PKN2 regulates cell-junctions to limit colitis and colon tumour formation

Background and Aims Epithelial barrier integrity in the gut must be tightly regulated to allow essential paracellular transport while limiting mucosal exposure to inflammatory microbes and toxins. Chronic inflammation in inflammatory bowel diseases is a major health burden and a key risk factor for colorectal cancer. Here, we identify the Rho-regulated kinase PKN2 as a non-redundant safeguard of epithelial barrier integrity and a candidate tumour suppressor in colorectal cancer.

Conditional PKN2 knockout mouse models were used to examine the impact of PKN2 loss on colitis severity and induction of colorectal adenomas. Intestinal organoid models, epithelial barrier assays and bioinformatic approaches explored PKN2 regulation of epithelial function. Human inflammatory bowel disease datasets are analysed for associations with PKN2 expression and signatures associated with compromised barrier integrity.

PKN2 deletion sensitises mice to inflammatory bowel injury and promotes colorectal adenoma formation in a colitis-associated colorectal cancer model. Tumour burden tightly correlates with colitis severity suggesting that enhanced inflammation-driven tissue injury underlies tumour promotion. Mechanistically, PKN2 localises to epithelial tight junctions and is required to stabilise barrier integrity during epithelial injury in mouse and organoid models. Transcriptomic changes associated with epithelial PKN2 loss mimic inflammatory bowel diseases and correlate with diseases severity and therapy response in human datasets.

PKN2 is a regulator of gut barrier integrity encoded at genomic loci previously associated with sensitivity to bowel inflammation and tumour suppression in both humans and mice. PKN2 loss recapitulates key features of human colitis and implicates PKN2 as a regulator tight junction integrity that shapes disease severity, treatment response and cancer risk. Competing Interest Statement The authors have declared no competing interest. Footnotes The manuscript has been revised following an internal review process aimed at improving clarity, rigor, and overall presentation. Sections 3 and 4 have been substantially revised to improve clarity, structure, and coherence. The presentation of methods and results has been refined, and the logical flow of the analyses has been improved. The introduction, discussion and abstract have been updated to accurately reflect the revised manuscript. The author list, references and acknowledgements have also been updated. Abbreviations - AOM - Azoxymethane - ATCC - American Type Culture Collection - BSA - Bovine serum albumin - CCs3 - Colon cancer susceptibility locus on mouse chromosome 3 - Cdcs1 - Cytokine deficiency-induced colitis susceptibility-1 - COAD - Colon adenocarcinoma (TCGA) - CreERT2 - Tamoxifen-inducible Cre recombinase - DAPI - 4⍰,6-diamidino-2-phenylindole - DAI - Disease Activity Index - DMEM - Dulbecco’s Modified Eagle Medium - DMSO - Dimethyl sulfoxide - DSS - Dextran sodium sulphate - EDTA - Ethylenediaminetetraacetic acid - EGTA - Ethylene glycol-bis(β-aminoethyl ether)-N,N,N⍰,N⍰-tetraacetic acid - FBS - Fetal bovine serum - FFPE - Formalin-fixed paraffin-embedded - FITC - Fluorescein isothiocyanate - GEO - Gene Expression Omnibus - GSVA - Gene Set Variation Analysis - GSEA - Gene Set Enrichment Analysis - H&E - Haematoxylin and eosin - HET - Heterozygous - HRP - Horseradish peroxidase - IBD - Inflammatory bowel disease - IBS - Irritable bowel syndrome - IL-1β - Interleukin-1 beta - IL-6 - Interleukin-6 - iPKN2KO - Inducible PKN2 knockout - JAM-A - Junctional adhesion molecule A - KD - Kinase-dead - KEGG - Kyoto Encyclopedia of Genes and Genomes - KO - Knockout - LPS - Lipopolysaccharide - PBS - Phosphate-buffered saline - PCA - Principal Component Analysis - PFA - Paraformaldehyde - PKN2 - Protein kinase N2 - PVDF - Polyvinylidene fluoride - READ - Rectal adenocarcinoma (TCGA) - SDS–PAGE - Sodium dodecyl sulphate–polyacrylamide gel electrophoresis - siRNA - Small interfering RNA - TAM - Tamoxifen / 4-hydroxytamoxifen - TCGA - The Cancer Genome Atlas - TBS - Tris-buffered saline - TNF-α - Tumour necrosis factor alpha - UMI - Unique molecular identifier - Vil1-CreERT2 - Villin promoter-driven inducible Cre line - WT - Wild-type - ZO-1 - Zona occludens-1