed using 16S rRNA gene sequencing, UPLC-MS and qRT-PCR.

Results

The microbiota and bile acid profile showed distinct changes at each timepoint. The inflammatory response became apparent at weeks 12 and 24. Moreover, significant correlations between individual taxa, cytokines and bile acids were detected. One co-abundance group (CAG) differed significantly between PBS- and AOM-treated mice at week 24. Correlation analysis also revealed significant associations between CAGs, bile acids and the bile acid transporter, ASBT. Aberrant crypt foci and adenomas were first detectable at weeks 24 and 48, respectively.

Conclusion

The observed changes precede host hyperplastic transformation and may represent early therapeutic targets for the prevention or management of CRC at specific timepoints in the tumorigenic process.

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Fig. 1: Alpha (α) and beta (β) diversity are altered across time in mice treated with either PBS or AOM.
Fig. 2: Histograms of the community composition of gut microbiota at the phylum level and co-abundance groups (CAGs).
Fig. 3: Taxa which differed significantly in their abundance between groups.
Fig. 4: Alterations in faecal bile acid profiles and transporter gene expression between AOM and PBS-treated mice.
Fig. 5: Alterations in immunoregulatory gene expression between AOM and PBS-treated mice.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol. 2019;16:713–32.

    Article  PubMed  Google Scholar 

  2. Yamagishi H, Kuroda H, Imai Y, Hiraishi H. Molecular pathogenesis of sporadic colorectal cancers. Chin J Cancer. 2016;35:4.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Flemer B, Lynch DB, Brown JM, Jeffery IB, Ryan FJ, Claesson MJ, et al. Tumour-associated and non-tumour-associated microbiota in colorectal cancer. Gut. 2017;66:633–43.

    Article  CAS  PubMed  Google Scholar 

  4. Scott AJ, Alexander JL, Merrifield CA, Cunningham D, Jobin C, Brown R, et al. International Cancer Microbiome Consortium consensus statement on the role of the human microbiome in carcinogenesis. Gut. 2019;68:1624–32.

    Article  CAS  PubMed  Google Scholar 

  5. Dulal S, Keku TO. Gut microbiome and colorectal adenomas. Cancer J. 2014;20:225–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. Diversity of the human intestinal microbial flora. Science. 2005;308:1635–8.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ahn J, Sinha R, Pei Z, Dominianni C, Wu J, Shi J, et al. Human gut microbiome and risk for colorectal cancer. J Natl Cancer Inst. 2013;105:1907–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mangifesta M, Mancabelli L, Milani C, Gaiani F, de’Angelis N, de’Angelis GL, et al. Mucosal microbiota of intestinal polyps reveals putative biomarkers of colorectal cancer. Sci Rep. 2018;8:13974.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ohigashi S, Sudo K, Kobayashi D, Takahashi O, Takahashi T, Asahara T, et al. Changes of the intestinal microbiota, short chain fatty acids, and fecal pH in patients with colorectal cancer. Dig Dis Sci. 2013;58:1717–26.

    Article  CAS  PubMed  Google Scholar 

  10. Alrawi SJ, Schiff M, Carroll RE, Dayton M, Gibbs JF, Kulavlat M, et al. Aberrant crypt foci. Anticancer Res. 2006;26:107–19.

    CAS  PubMed  Google Scholar 

  11. Hong BY, Ideta T, Lemos BS, Igarashi Y, Tan Y, DiSiena M, et al. Characterization of mucosal dysbiosis of early colonic neoplasia. NPJ Precis Oncol. 2019;3:29.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Keane JM, Joyce SA, Gahan CGM, Hyland NP, Houston A. Microbial metabolites as molecular mediators of host-microbe symbiosis in colorectal cancer. Results Probl Cell Differ. 2020;69:581–603.

    Article  CAS  PubMed  Google Scholar 

  13. Ocvirk S, O’Keefe SJ. Influence of bile acids on colorectal cancer risk: potential mechanisms mediated by diet-gut microbiota interactions. Curr Nutr Rep. 2017;6:315–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Earnest DL, Holubec H, Wali RK, Jolley CS, Bissonette M, Bhattacharyya AK, et al. Chemoprevention of azoxymethane-induced colonic carcinogenesis by supplemental dietary ursodeoxycholic acid. Cancer Res. 1994;54:5071–4.

