Commensal Bacteria- The Good, The Bad and The Unknown

Endeavors, Winter 2011

The Intestinal Microbiota and Colorectal Cancer

Temitope O. Keku, MSPH, PhD

ProfessorDivision of Gastroenterology and Hepatology

University of North Carolina, Chapel Hill

tokeku@med.unc.edu

Background

Gut Bacteria- who is there?

Functional studies- what are they doing?

Summary and conclusions

http://www.the- scientist.com/article/display/15649/

Outline

Colorectal cancer (CRC)

•Accounts for approximately 8.5% of all cancer deaths globally, with ~1.4 million new cases annually.

•In 2010, the global economic burden was ~ $14.1 billion and is expected to increase to $17.4 billion by 2020.

http://globocan.iarc.frSiegel et al. 2014 CA Cancer J. Clin. 2014:64

Male Female

Estimated age-standardized incidence and mortality rates per 100,000 among women and men worldwide.

Colorectal cancer (CRC) incidence varies by geography

CRC: Fourth most common cancer and the second leading cause of cancer death in the

USA

American Cancer Society, 2013

Estimated New Cases

Estimated Deaths

Lung 228,190 159,480

Colorectal 142,820 50,830

Breast 234,580 40,030

Prostate 238,590 29,720

Americans have ~ 5.6% lifetime risk of being diagnosed with CRC

Colorectal cancer is multifactorial

• Colorectal cancer is multistep process involving accumulation of genetic alterations and transformation of normal cells. • Adenomas are intermediate precursors.

Loss ofAPC

Mutationof K-ras

Deletion of 18q

Loss ofp53

Normal epithelium

Proliferation Intermediateadenoma

Early adenoma

(ACF)

Lateadenoma

Carcinoma

ENVIRONMENT

Gut Microbiome

Most bacteria reside in the intestinal communities (gut microbiota)

Stomach 0-102

Colon 1011

Distal Ileum 107-108

Duodenum 102

Jejunum 102

Proximal Ileum 103

Functions of the Gut Microbiota

Protective

Structural Metabolic

Commensal Gut Bacteria

Nutrient absorptionVitamin K, Folate, Biotin,Mg2+, Ca2+, SCFA

Development of Immune system

Protection against Pathogens, Homeostasis

Gut Microbiota are not innocent bystanders. They play a role in various diseases

•Necrotizing enterocolitis (NEC)

• Inflammatory Bowel Diseases (IBD)

•Ulcerative Colitis & Crohn’s Disease

•Colitis-associated cancer

•Colorectal cancer

• Stomach cancer

• Irritable bowel syndrome

• Infectious diarrheas

•Obesity & obesity-associated diseases

• Type I and Type II diabetes

•Asthma & other allergic diseases

•Autism

•Depression

Knock out Mouse

Inflammation Bacteria Incidence of Carcinoma

(Colon/Rectum)

Reference

Conventional Germ-free

IL-10 KO √ E. feacalis 60% 0 Berg et al. 1996

Rag2 KO

Rag2/Tgf1 DKO

√ H. hepaticus 100% 0 Erdman et al. 2003

TCR/P53 DKO √ Normal flora 70% 0 Kado et al. 2001

GPx1/GPx2 DKO

√ Normal flora-Helicobactersp.

28% 0 Chu et al. 2004

KO, knockout; DKO, double knockout; GPx, glutathione peroxidase; TCR, T cell receptor;

Evidence from animal models of inflammation support gut bacteria and CRC association

Gut Bacteria: Who is there?

16S ribosomal RNA gene (rRNA)

• Many members of the gut microbiota are not able to be cultured.

• Universal gene in bacteria – polymorphisms,

makes it reliable for inferring phylogenetic

relationships and characterizing microbial

community composition.

V1 V2 V3 V4 V6V5 V7 V8 V9

Conserved Regions

Variable Regions

16S ribosomal RNA (rRNA) Gene

Maximum likelihood tree generated from the top 371 OTUs using RaxXML EPA server (http://i12k-

exelixis3.informatik.tu-muenchen.de/raxml ). Branches are colored based on Phylum level assignments. Red colored

branches represent OTUs significantly different between cases and controls within each Phylum (at 10% FDR).

•Case: 87 taxa more abundant

•Control: 5 taxa more abundant

ISME J. 2012 Oct;6(10):1858-68

**T-tests on log-normalized abundances of genera in cases vs. controls Only top 11 taxa based

on p-values showing significant differences between cases and controls.

Bacterial Genus Direction in cases RankB-H corrected

p-value**Helicobacter UP 1 0.005Acidovorax UP 2 0.005Lactobacillus UP 3 0.008Cloacibacterium UP 4 0.006Lactococcus UP 5 0.005Stenotrophomonas UP 6 0.007Turicibacter UP 7 0.011Weissella UP 8 0.009Delftia UP 9 0.011

Acinetobacter UP 10 0.011

Streptococcus DOWN 11 0.04

Differences in bacterial abundance between cases and controls

Sanapareddy et al. 2012, ISME J. 6:1858-68

Specific bacteria are associated with the mucosal layer

57-58-comb

A

D E

x200

x400 x1800

G- ProteobacteriaFaecalibacterium

Ruminococcus Lactobacillus

Clostridia

x1800

x400

x400

x400

x400

x400

x400

B C

D E F

G H

Fluorescent in-situ hybridization analysis

Bacterial communities differ between CRC patients and healthy controls

Sobhani et al. PLoS One 2011

Bacteroides/Prevotella overabundance in CRC than controls

Sobhani et al. PLoS One 2011

Microbiota of CRC tissue and adjacent normal are different

Marchesi, PLoS One. 2011; 6(5): e20447

Genome Res. 2011 Oct 18.

Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.Kostic et al. Genome Res. 2011 Oct 18

Bacterial signatures of colorectal adenomas and cancer

Abundance of Fusobacterium in rectal mucosal biopsies from adenoma cases and non-adenoma controls. McCoy et al. 2013, PLoS One

Additional bacterial signatures associated with CRC

Bacteria Reference

Fusobacterium Burns et al. 2015Marchesi et al. 2011Castellarin et al. 2011Kostic et al. 2011

E. coli Shen et al. 2010Zackular et al. 2014

Bacteroides fragilis Sears et al. 2014, 2015

Acidovorax Sanapareddy et al. 2012

Porphyromonas Zackular et al. 2014

Summary

•Changes in gut bacteria community profiles (dysbiosis) are associated with colorectal adenomas and cancer.

•Few bacterial signatures.

Gut Bacteria: What are they doing?

The Metabolome

•Metabolome: group of metabolites produced during metabolic processes.

•Human metabolome consists of metabolites from human cells and microbial cells.

• Changes in the metabolome have recently been associated with many diseases.

» Inflammatory Bowel Disease

» CRC

• Limited studies on microbiota, metabolome and adenomas/CRC.

KEGG Reference Pathway: Metabolic Pathways

-10 -8 -6 -4 -2 0 2 4

Galactose

13,14-Dihydro-15-keto-PGE2

5-oxoproline

2,4-Diaminobutyric acid

Pentadecanoic acid

5-Hydroxyindoleacetic acid

Phosphoric acid, 2-aminoethanol

Dihydroceramide

Ornithine

Linoleic acid

Petroselinic acid

LysoPC(18:2(9Z,12Z))

Myo-Inositol

Diketogulonic acid

Prostaglandin E2

Methionine

2-aminobutyric acid

Oleamide

Glycine

Matitol

2-Phenylglycine

2-Phenylacetamide

N6-Acetyl-L-lysine

Fold Change

Me

tab

olit

es

Differential Metabolites: fold change for cases relative to controls

Diketogulonic acid

•Most biologically active metabolite of ascorbic acid (vitamin C)

•Vitamin C•Antioxidant - protects cells against oxidative stress from reactive

oxygen species.•Not synthesized by human cells•Obtained from the diet• Typically metabolized by bacteria in the GI tract.

• Low vitamin C -associated with oxidative stress and as such reduced levels of diketogulonic acid may be linked with increased oxidative stress.

• This is a potential link between the microbiome, metabolome, and pathogenesis of adenomas and CRC

Correlation between metabolites bacteria differ for cases and controls

Metabolome Summary •The metabolome is altered in

normal mucosa of patients with adenomas.

•Metabolome and microbiota are differentially correlated in the mucosa of adenoma patients compared to controls.

•Bacterial dysbiosis and altered metabolome may contribute to adenoma and CRC development.

• To develop bacterial signatures that could distinguish individuals with healthy colons from those with either colorectal adenomas or cancer

• To develop bacterial biomarkers or targets for screening and intervention

• Then we need……..

If the long term goal is…

IL10 −/− 129S6/SvEv 8 GF mice (Exp.)

&

129S6/SvEv GF 6 mice (Wild type)Bacteria

Cocktail ‘’pathogenic’ bacteria

OR

Cocktail ‘beneficial’ bacteria

Week 0 2 4 6 8 9

Fecal collection

Quantify bacteria

16S rRNA PCR/qPCR

1. Assess colon for tumors

2. Histology/Inflammation

3. Inflammation (Gene expression):

4. Quantification of bacteria via collection of

fecal/tissue samples

Gavaging ~109 poly-association of 5 bacteria/mouse

05/14 05/29 06/1207/17

Animal Experiments

06/27

National Gnotobiotic Rodent Resource Center, UNC, Chapel Hill

Sacrifice

WT Cecum (20X) IL10-/- Cecum (20X)

IL10-/- proximal Colon (20X)WT Proximal Colon (20X)

• Microbial dysbiosis and metabolome may contribute to the development of adenomas and colorectal cancer. (correlation studies)

• Mechanistic studies in animal models are needed.

Summary

Big Picture Questions

Dysbiosis Adenoma Cancer

Which bacteria are associated with adenoma/cancer development?

Is the gut bacteria a factor in progression of adenoma to cancer?

Can CRC patients treatment be optimized based on microbiota/metabolome profiles?

A Shift Towards Personalized Medicine

Current Medicine

• Reactive Treatment

• One size fits all

• Population-based

Personalized Medicine

• Preventative

• Targeted

• Customized

One Treatment

Treatment A

Treatment B

Treatment C

AcknowledgementsCollaboratorsKeku Lab

• DHS Study Participants

Carolyn SuittDylan PageKayla Ebner

Funding Sources: NIH P30 DK034987, NIH K01 CA093654, NIH P50 CA106991 and NIH R01 CA136887

Histology Core