Frank Ulrich Weiss

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 × 10−8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Retinoic Acid Receptor Antagonists Inhibit miR-10a Expression and Block Metastatic Behavior of Pancreatic Cancer

BACKGROUND & AIMS The infiltrating ductal adenocarcinoma of the pancreas is among the most lethal of all solid malignancies, largely owing to a high frequency of early metastasis. We identified microRNA-10a (miR-10a) as an important mediator of metastasis formation in pancreatic tumor cells and investigated the upstream and downstream regulatory mechanisms of miR-10a. METHODS Northern blot analysis revealed increased expression levels of miR-10a in metastatic pancreatic adenocarcinoma. The role of miR-10a was analyzed by Morpholino and short interfering RNA transfection of pancreatic carcinoma cell lines and resected specimens of human pancreatic carcinoma. Metastatic behavior of primary pancreatic tumors and cancer cell lines was tested in xenotransplantation experiments in zebrafish embryos. RESULTS We show that miR-10a expression promotes metastatic behavior of pancreatic tumor cells and that repression of miR-10a is sufficient to inhibit invasion and metastasis formation. We further show that miR-10a is a retinoid acid target and that retinoic acid receptor antagonists effectively repress miR-10a expression and completely block metastasis. This antimetastatic activity can be prevented by specific knockdown of HOX genes, HOXB1 and HOXB3. Interestingly, suppression of HOXB1 and HOXB3 in pancreatic cancer cells is sufficient to promote metastasis formation. CONCLUSIONS These findings suggest that miR-10a is a key mediator of metastatic behavior in pancreatic cancer, which regulates metastasis via suppression of HOXB1 and HOXB3. Inhibition of miR-10a expression (with retinoic acid receptor antagonists) or function (with specific inhibitors) is a promising starting point for antimetastatic therapies.

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model.

BackgroundAberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour.MethodsWe fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish.ResultsIn the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature.ConclusionOur results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

Recruitment of histone deacetylases HDAC1 and HDAC2 by the transcriptional repressor ZEB1 downregulates E-cadherin expression in pancreatic cancer

Objective Pancreatic cancer is characterised by invasive tumour spread and early metastasis formation. During epithelial–mesenchymal transition, loss of the cell adhesion molecule E-cadherin is frequent and can be caused by genetic or epigenetic modifications, recruitment of transcriptional activators/repressors or post-translational modifications. A study was undertaken to investigate how E-cadherin expression in human pancreatic adenocarcinoma and pancreatic cancer cell lines is regulated. Methods In 25 human pancreatic cancer resection specimens, the coding region of the E-cadherin gene (CDH1) was sequenced for somatic mutations. The tumour samples and 11 established human pancreatic cancer cell lines were analysed by immunohistochemistry, western blot analysis, chromatin immunoprecipitation and methylation-specific PCR. The role of specific histone deacetylase inhibitors (HDACi) on pancreatic tumour cell migration and proliferation was studied in vitro. Results Neither somatic mutations nor CDH1 promoter hypermethylation were found to be responsible for downregulation of E-cadherin in pancreatic cancer. In the transcriptionally active CDH1 promoter, acetylation of histones H3 and H4 was detected whereas HDAC1 and HDAC2 were found attached only to a silent promoter. Expression of ZEB1, a transcription factor known to recruit HDACs, was seen in E-cadherin-deficient cell lines in which ZEB1/HDAC complexes were found attached to the CDH1 promoter. Moreover, knockdown of ZEB1 prevented HDAC from binding to the CDH1 promoter, resulting in histone acetylation and expression of E-cadherin. HDACi treatment attenuated tumour cell migration and proliferation. Conclusions These findings imply an important role for histone deacetylation in the downregulation of E-cadherin in human pancreatic cancer. Recruitment of HDACs to the CDH1 promoter is regulated by the transcription factor ZEB1, and inhibition of HDACs may be a promising antitumour therapy for pancreatic cancer.

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

Improved non‐invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non‐coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non‐invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR‐1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR‐1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2‐1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2‐1f) and not from miR‐1246. In addition, we observed a remarkable stability of RNU2‐1f in serum and provide experimental evidence that hsa‐miR‐1246 is likely a pseudo microRNA. In a next step, RNU2‐1f was measured by qRT‐PCR and normalized to cel‐54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

Hereditary pancreatitis caused by mutation induced misfolding of human cationic trypsinogen - a novel disease mechanism

We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to ∼20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease‐sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers immunoglobulin‐binding protein (BiP) and the spliced form of the X‐box binding protein‐1 (XBP1s) were elevated in cells expressing mutant p.R116C. The results indicate that mutation‐induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis. Hum Mutat 0, 1–8, 2009.

Animal models for investigating chronic pancreatitis

Chronic pancreatitis is defined as a continuous or recurrent inflammatory disease of the pancreas characterized by progressive and irreversible morphological changes. It typically causes pain and permanent impairment of pancreatic function. In chronic pancreatitis areas of focal necrosis are followed by perilobular and intralobular fibrosis of the parenchyma, by stone formation in the pancreatic duct, calcifications in the parenchyma as well as the formation of pseudocysts. Late in the course of the disease a progressive loss of endocrine and exocrine function occurs. Despite advances in understanding the pathogenesis no causal treatment for chronic pancreatitis is presently available. Thus, there is a need for well characterized animal models for further investigations that allow translation to the human situation. This review summarizes existing experimental models and distinguishes them according to the type of pathological stimulus used for induction of pancreatitis. There is a special focus on pancreatic duct ligation, repetitive overstimulation with caerulein and chronic alcohol feeding. Secondly, attention is drawn to genetic models that have recently been generated and which mimic features of chronic pancreatitis in man. Each technique will be supplemented with data on the pathophysiological background of the model and their limitations will be discussed.