    CAS  PubMed  Google Scholar 

  15. Adam JK, Odhav B, Bhoola KD. Immune responses in cancer. Pharm Ther. 2003;99:113–32.

    Article  CAS  Google Scholar 

  16. Zuo T, Ng SC. The gut microbiota in the pathogenesis and therapeutics of inflammatory bowel disease. Front Microbiol. 2018;9:2247.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Arthur JC, Perez-Chanona E, Mühlbauer M, Tomkovich S, Uronis JM, Fan T-J, et al. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 2012;338:120–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lucas C, Barnich N, Nguyen HTT. Microbiota, inflammation and colorectal cancer. Int J Mol Sci. 2017;18:1310.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Mendes MCS, Paulino DSM, Brambilla SR, Camargo JA, Persinoti GF, Carvalheira JBC. Microbiota modification by probiotic supplementation reduces colitis associated colon cancer in mice. World J Gastroenterol. 2018;24:1995–2008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bent R, Moll L, Grabbe S, Bros M. Interleukin-1 beta-a friend or foe in malignancies? Int J Mol Sci. 2018;19:2155.

  21. Scheller J, Chalaris A, Schmidt-Arras D, Rose-John S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim Biophys Acta. 2011;1813:878–88.

    Article  CAS  PubMed  Google Scholar 

  22. Wang X, Lin Y. Tumor necrosis factor and cancer, buddies or foes? Acta Pharm Sin. 2008;29:1275–88.

    Article  Google Scholar 

  23. Claesson MJ, Jeffery IB, Conde S, Power SE, O’Connor EM, Cusack S, et al. Gut microbiota composition correlates with diet and health in the elderly. Nature. 2012;488:178–84.

    Article  CAS  PubMed  Google Scholar 

  24. Joyce SA, MacSharry J, Casey PG, Kinsella M, Murphy EF, Shanahan F, et al. Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut. Proc Natl Acad Sci USA. 2014;111:7421–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

  26. Rosenberg DW, Giardina C, Tanaka T. Mouse models for the study of colon carcinogenesis. Carcinogenesis. 2009;30:183–96.

    Article  CAS  PubMed  Google Scholar 

  27. Chen W, Liu F, Ling Z, Tong X, Xiang C. Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer. PLoS ONE. 2012;7:e39743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fang CY, Chen JS, Hsu BM, Hussain B, Rathod J, Lee KH. Colorectal cancer stage-specific fecal bacterial community fingerprinting of the Taiwanese population and underpinning of potential taxonomic biomarkers. Microorganisms. 2021;9:1548.

  29. Liu L, Yang M, Dong W, Liu T, Song X, Gu Y, et al. Gut dysbiosis and abnormal bile acid metabolism in colitis-associated cancer. Gastroenterol Res Pr. 2021;2021:6645970.

    Google Scholar 

  30. Le Gall G, Guttula K, Kellingray L, Tett AJ, Ten Hoopen R, Kemsley EK, et al. Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls. Oncotarget. 2018;9:33278–89.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ai D, Pan H, Li X, Gao Y, Liu G, Xia LC. Identifying gut microbiota associated with colorectal cancer using a zero-inflated lognormal model. Front Microbiol. 2019;10:826.

  32. Feng Q, Liang S, Jia H, Stadlmayr A, Tang L, Lan Z, et al. Gut microbiome development along the colorectal adenoma–carcinoma sequence. Nat Commun. 2015;6:6528.

    Article  CAS  PubMed  Google Scholar 

  33. Scanlan PD, Shanahan F, Clune Y, Collins JK, O’Sullivan GC, O’Riordan M, et al. Culture-independent analysis of the gut microbiota in colorectal cancer and polyposis. Environ Microbiol. 2008;10:789–98.

    Article  CAS  PubMed  Google Scholar 

  34. Zou J, Shen Y, Chen M, Zhang Z, Xiao S, Liu C, et al. Lizhong decoction ameliorates ulcerative colitis in mice via modulating gut microbiota and its metabolites. Appl Microbiol Biotechnol. 2020;104:5999–6012.

