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susing β-actin as normalizer protein showed the presence of MRP1, MRP4, and MRP5 in allestablished pancreatic cell lines, whereas MRP3 and BCRP protein were detectable only insome of these cell lines. We further studied the expression of MRP isoforms in parentalCapan-1 cells as compared to Capan-1 cells with acquired chemoresistance to 5-fluorouracil(5-FU). Both on mRNA and protein level, the 5-FU-resistant Capan-1 cells showed anupregulated expression of MRP3, MRP4, and MRP5 judged by normalization to RPL13AmRNA using QPCR and by immunoblot with β-actin protein, respectively. Further studiesusing RNA interference methods will show whether and which of the expressed MRP isoformscontribute to the observed chemoresistant phenotype in these pancreatic carcinoma cells.Thus cultured pancreatic carcinoma cell lines may provide useful model systems to studyand overcome drug resistance in human pancreatic carcinoma.

M2019

Expression of COX-2 Confers Growth Advantage to Pancreatic Cancer CellsAihua Li, Sascha Hasan, Eliane Angst, Howard A. Reber, Oscar J. Hines, Guido Eibl

Background and Aim: There is substantial evidence that cyclooxygenase-2 (COX-2), therate-limiting enzyme for prostanoid production, significantly contributes to the phenotypeof human malignancies. Targeting the COX-2/prostanoid pathway is considered an intriguingapproach for therapy and prevention of several human cancers. We have previously demon-strated that selective COX-2 inhibitors attenuates the growth of human pancreatic cancer(PaCa) in a xenograft mouse model and delays the progression of PaCa precursor lesionsin a genetically engineered mouse model. However, the precise molecular signals that underliethe pro-tumorigenic properties of COX-2 in PaCa are still poorly described. The aim of thepresent study therefore was to characterize the phenotype of COX-2 expressing human PaCacells. Methods and Results: The undifferentiated human PaCa cell line MIA PaCa-2, whichlacks COX-2 expression, was stably transduced with a third generation lentiviral constructharboring the full length human COX-2 cDNA and GFP as a marker. Lentiviruses encodingonly for GFP were used as a control. Transduced MIA PaCa-2 cells were sorted to yield >95%pure cell populations (MP2-COX2 and MP2+COX2). Western blot and immunofluorescenceconfirmed successful and stable COX-2 expression. Adding the COX-2 substrate arachidonicacid (AA, 10 mM) to the cell cultures for 1-24 hours lead to a marked time-dependentincrease in PGE2 production in MP2+COX2 cells (13-fold, 9-fold, and 3-fold increase after1, 3, and 6 hours, respectively, compared to MP2-COX2). Anchorage-dependent growthassays (cell count, MTT) demonstrated an increase in cell growth in MP2+COX2 comparedto MP2-COX2 cells in the presence of AA (110% increase after 6 days). Using a HumanApoptosis PCR Array comprising of 84 different cell death-related genes, significant differ-ences (>2 fold increase or decrease in gene expression) were detected between MP2+COX2and MP2-COX2 cells in several genes, including members of the bcl-2 family, caspase family,and TNF receptor superfamily. Conclusions: We have established a model to delineate thecontributions of COX-2 to the malignant phenotype of human pancreatic cancer cells. Stableoverexpression of COX-2 in pancreatic cancer cells leads to an elevated production ofprostanoids and increased cell growth. In addition, expression of COX-2 was associatedwith a differential expression of several cell-death related genes. These findings will providethe basis for more mechanistic studies on the role of COX-2 in pancreatic cancer and mayhelp to develop novel therapeutic strategies in pancreatic cancers aiming at the COX-2/prostanoid pathway.

M2020

Histone Deacetylation Regulates Loss of E-Cadherin Expression and PromotesTumorigenicity in Pancreatic CancerAli Aghdassi, Matthias Sendler, Frank U. Weiss, Julia Mayerle, Helmut Friess, Markus W.Buechler, Claus-Dieter Heidecke, Markus M. Lerch

Introduction: Dysfunction of the E-cadherin/catenin adhesion complex is found in manyhuman cancers and is believed to influence tumor spreading and metastasis formation.Complete loss of E-cadherin expression may result from somatic mutations, loss of heteroz-ygosity and also from epigenetic mechanisms. Here we investigated in pancreatic cancerresection specimen and pancreatic tumor cell lines whether promoter hypermethylation orhistone modifications play a role in E-cadherin silencing and how they affect tumor cellproliferation and migration. Methods: E-cadherin expression in 25 pancreatic cancer resectionspecimen was analyzed by western blot and immunhistochemical analysis and correlatedwith patient survival. In tumor tissues and pancreatic carcinoma cell lines lacking E-cadherinexpression CDH1 promoter hypermethylation was analyzed by methylation specific PCR(MSP). Total histone deacetylase (HDAC) activity was measured by a fluorometric assay.Influence of HDAC inhibition by Trichostatin A, Sodium butyrate and MS-275 on E-cadherinexpression as well as cell proliferation and migration were determined In Vitro. Results: Inour pancreatic tumor patients downregulation of E-cadherin expression correlated with poorsurvival. Hypermethylation of the E-cadherin promoter was only detectable in a singlepancreatic carcinoma cell line (MIA PaCa-2) but not in tumor tissues. E-cadherin could bere-expressed by inhibition of histone deacetylases in pancreatic carcinoma cells, whichcorrelated with decreased tumor proliferation and migration In Vitro. Total histone deacetylaseactivity was found to be increased in pancreatic cancer tissue compared to normal pancreasor chronic pancreatitis. Conclusions: E-cadherin is differently expressed in pancreatic cancerand its absence is associated with poorer patient survival. Promoter hypermethylation wasnot found to play a role in the repression of E-cadherin but histone modifications namelyhistone deacetylation appears to control E-cadherin expression in tumor formation. Histonedeacetylase activity also seems to correlate with decreased tumor cell proliferation andmigration and HDAC inhibition therefore may lead to decreased tumorigenicity. The potentialof histone deacetylase inhibitors for therapy of pancreatic carcinoma will have to be fur-ther investigated.

