Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-producing Escherichia coli in ApcMin/+ Mice.

Cécily Lucas, Laurène Salesse, My Hanh Thi Hoang, Mathilde Bonnet, Pierre Sauvanet, Anaïs Larabi, Catherine Godfraind, Johan Gagnière, Denis Pezet, Philip Rosenstiel, Nicolas Barnich, Richard Bonnet, Guillaume Dalmasso, Hang Thi Thu Nguyen
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Gastroenterology (New York, N.Y. 1943)
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BACKGROUND AND AIMS:Colibactin-producing Escherichia coli (CoPEC) colonize the colon mucosa of higher proportions 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 with and without 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 histology, immunoblot, and quantitative reverse transcription PCR analyses for markers of autophagy, DNA damage, cell proliferation and inflammation. We analyzed levels of mRNAs encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse transcription PCR. 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 6 (IL6) 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. ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, following 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, and decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in 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 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.