Bence Sipos

A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.

We performed a genome-wide association study of 19,779 nonsynonymous SNPs in 735 individuals with Crohn disease and 368 controls. A total of 7,159 of these SNPs were informative. We followed up on all 72 SNPs with P ≤ 0.01 with an allele-based disease association test in 380 independent Crohn disease trios, 498 Crohn disease singleton cases and 1,032 controls. Disease association of rs2241880 in the autophagy-related 16-like 1 gene (ATG16L1) was replicated in these samples (P = 4.0 × 10−8) and confirmed in a UK case-control sample (P = 0.0004). By haplotype and regression analysis, we found that marker rs2241880, a coding SNP (T300A), carries virtually all the disease risk exerted by the ATG16L1 locus. The ATG16L1 gene encodes a protein in the autophagosome pathway that processes intracellular bacteria. We found a statistically significant interaction with respect to Crohn disease risk between rs2241880 and the established CARD15 susceptibility variants (P = 0.039). Together with the lack of association between rs2241880 and ulcerative colitis (P > 0.4), these data suggest that the underlying biological process may be specific to Crohn disease.

MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. We used 377 feature microRNA (miRNA) arrays to investigate miRNA expression in normal pancreas, chronic pancreatitis, and PDAC tissues as well as PDAC-derived cell lines. A pancreatic miRNome was established comparing the data from normal pancreas with a reference set of 33 human tissues. The expression of miR-216 and -217 and lack of expression of miR-133a were identified as characteristic of pancreas tissue. Unsupervised clustering showed that the three pancreatic tissues types can be classified according to their respective miRNA expression profiles. We identified 26 miRNAs most prominently misregulated in PDAC and a relative quantitative reverse transcriptase-polymerase chain reaction index using only miR-217 and -196a was found to discriminate normal pancreas, chronic pancreatitis and cancerous tissues, establishing a potential utility for miRNAs in diagnostic procedures. Lastly, comparing differentially expressed genes from PDAC with predicted miRNA target genes for the top 26 miRNAs, we identified potential novel links between aberrant miRNA expression and known target genes relevant to PDAC biology. Our data provides novel insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development and offers new candidate targets to be exploited both for diagnostic and therapeutic strategies.

Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer

Physiological levels of Kras(G12D) are sufficient to induce pancreatic intraepithelial neoplasias (PanINs); the mechanisms that drive PanIN progression are unknown. Here, we establish that, in addition to oncogenic Kras(G12D), IL-6 transsignaling-dependent activation of Stat3/Socs3 is required to promote PanIN progression and pancreatic ductal adenocarcinoma (PDAC). Myeloid compartment induces Stat3 activation by secreting IL-6; consequently, IL-6 transsignaling activates Stat3 in the pancreas. Using genetic tools, we show that inactivation of IL-6 transsignaling or Stat3 inhibits PanIN progression and reduces the development of PDAC. Aberrant activation of Stat3 through homozygous deletion of Socs3 in the pancreas accelerates PanIN progression and PDAC development. Our data describe the involvement of IL-6 transsignaling/Stat3/Socs3 in PanIN progression and PDAC development.

Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4.

Abstract. The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.

A comprehensive characterization of pancreatic ductal carcinoma cell lines: towards the establishment of an in vitro research platform

There are a large number of stable pancreatic ductal carcinoma cell lines that are used by researchers worldwide. Detailed data about their differentiation status and growth features are, however, often lacking. We therefore attempted to classify commonly used pancreatic carcinoma cell lines according to defined cell biological criteria. Twelve pancreatic ductal adenocarcinoma cell lines were cultured as monolayers and spheroids and graded according to their ultrastructural features. The grading system was based on the integrity of membrane structures and on the presence of mucin granules, cell organelles, nuclear and cellular polymorphism, cell polarity, and lumen formation. On the basis of the resulting scores the cell lines were classified as well, moderately, or poorly differentiated. In addition, immunocytochemistry was performed for the markers cytokeratin 7, 8, 18, 19, carcinoembryonic antigen, MUC1 MUC2, MUC5, and MUC6. The population doubling time of monolayer cultures, determined by a tetrazolium salt based proliferation assay was correlated with the ultrastructural grade. The grading of the ultrastructural features of the monolayers, and particularly of the spheroids, revealed that Capan-1 and Capan-2 cells were well differentiated; Colo357, HPAF-2, Aspc-1, A818-4, BxPc3, and Panc89 cells were moderately differentiated and PancTu-I, Panc1, Pt45P1, and MiaPaCa-2 cells poorly differentiated. Membrane-bound MUC1 staining was a characteristic of well differentiated cell lines. The population doubling time of the monolayer cultures was related to the differentiation grade. No relationship was found between the p53, K-ras, DPC4/Smad4, or p16INK4a mutation status and the grade of differentiation. We conclude that the proposed ultrastructural grading system combined with the proliferative activity provides a basis for further comparative studies of pancreatic ductal adenocarcinoma cell lines.

