The nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. We provide evidence that proper innervation is critical at all stages of gastric tumorigenesis. In three separate mouse models of gastric cancer, surgical or pharmacological denervation of the stomach (bilateral or unilateral truncal vagotomy, or local injection of botulinum toxin type A) markedly reduced tumor incidence and progression, but only in the denervated portion of the stomach. Vagotomy or botulinum toxin type A treatment also enhanced the therapeutic effects of systemic chemotherapy and prolonged survival. Denervation-induced suppression of tumorigenesis was associated with inhibition of Wnt signaling and suppression of stem cell expansion. In gastric organoid cultures, neurons stimulated growth in a Wnt-mediated fashion through cholinergic signaling. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic acetylcholine M3 receptor suppressed gastric tumorigenesis. In gastric cancer patients, tumor stage correlated with neural density and activated Wnt signaling, whereas vagotomy reduced the risk of gastric cancer. Together, our findings suggest that vagal innervation contributes to gastric tumorigenesis via M3 receptor–mediated Wnt signaling in the stem cells, and that denervation might represent a feasible strategy for the control of gastric cancer.

BackgroundIn inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns.MethodsColon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores.ResultsGene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls.ConclusionsThere is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

Purpose: Low concentrations of citrate and high concentrations of choline-containing compounds (ChoCC) are metabolic characteristics observed by magnetic resonance spectroscopy of prostate cancer tissue. The objective was to investigate the gene expression changes underlying these metabolic aberrations to find regulatory genes with potential for targeted therapies.Experimental design: Fresh frozen samples (n ¼ 133) from 41 patients undergoing radical prostatectomy were included. Histopathologic evaluation was carried out for each sample before a metabolic profile was obtained with high-resolution magic angle spinning (HR-MAS) spectroscopy. Following the HR-MAS, RNA was extracted from the same sample and quality controlled before carrying out microarray gene expression profiling. A partial least square statistical model was used to integrate the data sets to identify genes whose expression show significant covariance with citrate and ChoCC levels.Results: Samples were classified as benign, n ¼ 35; cancer of low grade (Gleason score 6), n ¼ 24; intermediate grade (Gleason score 7), n ¼ 41; or high grade (Gleason score !8), n ¼ 33. RNA quality was high with a mean RNA Integrity Number score of 9.1 (SD 1.2). Gene products predicting significantly a reduced citrate level were acetyl citrate lyase (ACLY, P ¼ 0.003) and m-aconitase (ACON, P < 0.001). The two genes whose expression most closely accompanied the increase in ChoCC were those of phospholipase A2 group VII (PLA2G7, P < 0.001) and choline kinase a (CHKA, P ¼ 0.002).Conclusions: By integrating histologic, transcriptomic, and metabolic data, our study has contributed to an expanded understanding of the mechanisms underlying aberrant citrate and ChoCC levels in prostate cancer.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

SummaryAnti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the antimicrobial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1β and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.