Gastroenterology

Gastroenterology

Volume 158, Issue 5, April 2020, Pages 1373-1388
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice

https://doi.org/10.1053/j.gastro.2019.12.026Get rights and content
Under a Creative Commons license
open access

Background & Aims

Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC.

Methods

Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction.

Results

Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice.

Conclusion

Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Keywords

Microbiome
Pathogenic Bacteria
Toxin
Colon Cancer

Abbreviations used in this paper

AOM
azoxymethane
ApcMin/+/Atg16l1ΔIEC
ApcMin/+ mice deficient for Atg16l1 specifically in intestinal epithelial cells
CoPEC
colibactin-producing Escherichia coli
CRC
colorectal cancer
DSB
double-strand break
DSS
dextran sodium sulfate
IECs
intestinal epithelial cells
IL
interleukin
mRNA
messenger RNA
PBS
phosphate-buffered saline
pks
polyketide synthase
qRT-PCR
quantitative real-time reverse-transcription polymerase chain reaction
siRNA
small interfering RNA

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Conflicts of interest The authors disclose no conflicts.

This work was supported by the Ministère de la Recherche et de la Technologie, Inserm (Institut national de la santé et de la recherche médicale; UMR1071), INRAE (Institut national de recherche en agriculture, alimentation et environnement; USC 2018), the Agence Nationale de la Recherche of the French government through the program “Investissements d’Avenir” (16-IDEX-0001 CAP 20-25) (to Hang Thi Thu Nguyen), the European Union FP7 People Marie Curie International Incoming Fellowship (to Hang Thi Thu Nguyen) and the DFG (Deutsche Forschungsgemeinschaft) ExC Precision Medicine in Chronic Inflammation and the CRC1182 project C2 (to Philip Rosenstiel).

Author names in bold designate shared co-first authorship.