Bacterial Infection Promotes Colon Tumorigenesis in Apc min/+ Mice Joseph V. Newman, Takeo Kosaka, Barbara J. Sheppard, James G. Fox, and David B. Schauer

Background on Diet and Microflora Louis Pasteur ( ) 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 Anatomy Small 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 to 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 GlycolysisATP Pyruvate propionate Liver ATP Acetyl- CoA Gut EpitheliumSkin ATP H2H2 CO 2 + H 2 CH 4 methanogenesis butyrateacetate sulfidogenesis H2SH2S SO 2

Diet and its effect of gut flora Fermentation of SCFA  H 2 + CH 4 Efficient mechanism for H 2 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 Disease Chronic Inflammation Inflammatory Bowel Disease (IBD) Inflammation, Rigidity and Thickening of Colon Ulcerative Colitis Chronic Inflammation Diverticulitus Colon Develops a pocket Colon Caner

Relevance of studying Bacterial infection Helicobater pyloriIncreased gastric cancer H. hepaticusHepatocellular carcinoma, Liver Lawsonia intracellularisIntestinal epithelium proliferation (cancer biomarker) Group D StreptococcusInflammation, 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

Apc Min/+ 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 –Apc Min/+ w/ 100  L o/n culture –Apc +/+ w/ 100  L sterile media Confirmed infection 7 days post infection w/ CFU counts Sacrificed mice 10 days and 5 months post inoculation 10 days or 5 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 Apc Min/+ 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