    Article  CAS  PubMed  Google Scholar 

  35. Jones-Hall YL, Kozik A, Nakatsu C. Ablation of tumor necrosis factor is associated with decreased inflammation and alterations of the microbiota in a mouse model of inflammatory bowel disease. PLoS ONE. 2015;10:e0119441.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Rausch P, Steck N, Suwandi A, Seidel JA, Künzel S, Bhullar K, et al. Expression of the blood-group-related gene B4galnt2 alters susceptibility to salmonella infection. PLoS Pathog. 2015;11:e1005008–e.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Jia W, Rajani C, Xu H, Zheng X. Gut microbiota alterations are distinct for primary colorectal cancer and hepatocellular carcinoma. Protein Cell. 2021;12:374–93.

    Article  PubMed  Google Scholar 

  38. Hu Y, Le Leu RK, Christophersen CT, Somashekar R, Conlon MA, Meng XQ, et al. Manipulation of the gut microbiota using resistant starch is associated with protection against colitis-associated colorectal cancer in rats. Carcinogenesis. 2016;37:366–75.

    Article  CAS  PubMed  Google Scholar 

  39. Kemis JH, Linke V, Barrett KL, Boehm FJ, Traeger LL, Keller MP, et al. Genetic determinants of gut microbiota composition and bile acid profiles in mice. PLoS Genet. 2019;15:e1008073.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zarrinpar A, Chaix A, Xu ZZ, Chang MW, Marotz CA, Saghatelian A, et al. Antibiotic-induced microbiome depletion alters metabolic homeostasis by affecting gut signaling and colonic metabolism. Nat Commun. 2018;9:2872.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Grosser G, Müller SF, Kirstgen M, Döring B, Geyer J. Substrate specificities and inhibition pattern of the solute carrier family 10 members NTCP, ASBT and SOAT. Front Mol Biosci. 2021;8:689757.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tian Y, Gui W, Koo I, Smith PB, Allman EL, Nichols RG, et al. The microbiome modulating activity of bile acids. Gut Microbes. 2020;11:979–96.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Ju T, Kong JY, Stothard P, Willing BP. Defining the role of Parasutterella, a previously uncharacterized member of the core gut microbiota. ISME J. 2019;13:1520–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Devlin AS, Fischbach MA. A biosynthetic pathway for a prominent class of microbiota-derived bile acids. Nat Chem Biol. 2015;11:685–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Liu HX, Rocha CS, Dandekar S, Wan YJ. Functional analysis of the relationship between intestinal microbiota and the expression of hepatic genes and pathways during the course of liver regeneration. J Hepatol. 2016;64:641–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. He T, Cheng X, Xing C. The gut microbial diversity of colon cancer patients and the clinical significance. Bioengineered. 2021;12:7046–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bailey AM, Zhan L, Maru D, Shureiqi I, Pickering CR, Kiriakova G, et al. FXR silencing in human colon cancer by DNA methylation and KRAS signaling. Am J Physiol Gastrointest Liver Physiol. 2014;306:G48–58.

    Article  CAS  PubMed  Google Scholar 

  48. Fu T, Coulter S, Yoshihara E, Oh TG, Fang S, Cayabyab F, et al. FXR regulates intestinal cancer stem cell proliferation. Cell 2019;176:1098–112.e18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hagi T, Geerlings SY, Nijsse B, Belzer C. The effect of bile acids on the growth and global gene expression profiles in Akkermansia muciniphila. Appl Microbiol Biotechnol. 2020;104:10641–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Theriot CM, Bowman AA, Young VB. Antibiotic-induced alterations of the gut microbiota alter secondary bile acid production and allow for Clostridium difficile spore germination and outgrowth in the large intestine. mSphere. 2016;1:e00045-15.

  51. Ovadia C, Perdones-Montero A, Spagou K, Smith A, Sarafian MH, Gomez-Romero M, et al. Enhanced microbial bile acid deconjugation and impaired ileal uptake in pregnancy repress intestinal regulation of bile acid synthesis. Hepatology 2019;70:276–93.

    CAS  PubMed  Google Scholar 

  52. Wei W, Wang H-F, Zhang Y, Zhang Y-L, Niu B-Y, Yao S-K. Altered metabolism of bile acids correlates with clinical parameters and the gut microbiota in patients with diarrhea-predominant irritable bowel syndrome. World J Gastroenterol. 2020;26:7153–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Palm NW, de Zoete MR, Cullen TW, Barry NA, Stefanowski J, Hao L, et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Cell. 2014;158:1000–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Miyauchi E, Kim SW, Suda W, Kawasumi M, Onawa S, Taguchi-Atarashi N, et al. Gut microorganisms act together to exacerbate inflammation in spinal cords. Nature. 2020;585:102–6.

