Biomarkers associated with the progression from gastric intestinal metaplasia (GIM) to gastric adenocarcinoma (GA), i.e., GA-related GIM, could provide valuable insights into identifying patients with increased risk for GA. The aim of this study was to utilize multi-bioinformatics to reveal potential biomarkers for the GA-related GIM and predict potential drug repurposing for GA prevention in patients. The multi-bioinformatics included gene expression matrix (GEM) by microarray gene expression (MGE), ScType (a fully automated and ultra-fast cell-type identification based solely on a given scRNA-seq data), Ingenuity Pathway Analysis, PageRank centrality, GO and MSigDB enrichments, Cytoscape, Human Protein Atlas and molecular docking analysis in combination with immunohistochemistry. To identify GA-related GIM, paired surgical biopsies were collected from 16 GIM-GA patients who underwent gastrectomy, yielding 64 samples (4 biopsies per stomach x 16 patients) for MGE. Co-analysis was performed by including scRNAseq and immunohistochemistry datasets of endoscopic biopsies of 37 patients. The results of the present study showed potential biomarkers for GA-related GIM, including GEM of individual patients, individual genes (such as RBP2 and CD44), signaling pathways, network of molecules, and network of signaling pathways with key topological nodes. Accordingly, potential treatment targets with repurposed drugs were identified including epidermal growth factor receptor, proto-oncogene tyrosine-protein kinase Src, paxillin, transcription factor Jun, breast cancer type 1 susceptibility protein, cellular tumor antigen p53, mouse double minute 2, and CD44.

This brief communication presented a dissertation of PhD thesis on translational research using state-of-the-art methods of multi-disciplinary integrations between basic and clinical research and between dry- (computational) and wet- (experimental and clinical) investigations. The findings showed possible causal association between neural innervation and tumorigenesis of gastric cancer via Wnt signaling and metabolic reprogramming within the tumor microenvironment. Accordingly, selective vagotomy, Botulinum neurotoxin type A (injection into the tumor areas), RAD001 (also known as Everolimus), CPI-613 (devimistat) and ivermectin were found to be potential for treatment of gastric cancer. The assessment showed that this translational research approach guaranteed the efficient development of novel knowledge in fundamental medical sciences, and that the identification of the role of vagal innervation and the possibilities of its modulation during gastric tumorigenesis represented valuable contributions to the fields of gastroenterology and cancer research. Possible roles of angiogenesis vs neurogenesis and brain-gut axis would be worth exploring in further research.

Objective: The aim of the present study was repositioning of ivermectin in treatment of gastric cancer (GC) by computational prediction based on gene expression profiles of human and mouse model of GC and validations with in silico, in vitro and in vivo approaches. Methods: Computational drug repositioning was performed using connectivity map (cMap) and data/pathway mining with the Ingenuity Knowledge Base. Tissue samples of GC were collected from 16 patients and 57 mice for gene expression profiling. Additional seven independent datasets of gene expression of human GC from the TCGA database were used for validation. In silico testing was performed by constructing interaction networks of ivermectin and the downstream effects in targeted signaling pathways. In vitro testing was carried out in human GC cell lines (MKN74 and KATO-III). In vivo testing was performed in a transgenic mouse model of GC (INS-GAS mice). Results: GC gene expression “signature” and data/pathway mining but not cMAP revealed nine molecular targets of ivermectin in both human and mouse GC associated with WNT/β-catenin signaling as well as cell proliferation pathways. In silico inhibition of the targets of ivermectin and concomitant activation of ivermectin led to the inhibition of WNT/β-catenin signaling pathway in “dose-depended” manner. In vitro, ivermectin inhibited cell proliferation in time- and concentration-depended manners, and cells were arrested in the G1 phase at IC50 and shifted to S phase arrest at >IC50. In vivo, ivermectin reduced the tumor size which was associated with inactivation of WNT/β-catenin signaling and cell proliferation pathways and activation of cell death signaling pathways. Conclusion: Ivermectin could be recognized as a repositioning candidate in treatment of gastric cancer.

Tumors comprise cancer cells and the associated stromal and immune/inflammatory cells, i.e., tumor microenvironment (TME). Here, we identify a metabolic signature of human and mouse model of gastric cancer and show that vagotomy in the mouse model reverses the metabolic reprogramming, reflected by metabolic switch from glutaminolysis to OXPHOS/glycolysis and normalization of the energy metabolism in cancer cells and TME. We next identify and validate SNAP25, mTOR, PDP1/α-KGDH, and glutaminolysis as drug targets and accordingly propose a therapeutic strategy to target the nerve-cancer metabolism. We demonstrate the efficacy of nerve-cancer metabolism therapy by intratumoral injection of BoNT-A (SNAP25 inhibitor) with systemic administration of RAD001 and CPI-613 but not cytotoxic drugs on overall survival in mice and show the feasibility in patients. These findings point to the importance of neural signaling in modulating the tumor metabolism and provide a rational basis for clinical translation of the potential strategy for gastric cancer.

