Influence of intestinal microbiota on autophagy in colorectal cancer.
Authors: Nawfal Rashad , Noureldein Mohamed , Bitar Sara , Al Moussawi Sarah , Dia Batoul , El-Osta Assam , Khuri Fadlo Raja , Eid Assaad Antoine 
Published: June 2023

e15505

Background: Gut microbiota favors butyrate-forming bacteria and maintains homeostatic balance which is revoked during the development of colorectal cancer (CRC)-associated dysbiosis. Butyrate is a short chain fatty acid that acts as a lysine deacetylase inhibitor and is known to activate the AMPK pathway as well as inhibiting inflammation and ROS production. The role of AMPK activation in diabetes-associated complications including colorectal cancer is poorly understood. This project examines the role of dysbiosis and butyrate on CRC onset and disease progression. Methods: C57BL/6- Apctm1Tyj/J ( APC) mice that spontaneously develop CRC were used and the study included four groups of mice (n = 3 for each): (1) Control C57BL/6, (2) APC, (3) APC treated with probiotics rich in butyrate forming bacteria and (4) APC treated with butyrate. Probiotics and butyrate were administered by oral gavage and intraperitoneally, respectively. Feces were collected at early (when CRC is developed at 8 weeks of age) and late (at 16 weeks of age, day of sacrifice) timepoints, and microbial composition was identified by 16S rRNA sequencing. Mice colons following sacrifice were also harvested for molecular, anatomical, and histological analyses. Results: The butyrate kinase gene was assessed in fecal samples. Butyrate forming bacteria were significantly reduced in APC mice but elevated in APC receiving probiotics when compared to control mice. Anatomically, polyp number was reduced in groups receiving probiotics and butyrate when compared to APC mice without treatment (p < 0.01 for each). Histologically, butyrate and probiotics ameliorated the histological changes observed in in APC mice as assessed by PAS staining. Furthermore, we observed a decrease in goblet cells, and increased number of crypts on distal colon histology. The expression of NADPH Oxidase 4 (NOX4) and TIGAR, a P53 target protein expression were significantly increased in APC mice and restored following the administration of probiotics or butyrate. ROS production assessed by HPLC showed similar pattern across our 4 groups. The expression of the autophagy markers Beclin-1, ATG-12 and LC3-β were restored to control levels after probiotics and butyrate administration. 16S rRNA sequencing of fecal samples revealed significant differences in microbial composition between groups, most significantly at 16 weeks with Lachnospiraceae (ASV 175), Muribaculaceae (ASV 53) and Muribaculaceae (ASV 584) were decreased in APC when compared to control mice ( < -7 Log2 fold change each). Conclusions: Collectively, our data suggests colorectal cancer is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria. This disruption leads to over-expression of NOX4, and ROS production associated with alteration in autophagy, thus worsening gastrointestinal complications and CRC. Restoring butyrate forming bacteria might serve as an effective adjuvant to therapy in CRC.