    Article  CAS  PubMed  Google Scholar 

  55. Umar S. Citrobacter infection and Wnt signaling. Curr Colorectal Cancer Rep. 2012;8 https://doi.org/10.1007/s11888-012-0143-4.

  56. da Silva Duarte V, Dos Santos Cruz BC, Tarrah A, Sousa Dias R, de Paula Dias Moreira L, Lemos Junior WJF, et al. Chemoprevention of DMH-induced early colon carcinogenesis in male BALB/c mice by administration of Lactobacillus Paracasei DTA81. Microorganisms. 2020;8:1994.

  57. Zeng H, Ishaq SL, Liu Z, Bukowski MR. Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet. J Nutr Biochem. 2018;54:18–27.

    Article  CAS  PubMed  Google Scholar 

  58. Zhang W, An Y, Qin X, Wu X, Wang X, Hou H, et al. Gut microbiota-derived metabolites in colorectal cancer: the bad and the challenges. Front Oncol. 2021;11:739648.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Wang X, Huycke MM. Extracellular superoxide production by Enterococcus faecalis promotes chromosomal instability in mammalian cells. Gastroenterology 2007;132:551–61.

    Article  CAS  PubMed  Google Scholar 

  60. Liu Y, Zhang S, Zhou W, Hu D, Xu H, Ji G. Secondary bile acids and tumorigenesis in colorectal cancer. Front Oncol. 2022;12:813745.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Peters BA, Dominianni C, Shapiro JA, Church TR, Wu J, Miller G, et al. The gut microbiota in conventional and serrated precursors of colorectal cancer. Microbiome. 2016;4:69.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Rosshart SP, Vassallo BG, Angeletti D, Hutchinson DS, Morgan AP, Takeda K, et al. Wild mouse gut microbiota promotes host fitness and improves disease resistance. Cell. 2017;171:1015–28.e13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Leystra AA, Clapper ML. Gut microbiota influences experimental outcomes in mouse models of colorectal cancer. Genes (Basel). 2019;10:900.

  64. Wu Y, Jiao N, Zhu R, Zhang Y, Wu D, Wang AJ, et al. Identification of microbial markers across populations in early detection of colorectal cancer. Nat Commun. 2021;12:3063.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhang YK, Zhang Q, Wang YL, Zhang WY, Hu HQ, Wu HY, et al. A Comparison study of age and colorectal cancer-related gut bacteria. Front Cell Infect Microbiol. 2021;11:606490.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Lavelle A, Nancey S, Reimund JM, Laharie D, Marteau P, Treton X, et al. Fecal microbiota and bile acids in IBD patients undergoing screening for colorectal cancer. Gut Microbes. 2022;14:2078620.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Geller LT, Barzily-Rokni M, Danino T, Jonas OH, Shental N, Nejman D, et al. Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science. 2017;357:1156–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We acknowledge Pat Casey for his assistance with the animal studies. Graphical abstract was created with BioRender.com.

Funding

This work was supported by the APC Innovation Platform. APC Microbiome Ireland is a research institute funded by Science Foundation Ireland (SFI) through the Irish Governments National Development Plan (Grant SFI/12/RC/2273).

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JMK acquired data and played an important role in interpreting the results and drafted the manuscript. CJW, PC and KB acquired data. SM helped to design the work that led to the submission. PDC helped draft the manuscript, acquired data, and/or played an important role in interpreting the results. SAJ, CGMG, AH and NPH conceived and designed the work that led to the submission, played an important role in interpreting the results and drafted the manuscript. All authors approved the final version and agreed to be accountable for all aspects of the work.

Corresponding author

Correspondence to A. Houston.

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The authors are not aware of any competing interests that might be perceived as affecting the findings of this study.

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Animal experiments were conducted in accordance with the regulations and guidelines of the Irish Department of Health following approval by the University College Cork Animal Experimentation Ethics Committee (2011/023).

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Keane, J.M., Walsh, C.J., Cronin, P. et al. Investigation of the gut microbiome, bile acid composition and host immunoinflammatory response in a model of azoxymethane-induced colon cancer at discrete timepoints. Br J Cancer (2022). https://doi.org/10.1038/s41416-022-02062-4

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