A recombined allele of the lipase gene CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis.

Carboxyl ester lipase is a digestive pancreatic enzyme encoded by the CEL gene. Mutations in CEL cause maturity-onset diabetes of the young as well as pancreatic exocrine dysfunction. Here we describe a hybrid allele (CEL-HYB) originating from a crossover between CEL and its neighboring pseudogene, CELP. In a discovery series of familial chronic pancreatitis cases, we observed CEL-HYB in 14.1% (10/71) of cases compared to 1.0% (5/478) of controls (odds ratio (OR) = 15.5; 95% confidence interval (CI) = 5.1–46.9; P = 1.3 × 10−6 by two-tailed Fishers exact test). In three replication studies of nonalcoholic chronic pancreatitis, we identified CEL-HYB in a total of 3.7% (42/1,122) cases and 0.7% (30/4,152) controls (OR = 5.2; 95% CI = 3.2–8.5; P = 1.2 × 10−11; formal meta-analysis). The allele was also enriched in alcoholic chronic pancreatitis. Expression of CEL-HYB in cellular models showed reduced lipolytic activity, impaired secretion, prominent intracellular accumulation and induced autophagy. These findings implicate a new pathway distinct from the protease-antiprotease system of pancreatic acinar cells in chronic pancreatitis.

Drug Efflux Transporter Multidrug Resistance-Associated Protein 5 Affects Sensitivity of Pancreatic Cancer Cell Lines to the Nucleoside Anticancer Drug 5-Fluorouracil

Pancreatic adenocarcinoma is one of the malignancies that is highly resistant to therapy and among the leading causes of cancer-related death. Several factors may influence pancreatic cancer resistance, and expression of ATP-binding cassette transport proteins is one of the major mechanisms of drug resistance. Members of this familys C-branch, also referred to as multidrug resistance-associated proteins (MRPs), might be of particular interest because they are able to efflux nucleoside analogs used in the treatment of pancreatic cancer. Expression of MRP1, MRP3, MRP4, and MRP5 in human pancreas and pancreatic carcinoma has been reported. However, contributions of MRPs to chemoresistance of pancreatic cancer are not fully understood. MRP5 mRNA expression in pancreatic adenocarcinoma cell lines correlated significantly with cellular sensitivity to 5-fluorouracil (5-FU) (r = 0.738, p < 0.05). Long-term treatment with 5-FU increased expression of MRP5 by 2.4-fold and was associated with significant drug resistance [IC50 values for control and 5-fluorouracil (5-FU)-resistant Patu-T cell lines were 11.3 ± 5.3 and 33.2 ± 6.9 μM, respectively (p < 0.05)]. Consequently, overexpression of MRP5 in Colo-357 cells resulted in significantly reduced accumulation of 5-FU related radioactivity and 5-FU cytotoxicity. Knockdown of MRP5 significantly increased cellular cytotoxicity of 5-FU to Patu-02 cells and enhanced accumulation of radioactivity related to 5-FU and its metabolites. Our results suggest that MRP5 is expressed and functionally active and contributes to variable sensitivities of pancreatic adenocarcinoma cell lines to 5-FU. Further investigations using models that resemble human pancreas tumors are necessary to prove a causative relation between expression and activity of MRP5 and tumor resistance to 5-FU.

Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study

Objective Serum lipase activities above the threefold upper reference limit indicate acute pancreatitis. We investigated whether high lipase activity—within the reference range and in the absence of pancreatitis—are associated with genetic single nucleotide polymorphisms (SNP), and whether these identified SNPs are also associated with clinical pancreatitis. Methods Genome-wide association studies (GWAS) on phenotypes ‘serum lipase activity’ and ‘high serum lipase activity’ were conducted including 3966 German volunteers from the population-based Study-of-Health-in-Pomerania (SHIP). Lead SNPs associated on a genome-wide significance level were replicated in two cohorts, 1444 blood donors and 1042 pancreatitis patients. Results Initial discovery GWAS detected SNPs within or near genes encoding the ABO blood group specifying transferases A/B (ABO), Fucosyltransferase-2 (FUT2), and Chymotrypsinogen-B2 (CTRB2), to be significantly associated with lipase activity levels in asymptomatic subjects. Replication analyses in blood donors confirmed the association of FUT-2 non-secretor status (OR=1.49; p=0.012) and ABO blood-type-B (OR=2.48; p=7.29×10−8) with high lipase activity levels. In pancreatitis patients, significant associations were found for FUT-2 non-secretor status (OR=1.53; p=8.56×10−4) and ABO-B (OR=1.69, p=1.0×10−4) with chronic pancreatitis, but not with acute pancreatitis. Conversely, carriers of blood group O were less frequently affected by chronic pancreatitis (OR=0.62; p=1.22×10−05) and less likely to have high lipase activity levels (OR=0.59; p=8.14×10−05). Conclusions These are the first results indicating that ABO blood type-B as well as FUT2 non-secretor status are common population-wide risk factors for developing chronic pancreatitis. They also imply that, even within the reference range, elevated lipase activities may indicate subclinical pancreatic injury in asymptomatic subjects.