Gut microbiome : debugging the obesity and cancer link Overview

1. Gut microbiome: debugging the obesity and cancer linkOverview Carrie R. Daniel-MacDougall, PhD, MPH Department of Epidemiology Division of Cancer Prevention and Population Sciences

2. Biological mechanisms linking adiposity and cancer risk Cancer Adapted from Hoet al. Cancer Res 2012

3. Diet, energy balance, and gut microbiome Diet The composition of bacteria living within the gut can be linked to functional metabolic pathways in the host Energy balance regulation Secretion of leptin Hepatic insulin sensitivity and lipid synthesis Modulate intestinal environment and appetite signaling Figure of healthy gut (fecal) microbiome: NIH-HMP Consortium, Nature 2012

4. Diet may directly and indirectly modulate association between energy balance (EB) and cancer Satiety Insulin signaling Obesity or weight-loss Prebiotic fibers (e.g., legumes) resist digestion until they reach the colon; undergo bacterial fermentation Microbial energy harvest from the diet; fat storage by host; energy loss in feces Microbial production of SCFA Regulation of EB SCFA (Propionate, acetate, butyrate) act as signaling molecules on G-protein coupled receptors expressed in colon and adipocytes Secretion of leptin; hepatic insulin sensitivity and lipid synthesis; modulate intestinal environment and appetite signaling Endocrine

5. Diet, gut microbiome, obesity, and cancer Relevant exposures (e.g., diet, medications) Gut microbiome Energy balance Inflammation Insulin Resistance Obesity Cancer

6. Nadim Ajami, PhD, Baylor College of Medicine • Alkek Center for Metagenomics and Microbiome Research (Director: Joe Petrosino, PhD), Department of Molecular Virology and Microbiology • Cross-talk between Akkermansiamuciniphilaand intestinal epithelium controls diet-induced obesity (Everard 2013) Manasi Shah, MS, MD Anderson & UT-SPH • Decreased dietary fiber intake and structural alteration of gut microbiota in patients with advanced colorectal adenoma (Chen 2013)

7. Nadim J. Ajami, Ph.D. Alkek Center for Metagenomics and Microbiome Research Department of Molecular Virology and Microbiology Baylor College of Medicine Houston, TX

8. Key concepts • Akkermansia muciniphila • Gram-negative anaerobe • Degrades mucin • Resides in the mucus layer of the intestinal epithelium • Abundant in nutrient-rich environments • Represents 3-5% of the microbial community in healthy subjects • Abundance inversely correlates with body weight, severity of appendicitis, and T1D • Obesity and Type 2 Diabetes • Altered gut microbiota • Inflammation • Gut barrier disruption • Increased gut permeability -> endotoxemia & metabolic inflammation

9. Objective To define the physiological role of Akkermansia muciniphila in obesity. Hypothesis A. Muciniphila controls gut barrier function Experimental design • Administration of live or heat-killed A. muciniphila to mice fed a high-fat diet. • Gut barrier • Glucose homeostasis • Adipose tissue metabolism

10. Findings Akkermansia muciniphila treatment reverses high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance.

11. Abundance of A. muciniphila is decreased in obese and diabetic mice Prebiotic: Oligofructose 0.3gr/mouse/day

12. Prebiotic treatment restores A. muciniphila to basal levels and reverses metabolic endotoxemia

13. High fat (HF) diet increases macrophage infiltration and fat mass

14. Treatment with A. muciniphila does not induce changes in the gut microbiota MITChip V1/V6 16S rRNA gene

15. A. Muciniphila counteracts metabolic endotoxemia, diet induced obesity, adipose tissue macrophage infiltration, improves glucose homeostasis, and adipose tissue metabolism

16. A. Muciniphila colonization restores gut barrier function and increases intestinal endocannabinoids in diet-induced obese mice Intestinal acylglycerols previously demonstrated to reduce metabolic endotoxemia and systemic inflammation  gut barrier function 46% thinner mucus layer in HF-fed mice A. Muciniphila treatment counteracts this decrease

17. Heat-killed A. muciniphiladoes not counteract metabolic endotoxemia, diet induced obesity, oral glucose intolerance, gut barrier dysfunction or poor adipose tissue metabolism Endotoxemia Fat mass Plasma Glucose Markers of adipocyte differentiation Viable A. muciniphila is required

18. Conclusions and future directions • A. muciniphila: • Restores the mucus layer of the intestine • Restores gut barrier function and thereby contributes to normalize metabolic endotoxemia and adipose tissue metabolism • Improves glucose tolerance and decreases endogenous hepatic glucose production Development of a treatment that uses A. muciniphila for the prevention or treatment of obesity and its associated metabolic disorders?