Foxp3 Expression in Pancreatic Carcinoma Cells as a Novel Mechanism of Immune Evasion in Cancer

The forkhead transcription factor Foxp3 is highly expressed in CD4+CD25+ regulatory T cells (Treg) and was recently identified as a key player in mediating their inhibitory functions. Here, we describe for the first time the expression and function of Foxp3 in pancreatic ductal adenocarcinoma cells and tumors. Foxp3 expression was induced by transforming growth factor-beta2 (TGF-beta2), but not TGF-beta1 stimulation in these cells, and was partially suppressed following antibody-mediated neutralization of TGF-beta2. The TGF-beta2 effect could be mimicked by ectopic expression of a constitutively active TGF-beta type I receptor/ALK5 mutant. Down-regulation of Foxp3 with small interfering RNA (siRNA) in pancreatic carcinoma cells resulted in the up-regulation of interleukin 6 (IL-6) and IL-8 expression, providing evidence for a negative transcriptional activity of Foxp3 also in these epithelial cells. Coculture of Foxp3-expressing tumor cells with naive T cells completely inhibited T-cell proliferation, but not activation, and this antiproliferative effect was partially abrogated following specific inhibition of Foxp3 expression. These findings indicate that pancreatic carcinoma cells share growth-suppressive effects with Treg and suggest that mimicking Treg function may represent a new mechanism of immune evasion in pancreatic cancer.

Tumor Stroma Interactions Induce Chemoresistance in Pancreatic Ductal Carcinoma Cells Involving Increased Secretion and Paracrine Effects of Nitric Oxide and Interleukin-1β

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1β in an autocrine and nuclear factor-κB-dependent fashion. Because pancreatic ductal carcinoma contains many mesenchymal stromal cells, we further investigated how tumor–stroma interactions contribute to chemoresistance by using a transwell coculture model, including murine pancreatic fibroblasts and the chemosensitive human pancreatic carcinoma cell lines T3M4 and PT45-P1. If cultured with fibroblast-conditioned medium or kept in coculture with fibroblasts, both cell lines became much less sensitive toward treatment with etoposide than cells cultured under standard conditions. Furthermore, the secretion of IL-1β in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1β-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1β secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1β-inducing factor. Although both tumor cells secreted only little NO, which was in line with undetectable inducible nitric oxide synthase (iNOS) expression, fibroblasts exhibited significant iNOS expression and NO secretion that could be further induced by the tumor cells. Incubation of T3M4 and PT45-P1 cells with the NO donor S-Nitroso-N-acetyl-D,l-penicillamine up-regulated IL-1β secretion and conferred resistance toward etoposide-induced apoptosis. Conversely, the resistance-inducing effect of the fibroblasts was significantly abolished, when the specific iNOS inhibitor aminoguanidine was added during coculture. Immunohistochemistry on tissue sections from human pancreatic ductal carcinoma also revealed iNOS expression in stromal cells and IL-1β expression in tumor cells, thus supporting the in vitro findings. These data clearly demonstrate that fibroblasts contribute to the development of chemoresistance in pancreatic carcinoma cells via increased secretion of NO, which in turn leads to an elevated release of IL-1β by the tumor cells. These findings substantiate the implication of tumor–stromal interactions in the chemoresistance of pancreatic carcinoma.

Obesity accelerates epigenetic aging of human liver

Significance Because obese people are at an increased risk of many age-related diseases, it is a plausible hypothesis that obesity increases the biological age of some tissues and cell types. However, it has been difficult to detect such an accelerated aging effect because it is unclear how to measure tissue age. Here we use a recently developed biomarker of aging (known as “epigenetic clock”) to study the relationship between epigenetic age and obesity in several human tissues. We report an unexpectedly strong correlation between high body mass index and the epigenetic age of liver tissue. This finding may explain why obese people suffer from the early onset of many age-related pathologies, including liver cancer. Because of the dearth of biomarkers of aging, it has been difficult to test the hypothesis that obesity increases tissue age. Here we use a novel epigenetic biomarker of aging (referred to as an “epigenetic clock”) to study the relationship between high body mass index (BMI) and the DNA methylation ages of human blood, liver, muscle, and adipose tissue. A significant correlation between BMI and epigenetic age acceleration could only be observed for liver (r = 0.42, P = 6.8 × 10−4 in dataset 1 and r = 0.42, P = 1.2 × 10−4 in dataset 2). On average, epigenetic age increased by 3.3 y for each 10 BMI units. The detected age acceleration in liver is not associated with the Nonalcoholic Fatty Liver Disease Activity Score or any of its component traits after adjustment for BMI. The 279 genes that are underexpressed in older liver samples are highly enriched (1.2 × 10−9) with nuclear mitochondrial genes that play a role in oxidative phosphorylation and electron transport. The epigenetic age acceleration, which is not reversible in the short term after rapid weight loss induced by bariatric surgery, may play a role in liver-related comorbidities of obesity, such as insulin resistance and liver cancer.

TRAIL promotes metastasis of human pancreatic ductal adenocarcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.

DNA Methylation Analysis in Nonalcoholic Fatty Liver Disease Suggests Distinct Disease-Specific and Remodeling Signatures after Bariatric Surgery

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Liver samples from morbidly obese patients (n = 45) with all stages of NAFLD and controls (n = 18) were analyzed by array-based DNA methylation and mRNA expression profiling. NAFLD-specific expression and methylation differences were seen for nine genes coding for key enzymes in intermediate metabolism (including PC, ACLY, and PLCG1) and insulin/insulin-like signaling (including IGF1, IGFBP2, and PRKCE) and replicated by bisulfite pyrosequening (independent n = 39). Transcription factor binding sites at NAFLD-specific CpG sites were >1,000-fold enriched for ZNF274, PGC1A, and SREBP2. Intraindividual comparison of liver biopsies before and after bariatric surgery showed NAFLD-associated methylation changes to be partially reversible. Postbariatric and NAFLD-specific methylation signatures were clearly distinct both in gene ontology and transcription factor binding site analyses, with >400-fold enrichment of NRF1, HSF1, and ESRRA sites. Our findings provide an example of treatment-induced epigenetic organ remodeling in humans.