Figure A: Tumor-conditioned LECs express ENA-78 (a)Cytokine antibody array detected the level of human chemokines and inflammations expressed in the supernatants of tumor conditioned LECs (t-LECS) and normal LECs (n-LECs).(b,c,d) The set of c indicated the cytokines which express in t-LECs CM but not in TCM.ENA-78 is obviously overexpression in t-LECs than n-LECs.(e)ELISAs for human ENA-78 (R&D System) detected t-LECS CM obtained at Day 0, 1, 2, 3, 4 of TCM induction.(n=3).(f)ENA-78 concentration in each CM was determined at 72 h by ELISAs.TCM induced ENA-78 overexpression in t-LECs, compared to n-LECs (**P < 0.01) (n=3).(g,h)TCM (100 μL) and serum-free media (SFM) were administered into nude mice for 2 weeks.Lymph nodes from TCM treated animals showed mENA-78 expression around mouse lymphatic vessels.Scale bars, 50 μm.(** P<0.002, n=7) (i)The ELISA determined the expression of mENA-78 in the liver, lymph node and lung by tissue homogenate method.

Background: Pancreatic cancer (PanCa) remains one of the most challenging malignancies with a dismal outcome and limited therapeutic options.Accumulative findings support the notion that histone enzymes involved in lysine methylation represent an essential control mechanism in tumorigenesis and are conceived attractive targets for therapeutic intervention.However, their role in pancreatic cancer oncogenesis remains to be elucidated.Methods: DNA Methylation Sequencing and Gene Expression Microarrays were employed to investigate CpG methylation and expression patterns of Histone Lysine Methyltransferases and Histone Lysine Demethylases in PanCa tissues versus normal tissues.Targeted bisulfate sequencing was performed for the evaluation of DNA methylation at single-nucleotide resolution.Gene expression was assessed in extended cohorts of PanCa patients by qRT-PCR.Efficiency of knockdown experiments, performed by RNAi interference assays and shRNA-expressing lentiviral vectors, was evidenced by qRT-PCR and Western Blot Analysis.Ingenuity Pathway and molecular analyses, cell metabolism, proliferation and colony formation assays were conducted in genetically modified cell lines.Subcutaneous xenograft mouse models were used to evaluate tumor growth in vivo.Results: Lysine (K)-Specific Methyltransferase 2D (KMT2D) is identified as aberrantly hypermethylated and downregulated gene based on a combinatorial analysis of CpG methylation and gene expression profiling of PanCa versus normal tissues.Methylation of individual CpG motifs (nucleotide -29 and +45 relatively to KMT2D transcription start site) was further confirmed by targeted bisulfide sequencing.Significant suppression of KMT2D mRNA levels has been validated in three different cohorts of patients.Time and dose-dependent upregulation of KMT2D mRNA and protein levels is observed in a panel of demcitabine-treated PanCa cells.Microarray expression profiling on MIA PaCa-2 cells transiently depleted of KMT2D revealed its downstream targets.AMPK signaling was distinguished as the top canonical pathway according to bioinformatics prediction.Interestingly, KMT2D silencing results in attenuation of AMPK activation.Furthermore, stable depletion of KMT2D leads to an increased cellular preference for aerobic glycolysis, promotes cell proliferation, anchorage-independent growth, as well as tumor growth in vivo.Conclusions: Our findings demonstrate that single-site CpG methylation regulates KMT2D expression and silencing of the latter affects AMPK signaling, induces the glycolytic rate of PanCa cells and accelerates tumor growth both in vitro and in vivo.Collectively, we identified a novel epigenetic factor that is implicated in metabolism-associated pathways and PanCa growth.

with increasing tumour grade.Early stage CRC is often difficult to detect pathologically, especially in inflamed tissue from IBD patients.This study identifies caspases-4 and -5 as potential biomarkers for the diagnosis and staging of CRC, particularly in the context of IBD patient surveillance.

