MicroRNA Profiling Implies New Markers of Gemcitabine Chemoresistance in Mutant p53 Pancreatic Ductal Adenocarcinoma

Background: No 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. Methods: Gemcitabine-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. Results: Both 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. Conclusion: Consistent 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

SERPINB5 and AKAP12 -- Expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>Early 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.</p> <p>Methods</p> <p>Metastatic 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.</p> <p>Results</p> <p>In 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 <it>AKAP12 </it>mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher <it>SERPINB5 </it>mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased <it>SERPINB5 </it>methylation was associated with loss of mRNA and protein expression (P < 0.05). <it>SERPINB5 </it>methylation was also directly correlated to decreased metastasis scores (P < 0.05).</p> <p>Conclusions</p> <p><it>AKAP12 </it>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 <it>SERPINB5 </it>mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, <it>SERPINB5 </it>methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.</p

Postoperative pancreatic fistula affects recurrence-free survival of pancreatic cancer patients.

PurposePostoperative pancreatic fistula (POPF) with reported incidence rates up to 45% contributes substantially to overall morbidity. In this study, we conducted a retrospective evaluation of POPF along with its potential perioperative clinical risk factors and its effect on tumor recurrence.MethodsClinical data on patients who had received pancreatoduodenectomy (PD), distal pancreatectomy (DP), or duodenum-preserving pancreatic head resection (DPPHR) were prospectively collected between 2007 and 2016. A Picrosirius red staining score was developed to enable morphological classification of the resection margin of the pancreatic stump. The primary end point was the development of major complications. The secondary end points were overall and recurrence-free survival.Results340 patients underwent pancreatic resection including 222 (65.3%) PD, 87 (25.6%) DP, and 31 (9.1%) DPPHR. Postoperative major complications were observed in 74 patients (21.8%). In multivariable logistic regression analysis, POPF correlated with body mass index (BMI) (p = 0.025), prolonged stay in hospital (pConclusionBesides the known clinicopathological risk factors BMI and amylase in the drain fluid, the incidence of POPF correlates with high Picrosirius red staining score in the resection margins of the pancreatic stumps of curatively resected pancreatic ductal adenocarcinoma (PDAC). Furthermore, clinically relevant POPF seems to be a prognostic factor for tumor recurrence in PDAC

Clinical Impact of Epithelial-to-Mesenchymal Transition Regulating MicroRNAs in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma entities worldwide with early and rapid dissemination. Recently, we discussed the role of microRNAs as epigenetic regulators of Epithelial-to-Mesenchymal Transition (EMT) in PDAC. In this study, we investigated their value as diagnostic and prognostic markers in tissue and blood samples of 185 patients including PDAC, non-malignant pancreatic disorders, and age-matched healthy controls. Expression of the microRNA-200-family (microRNAs -141, -200a, -200b, -200c, -429) and microRNA-148a was significantly downregulated in tissue of PDAC Union internationale contre le cancer (UICC) Stage II. Correspondingly, stromal PDAC tissue showed strong expression of Fibronectin, Vimentin, and ZEB-1 (Zinc finger E-box-binding homeobox) versus low expression of E-cadherin. Transient transfection of microRNA-200b and microRNA-200c mimics resulted in the downregulation of their key target ZEB-1. Inversely, blood serum analyses of patients with PDAC UICC Stages II, III, and IV showed a significant over-expression of microRNA-200-family members, microRNA-148a, microRNA-10b, and microRNA-34a. Correspondingly, Enzyme-linked Immunosorbent Assay (ELISA) analyses revealed a significant over-expression of soluble E-cadherin in serum samples of PDAC patients versus healthy controls. The best diagnostic accuracy to distinguish between PDAC and non-PDAC in this patient collective could be achieved in tissue by microRNA-148a with an area under the receiver-operating-characteristic (ROC) curve (AUC) of 0.885 and in blood serum by a panel of microRNA-141, -200b, -200c, and CA.19-9 with an AUC of 0.890. Both diagnostic tools outreach the diagnostic performance of the currently most common diagnostic biomarker CA.19-9 (AUC of 0.834). Kaplan Meier survival analysis of this patient collective revealed an improved overall survival in PDAC patients with high expression of tissue-related microRNA-34a, -141, -200b, -200c, and -429. In conclusion, EMT-regulating microRNAs have great potential as liquid and solid biopsy markers in PDAC patients. Their prognostic and therapeutic benefits remain important tasks for future studies