Microbioma intestinale e carcinogenesi Maria Rosalia Pasca
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1 Microbioma intestinale e carcinogenesi Maria Rosalia Pasca Workshop Genetica e neoplasie del colon-retto: il ruolo delle analisi molecolari all interno del programma di screening 10 Novembre 2016, Grand Hotel Mediterraneo, Firenze
2 the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease Joshua Lederberg The Scientist 15[7]:8, Apr. 2, 2001 These microbes Microbial are not harmful communities to us, of Microbiota: they are essential for Bacteria; maintaining health; they produce some Archea; vitamins that we do not have the genes to make; break down our food Eukarya to extract Mycobiome; nutrients we need to survive; teach our immune systems Virus how Virome. to recognize dangerous invaders.
3 100 trillions Number of microbes in our gut The intestine is a preferred site over 70% of all bacteria are found in the colon. 90% of cells in and on our body are bacterial cells. 10:1 Ratio of nonhuman cells to human cells in our body Number of human genes in the human gene catalog > Number of genes in our microbiome
4 Composition of gut microbiota Phyla Genera There is wide spatial and temporal variation within the same intestine and between individuals, ages, cultures, diet (e.g.: vegetarian, vegan, etc.) and sexes. Although 4 phyla (Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria) dominate the human microbiota, the makeup of a person s microbiota can be considered as individual fingerprint. Bacteria Firmicutes (30-50%) Bacteroidetes (20-40%) Actinobacteria (3-15%) Proteobacteria (1-10%) Verrucomicrobia (0.1%) Ruminococcus Clostridium Peptostreptococcus Lactobacillus Enterococcus Bacteroidetes Bifidobacterium Desulfovibrio Escherichia Helicobacter Archea Euryarchaeota Methanobrevibacter smithii
5 Longitudinal Bacteria increase in number and composition changes from proximal to distal GI tract. Bacteroidetes + Actinobacteria (50%) Firmicutes (40%) Bacteroidetes + Actinobacteria (90%) Latitudinal Bacterial composition also differs between lumen, mucus, and attached to epithelium. Interact with host s immune system. Bakhtiar et al FEMS Microbiol Lett.; Gagnière et al., World J Gastroenterol Interact with food and digested metabolites.
6 From microbiome characterization to health impact Modification in the bacterial composition of microbiome could be alter our state of health. Healthy associated typical profile EUBIOSIS Bacterial groups are protective against pathogens; The intestinal microbial ecosystem is in balance. Disease associated typical profile DYSBIOSIS Bacterial groups are associated to several pathological conditions; There are qualitative and quantitative changes in the intestinal flora.
7 Balance Dysbiosis is the possible cause of intestinal, metabolic and autoimmune diseases. Treatment of dysbiosis Reduction of the risk of the disease Modified from Valdés et al Food Funct.
8 Colorectal Cancer (CRC) and microbiome dysbiosis Toxins Virulence factors Metabolites ROS and NOS production Induction of DNA damage, genome instability, inflammation; interferences of apoptosis and cellular cycle; alterations of cellular components. Gagnière et al., 2016 World J Gastroenterol
9 Putative bacterial species involved in colorectal carcinogenesis Increase of Escherichia coli (phylogroups: B2 and D) in CRC (in particular III and IV stage) prognostic factor? B2 E. coli toxin (colibactin) interferes with cell eukaryotic cycle and induces DNA damage and genomic instability (in mouse model). D E. coli could be potentially pathogenic via downregulation of DNA mismatch repair system. Increase of Enterotoxigenic Bacteroides fragilis in CRC; It was demonstrated in vitro and in murine model that BFT toxin increased cell proliferation and DNA damage. Increase of Enterococcus faecalis in CRC; It could produce pro-oxidative reactive oxygen species (ROS). Increase of Fusobacterium nucleatum in adenomas and CRC (> 80%) CRC promotion. This effect may be mediated by FadA adhesion and activates the Wnt-βcatenin pathway (in mouse model). Sears and Garrett Cell Host Micr; Gagnière et al World J Gastroenterol.
10 Driver and Passenger Bacteria Driver bacteria iperproliferation and neoplastic transformation Passenger bacteria last neoplastic transformation Specific microbial associations can be potentially used as biomarkers during neoplastic progression Sears and Garrett Cell Host Micr.; Tjalsma et al Nature Rev. Microbiol.
