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Background & Aims Tumor-suppressor proteins are inactivated by many different mechanisms, including nuclear exclusion by chromosome region maintenance (CRM)-1. Increased tumor levels of CRM-1 have been correlated with poor prognosis of patients with pancreatic cancer, making it a therapeutic target. Selective inhibitors of nuclear export (SINEs) bind to CRM-1 to irreversibly inhibit its ability to export proteins; we investigated a new class of SINEs in pancreatic cancer cells. Methods We studied the effects of SINE analogs in a panel of pancreatic cancer cell lines and nontransformed human pancreatic ductal epithelial cells using proliferation, apoptosis, immunoblot, co-immunoprecipitation, small inhibitor RNA, and fluorescence microscopy analyses. The effects of the SINEs also were investigated in mice with subcutaneous and orthotopic tumors. Results SINEs (KPT-185, KPT-127, KPT-205, and KPT-227) inhibited proliferation and promoted apoptosis of pancreatic cancer cells, but did not affect human pancreatic ductal epithelial cells. The nuclei of cells incubated with KPT-185 accumulated tumor-suppressor proteins (p27, FOXO, p73, and prostate apoptosis response-4 [PAR-4]) and inhibited interactions between CRM-1 and these proteins. Mutations in the region of CRM-1 that bind to SINEs (Cys-528), or small inhibitor RNA knockdown of PAR-4, prevented the ability of KPT-185 to block proliferation and induce apoptosis of pancreatic cancer cells. Oral administration of KPT-330 to mice reduced growth of subcutaneous and orthotopic xenograft tumors without major toxicity. Analysis of tumor remnants showed that KPT-330 disrupted the interaction between CRM-1 and PAR-4, activated PAR-4 signaling, and reduced proliferation of tumor cells. Conclusions We identified SINEs that inhibit CRM-1 and promote nuclear accumulation of tumor-suppressor proteins in pancreatic cancer cells. Oral administration of the drug to mice reduces growth of xenograft tumors.

Background & AimsRAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis.MethodsWe used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium.ResultsWe observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (Pcombined UC = 3.3 × 10−8, odds ratio = 1.43 [95% confidence interval: 1.26–1.63]) and rs4720672 (Pcombined UC = 4.7 × 10−6, odds ratio = 1.36 [95% confidence interval: 1.19–1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium–induced colitis.ConclusionsHuman studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

Background & Aims: Enteric infections have been implicated in the pathogenesis of both food intolerance and autoimmune diseases secondaryto the impairment of the intestinal barrier. On the basis of our recent discoveryof zonulin, a modulator of small-intestinal tight junctions, we asked whether microorganisms might induce zonulin secretion and increased small-intestinal permeability. Methods: Both ex vivo mammalian small intestines and intestinal cell monolayers were exposed to either pathogenic or nonpathogenic enterobacteria. Zonulin production and changes in paracellular permeabilitywere monitored in Ussing chambers and microsnapwells. Zonula occludens 1 protein redistribution after bacteria colonization was evaluated on cell monolayers. Results: Small intestines exposed to enteric bacteria secreted zonulin. This secretion was independent of either the species of the small intestines or the virulence of the microorganisms tested, occurred only on the luminal aspect of the bacteria-exposed smallintestinal mucosa, and was followed bya decrease in small-intestinal tissue resistance (transepithelial electrical resistance). The transepithelial electrical resistance decrement was secondaryto the zonulin-induced tight junction disassembly, as also shown by the disengagement of the protein zonula occludens 1 protein from the tight junctional complex. Conclusions: This zonulindriven opening of the paracellular pathwaymayrepresent a defensive mechanism, which flushes out microorganisms and contributes to the host response against bacterial colonization of the small intestine.

Background & Aims The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. Methods We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22 phox protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91 phox ; neutrophil cytosol factor 1 and encodes p47 phox protein; neutrophil cytosol factor 2 and encodes p67 phox protein; neutrophil cytosol factor 4 and encodes p40 phox protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2 NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. Results Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. Conclusions We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2 NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.