bacterial infection promotes colon tumorigenesis in apc min/+ mice joseph v. newman, takeo kosaka,...

Bacterial Infection Promotes Colon Tumorigenesis in

Apcmin/+ Mice

Joseph V. Newman, Takeo Kosaka, Barbara J. Sheppard, James G. Fox,

and David B. Schauer

Background on Diet and Microflora

• Louis Pasteur (1822-1895)

• Adult Humans have more prokaryotic than eukaryotic cells

• Symbiotic relationship in GI tract for more efficient nutritional benefit

• Epidimiological studies have linked high incidence rates for colon cancer to a western diet (high in animal products)

Digestive AnatomySmall vs. Large intestine:

• Different developmental layers• Epithelium structure• Commensal digestive flora• The occurrence of intestinal tumors in mice vs. humans

Common Digestive Flora

• Stomach– (Heliobacter sp.)

• Small Intestine– Enterococci– Lactobacilli– (E. coli,

Psesudomonads)

• Colon– Enterobacteria– Enterococcus faecalis– Bacteroides *– Bifidobacterium *– Clostridium– Lactobacillus *– Streptococcus– Staphylococcus– Ruminococcus– Peptostreptococcus– Peptococcus

Large Intestine

• 3 distinct regions– Cecum– Colon– Rectum

• 1011 to 1012 cells/g wet feces• >500 species• Lactobacilli• Bacteria have a few key

roles:– Nutrient breakdown

– Preventing pathogenic colonization

– Maintaining overall physiological conditions

Dietary Fiber breakdown Sloughed cells and dietary fiber

Fermentation

Glycolysis ATP

Pyruvate propionate Liver

ATP

Acetyl- CoA Gut EpitheliumSkin

ATP ATP

H2 CO2 + H2 CH4methanogenesis

butyrateacetate

sulfidogenesisH2S

SO2

Diet and its effect of gut flora

• Fermentation of SCFA H2 + CH4

• Efficient mechanism for H2 disposal has evolved along 2 major pathways:– Methanogenic achea

– SRB

– Acetogenic*

• Low in colon cancers: high levels of methanogens• western diet: higher levels of colon cancer

• Difference: Meat in the diet leads to an increase in SRB • The composition of diet not only impacts the substrates for gut

flora, but also sets up a predictable competitive relationship

Large Intestine

• Colon mucosa has flat epithelium with crypt complexes– Differentiated cells

– Proliferative stem and precursor cells

• SCFA degredation: proliferation differentiation

Intestinal Epithelium

• Mucosal epithelium are bound by tight junctions, the most luminal cell-cell junctions

• 2 major functions:– Permeability barrier– Protein Separation

• Tight junctions– Occludin– Claudin

Bacteria, inflammation, …

• Analyze KO mice to germ-free conditions– TCR/p53 Dbl. KO– IL-10 deficient mice– Apc Min mice 50%

tumor

• Hosting a bacterial population is not without consequence– Maintain gene to protect

against bacterial stress: peroxidative stress, bacterial antigen, inflammation

– Intact mucosal barrier

Possible models of tumorigenesis

• Inflammation/cancer depends on aggregate interactions– Quorum sensing– Alterations in flora due to diet

• Weak genetic defects and polymorphisms in hosts might allow normal flora to induce tumors over extended period of time

Diseases of the Colon/Large Intestine

Crohn’s DiseaseChronic Inflammation

Inflammatory Bowel Disease (IBD)Inflammation, Rigidity and

Thickening of Colon

Ulcerative ColitisChronic Inflammation

DiverticulitusColon Develops a

pocket

Colon Caner

Relevance of studying Bacterial infection

Helicobater pylori Increased gastric cancer

H. hepaticus Hepatocellular carcinoma, Liver

Lawsonia intracellularis Intestinal epithelium proliferation (cancer biomarker)

Group D Streptococcus Inflammation, dysplasia, rectal carcinoma

Introduction

• C. rodentium - naturally gram (-) occurring bacterial pathogen of lab mice

• Infection: – Epithelial cell hyperproliferation (IBD, Chron’s,

colitis) & thickening/rigidity of colon– Diarrhea and weight loss (suckling mice)– Colonic hyperplasia and limited inflammation

(adult)

Transmissible Murine Colonic Hyperplasia (TMCH)

• Colonic crypts are 2 to 3 times longer compared to normal mice

• Epithelium contain twice the number of dividing cells

• No direct evidence linking C. rodentium to tumorigenesis

• Increased colonic adenoma counts in presence of carcinogens

AE Lesions

• Attaching and Effacing lesions during colon infection– Dissolution of brush border, – cupping of adherent bacteria, – cytoskeleton rearrangements of epithelium

• Enteropathogenic and Enterohemorrhagic E. coli (EPEC & EHEC) infections

• Similar gene locus is required for AE formation C. rodentium animal model of infection

AE Lesions (Chicken and the egg)

• Is inflammation causing the altered epithelium, which allows for bacterial association?

• Does bacterial attachment cause these lesions, which then induce inflammation?

• AE pathogens have been shown to attach to surface epithelial cells via type III secretion pathway, possibly causing the release of some inflammatory mediators– 7 day post infection– 21 days post infection

ApcMin/+ Mouse

• Nonsense mutation of adenomatous polyposis coli gene

• Apc:– Regulates cellular division frequency– Regulates cellular attachment/movement

• Mice are pre-disposed to multiple intestinal neoplasms (Min)

Methods

• Inoculated 4 week old mice– ApcMin/+ w/ 100L o/n

culture– Apc+/+ w/ 100L sterile

media

• Confirmed infection 7 days post infection w/ CFU counts

• Sacrificed mice 10 days and 5 months post inoculation

10 days or5 months

100 L sterile media

100 L Culture

or

the messy steps

Pathology• The colon was removed

and examined for hyperplasia

• Adenomas were counted and measured

• Grossly altered tissue was excised and mounted for histological analysis

Immunohistochemistry• Representative samples

were frozen and stained for: smooth muscle

actin– F4/80 (macrophage

marker)– COX-2

Results

• A: Mucosal epithelium,10 days post infection

• B: Intact basement membrane and hyperplasia

• C: Dysplastic tissue with adenoma

Colonic Adenoma from infected Min mouse

• D. COX-2• E. COX-2 + F4/80• F. COX-2 + actin

High magnification of Adenoma

G. COX-2

H. COX-2 + F4/80

I. COX-2 +Actin

Results

ApcMin/+ mice• Visible thickening

and rigidity of colon (10 days post infection)

• Mean crypt column height 2x that of uninfected Min mice (significant)

Wt mice• Visible thickening

and rigidity of colon (10 days post infection)

• Mean crypt column height 2x that of uninfected Min mice (significant)

No significant difference

Discussion

• Infection promotes adenoma formation in Min mice

• Could promotion be due to hyperproliferative state induced by infection?

• COX-2 levels were not detected in colon tissue from infection (10 day post-infection) Is COX-2 involved in earliest stages of tumor promotion in Min mice?

Future Direction

• Is microbiota required for colon tumorigenesis?

• Do A/E pathogens produce alterations of epithelial cell cytokinetics?

• What “chemical signals” are secreted by bacteria associated with aberrant crypts?

• Do bacterial signals influence gene activities of colon mucosal cells?

• Heat-stable enterotoxin

• GC-C pathway specific to intestinal epithelium

• Carcinoma cell proliferation was inhibited