Abstract
Background
Crohn’s disease (CD) and ulcerative colitis (UC) are autoimmune-mediated conditions of chronic inflammation affecting the intestinal mucosa, that pose a lifelong risk to patients to develop a colitis-associated carcinoma (CAC). Much of current knowledge on mechanisms in CAC development result from murine CAC models, while studies on human CAC carcinogenesis are scarce. Thus, in our present study, we aimed to contribute to the understanding of colitis-associated carcinogenesis by comparative analysis of gene expression in human samples and further validation of these findings in colon organoids.
Methods
RNA isolation was done from microdissected surgical colon specimen from 60 patients that suffered from either UC, CD, UC-CAC, CD-CAC or inflammation-free healthy controls (10 patients per group). Nanostring nCounterTM technology with a gene panel comprising >630 genes was performed to examine genes focusing on mucosal immunology, epithelial barrier/polarity. nSolverTM data analysis software was used for primary data analysis and statistics. Patients were grouped using expression patterns (by heat maps), differential expression and pathway analysis. For organoid cultures, crypts were isolated from mouse colon and cultured using the R-Spondin method. Organoids were subjected to osteopontin treatment. Consecutively, RNA was isolated. This was followed by gene expression analysis of core EMT (epithelial to mesenchymal transition) transcription factors (TFs) focusing on Twist1, Snai1, Snai2 by RT-PCR.
Results
Heat maps generated from the expression data revealed close-to-optimal grouping recapitulating the clinical subgroups. Differential expression comparing CD with CD-CAC and UC with UC-CAC identified 203 and 271 differentially expressed genes, respectively. Genes most significantly upregulated included SPP1/Osteopontin (OPN), GRHL2 and EMT signature gene as fibronectin1 and ZEB1, generating the hypothesis of OPN driving EMT and thereby inducing inflammation-associated carcinogenesis. Furthermore, mouse colon organoids treated with OPN (50ng/µl) revealed an increased expression by 3fold of core the EMT TFs Twist1, Snai1, Snai2.
Conclusion
We provide a comprehensive quantitative gene expression analysis for CAC with comparison of gene expression in CAC to the respective underlying IBD, CD and UC. Identified upregulated or downregulated genes allow to allocate signal transduction pathways important for CAC carcinogenesis. OPN as the most upregulated gene in CAC in our gene panel might be crucial for regulating EMT in CAC carcinogenesis. Thus, our findings uncover the role of OPN in CAC carcinogenesis.
OP13 Molecular response to ustekinumab in moderate-to-severe ulcerative colitis by serum protein and biopsy gene expression analysis: Results from the UNIFI Phase 3 induction study