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
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