David T. Asuzu

Loss of Kitlow progenitors, reduced stem cell factor and high oxidative stress underlie gastric dysfunction in progeric mice

Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing‐associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti‐ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing‐associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3‐dimensional reconstruction, flow cytometry, quantitative RT‐PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT‐PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho‐deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho‐deficient mice were marantic and had low insulin, insulin‐like growth factor‐I and membrane‐bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho‐deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging.

Background  Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging‐associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make‐up of the small intestinal and colonic neuromuscular apparatus.

FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer‐initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome‐activating protein and investigate its potential pro‐apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two‐hybrid screen and further studied by deletion mutants, glutathione‐S‐transferase pull‐down, co‐immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock‐down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), “fibroblast‐like cells” (FLCs) and ICC stem cells (ICC‐SCs) was investigated by Northern blotting, reverse transcription—polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC‐SCs stably transduced to re‐express FAM96A was studied by xeno‐ and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC‐SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC‐SCs. Re‐expression of FAM96A in GIST cells and transformed ICC‐SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro‐apoptotic tumor suppressor that is lost during GIST tumorigenesis.

Epigenetics and chromatin dynamics: a review and a paradigm for functional disorders.

Background  Motility and functional gastrointestinal disorders have high prevalence in the community, cause significant morbidity, and represent a major health care burden. Despite major advances in our understanding of the cellular and molecular basis of gastrointestinal neuromuscular functions, many of these diseases still defy mechanistic explanations. The biopsychosocial model underlying the current classification of functional gastrointestinal disorders recognizes and integrates the pathogenetic role of genetic, environmental, and psychosocial factors but has not been associated with specific molecular mechanisms.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes.

10 The Stem Cell-Targeting Drug Salinomycin Inhibits Proliferation of Normal, Benign Hyperplastic, and Transformed Interstitial Cells of Cajal and Their Stem Cells (ICC-SC) by Increasing the Expression of Cell Cycle Restriction Checkpoint Regulators

the index colonoscopy to the first of the following: 10 years follow-up; performance of another colonoscopy without CRC; termination of membership; diagnosis of CRC; or January 31, 2011. CRCs occurring within 6 months after the index colonoscopy were excluded. Analyses used multi-level Cox proportional hazards models with clustering on physician. Potential predictors of subsequent CRC risk included physician ADR, exam type, and patient sex, age, race/ethnicity, family history of CRC, and Charlson comorbidity score. Hazard ratios (HR) for subsequent CRC risk were calculated separately by patient sex and both sexes combined, and by distal and proximal CRC location (both sexes combined). Results: We identified 316,334 colonoscopy exams and 716 subsequent post-colonoscopy CRC cases for the analyses. Patients (both sexes combined) whose colonoscopies were performed by endoscopists with lower ADRs were more likely to be diagnosed with subsequent CRC than patients whose endoscopists had a higher ADR. The adjusted HRs (and CIs) for ADR quartiles of ,20.3%, 20.3%-25.2%, and 25.3%-32.0%, as compared to ≥32.1%, were 1.74 (1.36, 2.24), 1.52 (1.14, 2.04), and 1.31 (1.00, 1.73), respectively (Table). The relationships between ADR and subsequent CRC risk by proximal and distal location were similar (Table). Finally, the linear relationship between ADR and CRC risk did not vary by patient sex. Conclusions: Physician ADR is an independent predictor of subsequent CRC risk following a negative colonoscopy, the relationship does not vary by patient sex, and the relationships were similar for proximal and distal cancers. The use of ADR as a quality measure of colonoscopy’s ability to prevent subsequent cancers appears warranted; cancer risk increases linearly with decreasing physician ADR and there was no clear threshold above which there was no further improvement.

8 KitLow Interstitial Cell of Cajal Stem Cells are a Cell of Origin of Gastrointestinal Stromal Tumors

BACKGROUND: We previously showed that the mast cell stabilizer ketotifen reduces IBS symptoms and increases the threshold of discomfort in hypersensitive IBS-patients. However, as ketotifen also has Histamine 1-receptor antagonistic properties and is known to penetrate the blood-brain barrier, its beneficial effect observed in this trial could also result from H1 receptor blockade, either at the peripheral or central level. AIM: To establish whether fexofenadine, a peripherally restricted 2nd generation Histamine 1-receptor antagonist, can mimic the effect of ketotifen and A) reverse stress-induced, mast cell dependent visceral hypersensitivity in a rat model of maternal separation (MS), and B) modulate gastrointestinal transit in MS and non-handled (NH) rats. METHODS: Adult MS and NH rats (n=9 in all groups) were subjected to acute stress (1 hr water avoidance,WA). The visceromotor response (VMR) to colorectal distension (1, 11⁄2, 2ml) was established preand 24 hours post-WA and expressed as area-under-curve (AUC, volume-vs-response, significant difference when P<0.05: Wilcoxon). Rats were then treated with fexofenadine (1,8 mg/kg or 18 mg/kg) or vehicle and re-evaluated 48 hrs post-WA. Pre-WA transit was measured in NH (n=4) and MS (n=5) rats treated with fexofenadine or vehicle by evaluating the transit (geometric centre or GC) of orally administered fluorescein-labeled dextran (MW=70.000) (1 is stomach, 2 most proximal small bowel, 11 most distal, 12 cecum, 13-15 colon). RESULTS: WA induced an increased VMR to distension in MS rats (pre-WA vs post-WA, 64±4 vs 98±6 (mean±SEM), P=0.012) which was reversed by 1.8 mg/kg fexofenadine (post-WA vs post-fexofenadine, 98±6 vs 69±5, P=0.012). High concentration fexofenadine (18 mg/kg) showed similar results (pre-WA vs post-WA, 72±4 vs 97±6, P=0.012 and post-WA vs post fexofenadine 97±6 vs 60±6, P=0.008). NH rats remained normo-sensitive under all conditions. Baseline pre-WA transit times of NH and MS were equal (10±0.2 vs 9.6±0.4). Administration of high-dose fexofenadine decreased transit time in NH (vehicle vs fexofenadine, 10±0.2 vs 6.4±1.1, P= 0.028) and MS-rats (9.6±0.4 vs 6.9±0.2, P=0.008). CONCLUSION: The peripheral H1 receptor antagonist fexofenadine effectively reverses stress-induced visceral hypersensitivity and decreases intestinal transit. As low dose fexofenadine does not cross the blood brain barrier, these data justify future IBS patient trials with 2nd generationH1-receptor antagonists.