11 Studying microbiome: 16S rrna gene sequencing First step is to obtain total bacterial DNA. DNA from all bacteria present in a sample (faeces, biopsies) need to be recovered. All the organisms possess at least one copy of such gene. Essential and conserved gene. The 16S sequence is known for every known bacterial species. Large public databases available for comparison. It is characterized by 9 high conserved 16S regions rrna interspersed gene with 9 hypervariable regions (V1-V9). Variable sequence can be thought of as a molecular fingerprint. The best phylogenetic marker for microbiota characterization Can be used to identify bacterial genera and species. Modified from Petrosino et al., 2009 Clin. Chem. V4 16S gene libraries and Illumina sequencing
12 NEXT GENERATION SEQUENCING Bioinformatic analysis OTUs (Operational Taxonomic unit) It is an arbitrary definition of a taxonomic unit based on sequence divergence. OTUs are number of clusters of similar sequences (reads). Generally, when 16S sequences are clustered at 97% identity ~ species. Modified from Stewart and Yigit, New England Biolabs, Inc.
13 Samples CancerType Healthy CRC CRC Healthy CRC #OTU ID SC255 SC131 SC276 SC104 SC250 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 Actinomyces 0, , ,82E-05 0,0001 4E-05 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 Actinotignum D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 Arcanobacterium D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 Mobiluncus 3,243E ,6E-05 0 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 Varibaculum ,5E-05 0 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Actinomycetales;D_4 Actinomycetaceae;D_5 uncultured 0 4,7E D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Bifidobacteriales;D_4 Bifidobacteriaceae;D_5 Aeriscardovia D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Bifidobacteriales;D_4 Bifidobacteriaceae;D_5 Alloscardovia 1,621E-05 4,7E D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Bifidobacteriales;D_4 Bifidobacteriaceae;D_5 Bifidobacterium 0, , , , ,0093 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Bifidobacteriales;D_4 Bifidobacteriaceae;D_5 Gardnerella 0 9,5E E-05 5E-06 D_0 Bacteria;D_1 Actinobacteria;D_2 Actinobacteria;D_3 Bifidobacteriales;D_4 Bifidobacteriaceae;D_5 Pseudoscardovia 0 4,7E D0 = kingdom D1 = phylum D2 = class D3 = order D4 = family D5 = genus OTU Table Bioinformatic and Statistical analysis Taxonomy charts Relative abundance of taxonomic groups Percentage distribution of taxa Samples
14 Gut microbiome and colorectal adenomacarcinoma sequence Ito et al Int J Cancer. F. nucleatum expression Feng et al Nature Comm. Flemer et al Gut.
15 Study of gut microbiome dysbiosis in the progression of neoplasia in sporadic colorectal cancer (CRC) Stage of neoplastic progression Istituto Scientifico Romagnolo per la Cura e lo Studio dei Tumori (IRST), Meldola Department of Biology and N Biotechnology «L. Spallanzani», University of Pavia, patients Pavia Centre for Integrative Biology, CIBIO, University of Trento, Trento. Healthy (negative colonscopy) 19 Hyperplastic polyps (HPs) 14 Low risk adenomas (LRAs) 20 High risk adenomas (HRAs) 21 Carcinomas (ADKs) 13 ADKs treated with chemotherapy/radiotherapy 9 96 stool samples Bacterial DNA extraction V4 16S rrna libraries Illumina sequencing Bioinformatic analysis
16 In progress We are waiting for sequencing results and bioinformatic analysis of all samples. Microbiome composition will be characterized for entire colorectal cancerogenesis progression. We will put in correlation patients microbiome with their clinical data. FINAL GOALS To identify microbial population that are specific to early time points prior to disease development. Dysbiotic patients can be treated to restore eubiosis (personalized medicine). Future perspectives Metabolomics and mycobiome study. Correlation with diet and nutrition. Microbiome dysbiosis in colorectal cancer hereditary syndromes.
17 Acknowledgements Department of Biology and Biotechnology, University of Pavia Prof. MR Pasca Dr. G Mori Prof. A Albertini Dr. G Barbieri Prof. N Ranzani Prof. D. Sassera Dr. F. Comandatore Centro per la Biologia Integrata Dr. N Segata Dr. F Armanini Istituto Scientifico Romagnolo per la Cura e lo Studio dei Tumori Dr. D Calistri Dr. C Rengucci Dr. G DeMaio
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Submit a Manuscript: http://www.wjgnet.com/esps/ Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx DOI: 10.3748/wjg.v22.i2.501 World J Gastroenterol 2016 January 14; 22(2): 501-518 ISSN 1007-9327 (print)
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