Microbes Microbiota and colon tumorigenesis 2018 05 10

Microbes, Microbiota, and colon tumorigenesis 2018. 05. 10. R 3 이철형 / Pf. 이동호

Introduction l CRC (Colorectal cancer) - Lifetime risk of developing CRC, approximately 5% (adenoma 20%) - Ranking top three causes of cancer-related death around the globe l Microbiota in large intestine - Most densely populated microbial ecosystem in human - 1 g = 1011 (large intestine) - Curiosity have arisen the role of bacteria in colon carcinogenesis - Evolution of microbial meta’omics facilitates ample of researches

Introduction − Gradual accumulation of oncogenic mutations is responsible for CRC tumorigenesis − It remains unknown with precision what triggers the initial carcinogenesis Cynthia L. S. , Wendy S. G. , Cell host & Microbes, 2014

Introduction l Colon microbiome in CRC pathogenesis − Difficult to prove causation (cf> H. pylori in gastric cancer) − “noncultivable” − Importance of utilizing multiple approaches Cynthia L. S. , Wendy S. G. , Cell host & Microbes, 2014

Introduction l Several microorganisms proposed as culprit − Enterococcus faecalis − Enterotoxigenic Bacteroides fragilis (ETBF) − B. fragilis toxin (BFT) alters CEC (colonic epithelial cell) structure − Escherichia coli − Polyketide synthase (pks) : genotoxin induces DNA damage − Fusobacterium spp. l Host mucosal environment - Pro-inflammatory cytokines (IL-17, IL-6) - CEC signaling (NF-k. B, Stat 3)

Hussan H et al. Fusobacterium and CRC development, World J. of Gastroenterology (2017)

Science, 2018

Background l Familial adenomatous polyposis (FAP) − Onset and frequency of polyp formation differs within families − Previous study of sporadic CRC patients shows biofilms of normal mucosa associated with a pro-oncogenic state

Methods – identify microbiota l Colon samples of clinical FAP phenotype patients (n=20) undergone surgery l Colonoscopic Bx or surgical resections without disease serves as control (n=20) l Panbacterial 16 S ribosomal RNA : fluorescent in situ hybridization (FISH) => Additional probes for identify predominant biofilm spp. using q. FISH

Result : FISH and microbiology culture analysis of FAP mucosal tissues Panel A B. fragilis : green E. coli : red Panel B Enterobacteriaceae : yellow E. coli : red Christine M. Dejea et al. Science 2018; 359: 592 -597

Methods – ETBF and pks+ E. coli l E. coli containing polyketide synthase(pks) – encodes colibactin genotoxin – induces DNA damage with colon tumorigenesis in AOM treated mice l Enterotoxigenic B. fragilis induces colon tumorigenesis in Apc. Min/+ mice via bft l 3 mm diameter punch Bx and culture from FAP patients (n=25) and control(n=23) colon mucosa to identify B. fragilis and E. coli l PCR assay to identify bft and clb. B

Result : FISH and microbiology culture analysis of FAP mucosal tissues Christine M. Dejea et al. Science 2018; 359: 592 -597

Methods l Mouse model − Specific pathogen free C 57 BL/6 J mice : AOM treatment − Apc. Min/+ mice : human FAP equivalent − Monoinoculated or coinoculated with pks+ E. coli or ETBF for 15 wks and assessed tumor #, hyperplasia score, inflammation score − To determine type of inflammation, analyzed immune cell infiltration of lamina propria with flow cytometry − Statistical analysis : Kruskal-Wallis test, Mann-Whitney U

Cocolonization by pks+ E. coli and ETBF increases colon tumor onset and mortality AOM treat Apc. Min/+ Christine M. Dejea et al. Science 2018; 359: 592 -597

Cocolonization by pks+ E. coli and ETBF related to inc. inflammation AOM mice Apc. Min/+ Christine M. Dejea et al. Science 2018; 359: 592 -597

Cocolonization by pks+ E. coli and ETBF related to inc. inflammation AOM mice Christine M. Dejea et al. Science 2018; 359: 592 -597

IL-17–induced inflammation is necessary for bacterial-driven tumorigenesis. AOM mice C 57 BL Christine M. Dejea et al. Science 2018; 359: 592 -597

ETBF enhances pks+ E. coli colonization and colonic epithelial cell DNA damage AOM mice Christine M. Dejea et al. Science 2018; 359: 592 -597

ETBF enhances pks+ E. coli colonization and colonic epithelial cell DNA damage Christine M. Dejea et al. Science 2018; 359: 592 -597

Conclusion l ETBF and pks+ E. coli cocolonization is found more than half of FAP patients (less than 25% of controls) l ETBF and pks+ E. coli cocolonization promotes enhanced carcinogenesis via two distinct steps – Mucus degradation, enabling pks+E. coli adherence, inducing increased CEC DNA damage by colibactin – IL-17 induction promoted (by ETBF) with early augmentation with pks+ E. coli l Analysis of coexpression of bft and clb. B may have value in screening and prevention of CRC

Cell Host & Microbe, 2018

Background l Apc. Min mice colonized with ETBF : model for de novo microbial induced colon tumorigenesis l Key virulence factor, B. fragilis toxin (bft) : zinc-dependent metalloproteinase targets tight junction of CEC l IL-17, pro-inflammatory signaling pathway Stat 3 has been proposed important role in colon tumorigenesis in animal models.

Methods l Animal model : Apc. Min mice and KO mice (IL-17 -/-, IL-17 R-/-) l BM chimera Apc. Min mice for elucidating the role of immune cells in tumorigenesis l Colonization with ETBF/ETBF(∆bft) for 3 months and evaluate tumorigenesis l Using IF technique to evaluate tissue IL-17 R expression l Statistical analysis : Mann-Whitney U test

Methods IL-17 R-/- Apc. Min IL-17 R-/- Apc. Min

Result - BFT and IL-17 R are required for ETBF-triggered tumorigenesis Cell Host & Microbe 2018 23

Methods l Marked distal colon tumorigenesis in ETBF model l Compared m. RNA expression of inflammatory related (Ir) genes – ETBF colonized WT // 17 -/- mice (instead of Min mice) l CD 45 -Ep. CAM+ CECs were cell sorted, evaluate after 7 dcolonization l Colons divided into distal(C 1) to proximal(C 6) sections l Taqman-based RT-PCR was used to evaluate RNA expression l Immunoblotting was used to evaluate protein level

Result - IL-17 dependent NF-k. B signaling triggers chemokine gradient Cell Host & Microbe 2018 23

Methods l Does response of CECs to IL-17 affects myeloid population and distribution along the axis of colon? l Assess myeloid population of lamina propria by enzymatic digestion and flow cytometry, after 7 days of ETBF colonization of Apc. Min mice l Using Cx. CR 2 -/- mice (Rc for CXCL 1, 2, 5)

Result - IL-17 dependent Accumulation of IMCs in distal colon Distal colon, ETBF+ Cell Host & Microbe 2018 23 Distal colon, sham

Cxcr 2 -/- Apc. Min WT Apc. Min

Result - Myeloid Recruitment and Colon Tumorigenesis dependent on chemokine signal Pepducin uncouples Cx. CR 2 ligands Cell Host & Microbe 2018 23

Result Cell Host & Microbe 2018 23

Discussion l Restricted in relatively simplified animal models l Elucidating the interaction of microbes, host, and the real tumorigenesis (“causation”) l Emphasize the importance of tumor microenvironment l Microbial-microbial communication – E. coli & A. Muc vs BFTS l Possibility of microbial control and postpone tumorigenesis