Epigenetic alterations with upregulated CD40-targeting miR-224 and miR-486 are related to downregulated CD40 protein expression at cell surfaces in highly invasive and metastatic PDAC. Thus, miRNA-regulated CD40 expression seems to play an important role in progression of PDAC. These data suggest a diagnostic and therapeutic potential for CD40 and/or its targeting miRNAs in PDAC.

Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDACs) are well known. This study investigates genetic and epigenetic data together with tumor biology to find specific alterations responsible for metastasis formation. Using 16 human PDAC cell lines in a murine orthotopic PDAC model, local infiltration and metastatic spread were assessed by standardized dissemination scores. The cell lines were further classified into 3 hierarchical groups according to their metastatic potential. Their mRNA and microRNA (miRNA) expression was profiled via mRNA-microarray as well as Taqman Low Density Array, and validated by single quantitative RT-PCR and Western blotting. In the highly metastatic group, a significant induction of EP300 targeting miRNAs miR-194 (fold change: 26.88), miR-200b (fold change: 61.65), miR200c (fold change: 19.44) and miR-429 (fold change: 21.67) (p < 0.05) was detected. Corresponding to this, decreased expression of EP300 mRNA (p < 0.0001) and protein (p < 0.05) were detected in the highly metastatic PDAC cell lines with liver metastases compared to the nonmetastatic or marginally metastatic cell lines, while no correlation with local tumor growth was found. In conclusion, epigenetic alterations with upregulated EP300 targeting miRNAs miR-194, miR-200b, miR200c and miR-429 are related to reduced EP300 mRNA and protein in PDAC. These results demonstrate that miRNAs might be able to modulate the expression of metastasis-specific suppressor genes and metastatic behavior in PDAC, suggesting diagnostic and therapeutic opportunities for EP300 and its targeting miRNAs in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is among the most malignant tumors with a highly unfavorable prognosis. The 5-year survival rate of all patients is below 5%, and the median survival time after diagnosis is $6 months. Even after operation with curative intention, the 5-year survival rate in specialized centers is below 15%, increasing to only 25% when adjuvant chemotherapy is applied. 1,2 The cancer's aggressive nature, the lack of methods for early detection and the limited response to available treatments contribute to its high mortality rate. Pancreatic cancer is characterized by modifications in gene expression due to mutations, deletions and amplifications of genes critical for tumor development and progression. Although these alterations are purely genetic, epigenetic mechanisms such as shifts in DNA methylation patterns can also contribute to the induction and maintenance of pancreatic cancer. 3,4 Epigenetic mechanisms are modifiers of gene expression that are heritable but potentially reversible and do not involve changes in the DNA sequence. At the post-transcriptional level, epigenetic regulation can take place via microRNAs (miRNAs). miRNAs are small noncoding RNAs that are cleaved from 70-to 100-nucleotide hairpin pre-miRNA precursors in the cytoplasm by RNaseIII Dicer into their mature form of 18-23 nucleotides. 5 Single-stranded miRNAs bind messenger RNAs (mRNA) of potenti...

BackgroundNo reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma (PDAC). MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses chemoresistant PDAC for its specific miR expression pattern.MethodsGemcitabine-resistant variants of two mutant p53 human PDAC cell lines were established. Survival rates were analyzed by cytotoxicity and apoptosis assays. Expression of 1733 human miRs was investigated by microarray and validated by qRT-PCR. After in-silico analysis of specific target genes and proteins of dysregulated miRs, expression of MRP-1, Bcl-2, mutant p53, and CDK1 was quantified by Western blot.ResultsBoth established PDAC clones showed a significant resistance to gemcitabine (p<0.02) with low apoptosis rate (p<0.001) vs. parental cells. MiR-screening revealed significantly upregulated (miR-21, miR-99a, miR-100, miR-125b, miR-138, miR-210) and downregulated miRs (miR-31*, miR-330, miR-378) in chemoresistant PDAC (p<0.05). Bioinformatic analysis suggested involvement of these miRs in pathways controlling cell death and cycle. MRP-1 (p<0.02) and Bcl-2 (p<0.003) were significantly overexpressed in both resistant cell clones and mutant p53 (p = 0.023) in one clone.ConclusionConsistent miR expression profiles, in part regulated by mutant TP53 gene, were identified in gemcitabine-resistant PDAC with significant MRP-1 and Bcl-2 overexpression. These results provide a basis for further elucidation of chemoresistance mechanisms and therapeutic approaches to overcome chemoresistance in PDAC.

MiR-200 family members and their targets are significantly deregulated in HCC and liver cirrhosis. The miR-200 family is able to distinguish between cirrhotic and HCC tissue and could serve as an early marker for cirrhosis-associated HCC.

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains a highly chemoresistant tumor entity for which no reliable molecular targets exist to predict or influence the success of chemotherapy. Recently, we identified a panel of microRNAs associated with induced gemcitabine chemoresistance in human PDAC cell lines. This clinical study evaluates these microRNAs and associated molecular markers as prognostic markers of outcome in 98 PDAC patients Union Internationale Contre le Cancer (UICC) stage II undergoing curative surgery with adjuvant gemcitabine chemotherapy. The primary end points of this study are recurrence-free survival and overall survival.ResultsPoor response to chemotherapy was significantly correlated to overexpression of microRNA-21 (p = 0.029), microRNA-99a (p = 0.037), microRNA-100 (p = 0.028), and microRNA-210 (p = 0.021) in tissue samples of PDAC patients UICC stage II. Upregulation of these microRNAs was associated with a significantly shorter overall survival and recurrence-free survival (p < 0.05). Overexpression of phosphatase and tensin homolog (PTEN) (p = 0.039) and low expression of multidrug resistance (MDR)-1 (p = 0.043) and breast cancer resistance protein (BCRP)-1 (p = 0.038) were significantly correlated to improved response to adjuvant chemotherapy. Adjuvant gemcitabine treatment (p < 0.0001) and low tumor grading (p = 0.047) were correlated to better outcome. MicroRNA-100, microRNA-21, and its targets PTEN and MDR-1 were independent factors of survival in multivariate analysis.ConclusionsMultivariate survival analyses identified microRNA-21 and microRNA-100 as unfavorable prognostic factors in resected and adjuvant treated PDAC UICC stage II patients.

Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis, making tools for early diagnosis and new therapeutic modalities urgently needed. MicroRNAs (miRNAs), endogenous noncoding RNA molecules of approximately 22 nt, have gained attention as an epigenetic component involved in the development of many cancers, including PDAC. miRNA expression profiles of varying pancreatic tissues have identified a number of differentially expressed miRNAs and seem to be able to differentiate between three tissues of clinical importance: normal pancreas, chronic pancreatitis, and PDAC. This article gathers our current knowledge of differentially expressed miRNAs in pancreatic tissues with relevance to PDAC and presents potential diagnostic and therapeutic opportunities.

BackgroundEarly metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool.MethodsMetastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot.ResultsIn general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05).ConclusionsAKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

Our data demonstrate that liver resection in the presence of compensated liver cirrhosis is feasible but associated with a significantly impaired prognosis for overall and recurrence-free survival. The management of cirrhotic patients with compensated liver function and HCC therefore also requires the opportunity for transplantation.