19. Thank you for your attention Nadim J. Ajami, Ph.D. Alkek Center for Metagenomics and Microbiome Research nadim.ajami@bcm.edu

20. Gut microbiome: debugging the obesity and cancer link Manasi Shah, MS Department of Epidemiology Division of Cancer Prevention and Population Sciences

21. The Gut Microbiome

22. Background • Diet may influence colon cancer risk via the microbiota and its metabolites • Dietary habits may influence early events in the colon carcinogenic process • Fiber Short Chain Fatty Acids (SCFA) in colon lower risk of CRC • Colon cancer risk influenced by the balance between microbial production of health-promoting metabolites, such as butyrate; and potentially carcinogenic metabolites, such as secondary bile acids

23. Gut Microbiome and Colon Cancer- The Balancing Act Fig: Jobin, Inflamm Bowel Dis 2011

24. Gut Microbiome and Colorectal Neoplasia Four high resolution maps of colonic dysbiosis have been independently reported: • CRC tissue as compared to adjacent non-malignant mucosa: Potential pathogenic bacteria in CRC tissue Coriobacteridae, Roseburia, Fusobacterium and Faecalibacterium(Marchesi, PLoS2011) • Fusobacterium sequences were enriched in carcinomas while the Bacteroidetes and Firmicutes phyla were depleted (Kostic, Genome Res 2012) • Overabundance of Fusobacterium sequences in tumor vs matched control tissue ( Castellarin, Genome Res 2012) • Significantly lower level of Lachnospiraceace, Ruminococcaceaeand Lactobacillaceaein cancerous tissues compared to normal intestinal lumen. Relative abundance of Bacteroidaceae, Streptococcacea, Fusobacteriaceaewas also higher in the cancerous tissue (Chen, PLoS One 2012)

25. Cross Sectional Study Flow:

26. Subjects and Methods • Consecutive patients who had undergone colonoscopy in 5 medical centers in China, ≥ 50 yrs of age • NO previous history of colorectal adenoma or carcinoma or IBD, normal bowel movement • Pathological confirmation of advanced colorectal adenoma (A-CRA) • Patients with no obvious abnormalities allotted in the healthy control (HC) group • Data on demographics, colonoscopy results, lifestyle factors and dietary intake were collected via interview-administered questionnaires • Fresh stool samples were collected from all the participants. • Fecal SCFA content analyzed by gas chromatography • Analysis of 16s rRNA sequences done by 454 pyrosequencing of fecal samples

27. Results

28. Multivariate logistic analysis identified four statistically significant factors associated with advanced colorectal adenoma (A-CRA)

29. Significantly lower yields of fecal SCFAs were found in the A-CRA group (n = 47) than in the HC group (n = 47).

30. Separation between the microbiota genus in the HC (n = 47) and A-CRA (n = 47) groups was significant PC analysis plots based on the unweightedUniFrac. p = 0.001, t test of permutation

31. Differences in butyrate and butyrate-producing bacteria:

32. Summary and Discussion Main Findings: • Structural imbalance in the fecal gut microbiota and difference in SCFA concentrations was defined in the A-CRA, as compared to HC • Butyrate and other SCFA’s associated with reduced risk of CRC: 3 genera of butyrate-producing bacteria (Clostridium, Roseburia, and Eubacterium) were lower in the A-CRA group than in the HC group • Significant higher levels of opportunistic pathogens: Enterococcus and Streptococcus in A-CRA Implications: • Microbial community may play an important role in pathogenesis of the progression from A-CRA to CRC via dietary SCFA products • Persistent deficiency in substrate dietary fiber may result in a deficiency in butyrate-producing bacteria  fermentation of SCFAs  increased risk of A-CRA

33. Limitations • Cross-sectional design: no cause-effect • Prospective human studies are required to determine whether an altered gut microbiome increases risk of colorectal cancer/adenoma or is an artifact of pre-existing disease • 454 pyrosequencing identifies bacteria at genus, but not species level. Higher depth genomic techniques may be required to fully differentiate the colonizing groups and their subsequent functions • Fecal SCFA concentrations correlate with both formation and uptake throughout the gut, but do not necessarily reflect SCFA production

34. ‘Food’ for Thought: Future Directions Obesity • Transmissible and modifiable interactions between diet and microbiota influence host biology and cancer development in mice: • Intact uncultured and culturable bacterial component of Obese co-twin’s fecal microbiota caused significantly greater increases in body mass and adiposity than those of lean communities (Ridaura, Science 2013) • Diet- or gene-induced obesity alters gut microbiota, thereby increasing the levels of metabolites that secrete inflammatory and tumor-promoting factors in the liver facilitating development of liver cancer (Yoshimoto, Nature 2013) • Potential for translation to humans… Gut Microbiome Colorectal Cancer Diet Inflammation

35. Thank You!

36. In Conclusion Gene Induced Obesity Mutation or polymorphism Increased Energy Harvest Microbe-dependent effects Diet-Induced Obesity High fat/high calorie diet Altered Metabolic Signaling Microbiome Induced Obesity Disrupted microbial community Altered Inflammation • Microbe involvement in SCFA Metabolism & GPCR recognition • High Fiber diet- reduces insulin stimulation, increases gastric transit time Increased Adipose Tissue Figure adapted from Blaser et al, Cell Press (2013)