Abstract Background/aim: Cholinergic nerves have been shown to regulate gastric WNT/ß-catenin signaling in leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive stem cells and have recently been implicated in gastric tumorigenesis. Denervation by local vagotomy or botulinum neurotoxin type A injection or the muscarinic acetylcholine receptor 3 antagonist limit gastric cancer growth. The aim of this study was to further unravel the metabolic pathways underlying the vagotomy-induced suppression of gastric tumorigenesis. Methods: Unilateral subdiaphragmatic truncal vagotomy (VT) was performed in wild-type (WT) and the INS-GAS mice, a genetic mouse model of spontaneous gastric cancer. Metabolic profiling and gene expression profiling in gastric tissues at 6 months post-surgery were performed using liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry, high resolution magic angle spinning NMR spectroscopy, and microarray gene expression (Illumina). Results: VT-induced suppression of tumorigenesis was manifested by reduced proliferation rate and increased apoptotic and autophagic signaling pathways, leading to reduced tumor size and prolonged survival. Principal component analysis showed four distinct clusters among 343 metabolic compounds: WT without VT, INS-GAS without VT, WT with VT and INS-GAS with VT. In the gastric tumor, the metabolic pathways that regulate stem cell homeostasis were downregulated after VT. Glutaminolytic pathway, including glutamine, glutamate, glycine and glutathione-S-S-glutathione, was down-regulated. The tricarboxylic acid cycle (TCA), including citrate, cis-aconitate, acetyle-CoA, threonine and glycine, was also down-regulated. However, glycolytic pathway, including glucose, glucose 6-phosphate, fructose 6-phosphate and lactate, was not significantly down-regulated. Signaling pathways that regulate glutamine metabolism, such as WNT/ß-catenin signaling, WNT target genes Cyclin D1, Axin2, Myc, Lgr5 and Cd44, p53 signaling, and mTOR signaling were down-regulated. The central carbon metabolism in cancer (“The Warburg effect” signaling) and lactate production were unchanged after VT. In addition, the choline metabolism, the lipid-derived eicosanoids and prostaglandins were reduced after VT. Conclusions: The denervation-induced suppression of gastric tumorigenesis was associated with the inhibition of WNT/ß-catenin signaling-related glutamine metabolism but not the Warburg effect. We suggest that glutamine and choline phospholipid metabolisms can be used for metabolism-based tumor detection with MRS and/or positron emission tomography (PET) for gastric cancer diagnosis and that blocking these metabolic functions can be a therapeutic approach for gastric cancer treatment. Citation Format: Gøran Andersen, Riyas Vettukattil, Yoku Hayakawa, Jon Erik Grønbech, Timothy C. Wang, Duan Chen, Chun Mei Zhao. Inhibition of WNT/ß-catenin signaling-related glutamine metabolism but not the Warburg effect in denervation-induced suppression of gastric tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2015-1167

which are better than conventional tumor markers.Also additional analysis showed good separation between early stage of gastric cancer (stage Ia and stage I) and normal group (Q 2 of 62.1% and 66.0%, respectively).The S-TOCSY plot showed multiple identified markers for the separation.The analysis revealed elevated level of glucose, citrate, lactate, alanine, hippurate, phenylalanine, dimethylamine, 3-aminoisobutyrate, 3-indoxylsulfate, while downregulation of glycerol and oxalacetate in gastric cancer patients regardless of stage.Conclusion Our findings suggest that the NMR-based metabolomics approach showed an effective and non-invasive way to discriminate the patients not only with all stages but also with early stage of gastric cancer from healthy person.The established prediction model had high quality of sensitivity and specificity and it can be a novel screening tool for early gastric cancer.

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.

Background/aim: Innervation plays an important role in the regulation of epithelial homeostasis as well as tumorigenesis. Botulinum neurotoxin type A (Botox) is known to block both afferent and efferent nerve fibers by binding to SNARE proteins. The aim of this trial was to evaluate the effectiveness and underlying mechanism of local injection of Botox in the treatment of gastric cancer. Methods: 268 genetically-manipulated INS-GAS male and female mice with spontaneous gastric cancer were subjected to Botox injection or subdiaphragmatic unilateral vagotomy (UVT), with or without chemotherapy (5-FU, oxaliplatin, or 5-FU + oxaliplatin). Botox was injected subserosally along the greater curvature on the anterior side of the stomach and the injection was repeated every 2nd month. Chemotherapy was given by intraperitoneal injection once per week for 4 weeks in a two-cycle course with a 1-week rest. Clinical endpoints included tumor size, histopathological score, and survival. Gastric organoid cultures with or without Botox, neurons, or a muscarinic receptor agonist or antagonist were used. Results: Botox treatment was given to INS-GAS mice at 6 months of age when gastric cancer started to develop. Six months later, tumor size, cell proliferation rate, scores of inflammation, epithelial defects, atrophy, hyperplasia, dysplasia and metaplasia were markedly reduced in comparison with the uninjected posterior area of the stomach. When Botox treatment was given at 14 months of age in combination with 5-FU+oxaliplatin, tumor size was significantly reduced as early as 2 months after starting treatment, particularly in the area of Botox injection or in the surgically denervated stomach following UVT. The combination of either Botox or UVT with chemotherapy led to a significant increase in survival compared to chemotherapy alone. In vitro studies showed that neurons stimulated gastric organoid growth when compared to gastric organoids cultured in the absence of neurons, and that the addition of either Botox or scopolamine inhibited this stimulatory effect, whereas pilocarpine increased organoid growth. Furthermore, pilocarpine upregulated expression of stem cell markers and Wnt target genes, such as Lgr5, CD44, and Sox9, and both pilocarpine and neurons could substitute for Wnt3a in gastric organoid cultures that were otherwise strictly dependent on the addition of Wnt ligands. Conclusions: This preclinical trial demonstrates the efficacy of local Botox injection with or without chemotherapy in the treatment of gastric cancer. The therapeutic effect is likely mediated by acetylcholinemediated Wnt signaling in gastric stem cells. We may further suggest that greater consideration should be given to novel denervation approaches in combination with other therapies for gastric cancer and likely other solid malignancies.