The interplay of ATG16L1 and XBP-1 crucially coordinates epithelial DNA damage responses and protects from intestinal carcinogenesis

Introduction The unfolded protein response (UPR) and macro-autophagy are key cellular homeostatic pathways and mutations of genes involved in these pathways are associated with intestinal bowel disease (IBD)^1,2. We have previously shown that deficiency in the enzyme RNaseH2b^3,4 leads to impaired ribonucleotide excision repair and induced spontaneous DNA damage with subsequent suppression of stem cell proliferation.

The aim of this study was to test the role of macro-autophagy (ATG16L1) and unfolded protein response (XBP-1) and their interplay in coordinating epithelial stem cell fate in the context of DNA damage (RNaseH2b).

Methods H2b^dIEC mice, ATG16L1^dIEC mice and XBP-1^dIEC mice were established and crossed to generate ATG16L1/RNaseH2b^dIEC mice, XBP-1/RNaseH2b^dIEC mice and ATG16L1/XBP-1/RNaseH2b^dIEC mice. In vivo we performed Immunohistochemistry (IHC) and Immunofluorescence of young and aged mice. We generated organoids from small intestinal crypts to describe in vitro the direct molecular effects of ATG16L1 and XBP-1 on epithelial DNA damage. Western Blot, Propidium iodide staining, flow cytometry and colony forming assay were carried out to measure stem cell suppression and cell death.

Results Upon inhibition of autophagy epithelial cell death was increased under DNA-damage conditions. Using IHC, we observed that ATG16L1/RNaseH2b^dIEC mice and ATG16L1/XBP-1/RNaseH2b^dIEC mice displayed increased DNA damage (ɣH2Ax) and epithelial cell death (Tunel) compared to RNaseH2b^dIEC mice and XBP-1/RNaseH2b^dIEC mice. Intestinal organoids with deficient autophagy were more prone to cell death in response to DNA-damage. Despite ongoing cell death, BrdU- and Ki67- IHC revealed that defect autophagy significantly increased epithelial proliferation in ATG16L1/RNaseH2b^dIEC mice compared to RNaseH2b^dIEC mice. Prevalence of malignancies was two times increased from 50 % of all aged XBP-1/RNaseH2b^dIEC mice (n=12) to 90 % of all aged ATG16L1/XBP-1/RNaseH2b^dIEC mice (n=10).

Colclusion We conclude that dysfunctional macro-autophagy which is associated with IBD can promote DNA-damage induced cell death but also increase epithelial proliferation. In addition we show that UPR and autophagy mutually fine tune p53-dependent DNA damage response to protect from intestinal carcinogenesis.

Publication History

Article published online:
08 September 2020