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M2021

Metformin Inhibits the Synergistic Effect of Insulin On Early and LateCellular Events Induced By G-Protein Coupled Receptor Agonists in HumanPancreatic Cancer CellsKrisztina Kisfalvi, Enrique Rozengurt

Background: Hyperinsulinaemia, a major characteristic of latent Type2 diabetes, obesityand metabolic syndrome, increases the risk of cancers, including pancreatic cancer. Recentlywe identified a novel crosstalk between insulin and G-protein-coupled receptor (GPCR)signaling pathways in human pancreatic cancer cells. By this crosstalk insulin strikinglyenhances GPCR signaling, including intracellular Ca2+ release, IP3 generation and cell prolif-eration. The potentiating effect of insulin on GPCR signaling occurs through an mTOR-dependent pathway. Metformin, the most widely used drug in treating Type2 diabetes,activates AMP kinase, which negatively regulates mTOR. Epidemiological studies show thatmetformin lowers the risk of cancers in Type2 diabetes. However, the mechanism by whichmetformin has beneficial effects on cancers remain unclear. We hypothesized that metformin,acting through AMPK-TSC2-mTOR pathway, reverses the enhancing effect of insulin onGPCR signaling in pancreatic cancer cells. Results: Human pancreatic cancer cells (Panc-1, MiaPaCa-2, BxPc-3) were pretreated with insulin (10ng/ml). 5-min exposure to insulinmarkedly enhanced the increase in intracellular [Ca2+] induced by GPCR agonists (neuroten-sin, bradykinin, angiotensin and vasopressin). Furthermore, insulin synergistically enhancedGPCR agonist-induced growth, measured by DNA synthesis, and cell numbers culturedin adherent (plain dishes) or non-adherent conditions (anchorage-independent growth inPolyHEMA-coated dishes). Metformin pretreatment (5mM, 1h) completely abrogated theinsulin-induced potentiation of [Ca2+]i increase but did not interfere with the effect of GPCRagonists alone. Low doses of metformin (0.1-0.5mM), which are in the range present inblood in patients treated with the drug, completely eliminated the synergistic effect ofinsulin in the insulin+GPCR agonists group on DNA synthesis, anchorage-dependent and-independent growth, but did not affect the growth-promoting effect of insulin or GPCRagonists alone. Higher doses of metformin (2.5-5mM), however, inhibited the growth-promoting effect of insulin in a dose-dependent manner. Selective AMPK inhibitor (com-pound C, 0.5-5µM) dose-dependently reversed the effects of metformin on [Ca2+]i releaseand DNA synthesis, indicating that metformin acts through AMPK activation. Conclusion:Metformin significantly inhibits the growth of pancreatic cancer cells. This effect could bepartially explained by the elimination of the synergistic crosstalk between the insulin andGPCR signaling pathways. Thus, metformin could be a potential candidate in novel strategiesfor prevention and treatment of human pancreatic cancer.

M2022

Integrin α6β4 Promotes Migration, Invasion Through Tiam1 Upregulation andSubsequent Rac ActivationKathleen L. O'Connor, Zobeida Cruz-Monserrate

Pancreatic adenocarcinoma is one of the most lethal human cancers due to an elevatedincidence of tumor cell invasion and metastasis. The general mechanisms and moleculesinvolved in these processes are not yet understood. Our study focuses on the pro-invasiveintegrin α6β4 which is highly upregulated in human pancreatic adenocarcinomas. Wehave previously shown that the α6β4 integrin is overexpressed in over 93% of pancreaticadenocarcinomas and hypothesize that this integrin contributes to the aggressive nature ofpancreatic cancers. To assess the importance of the integrin α6β4 expression in pancreaticcancer cell migration and invasion, cell lines were screened for the presence of integrinα6β4 by immunoblotting, and fluorescence activated cell sorting (FACS) and their abilityto migrate towards hepatocyte growth factor (HGF). We found that cell surface expressioncorrelated with the cells' ability to migrate and invade toward HGF, a known mitogenic andmotility factor of pancreatic carcinomas. When cells expressing high levels of integrin α6β4were treated with siRNA targeting the α6 or β4 integrin subunits, we observed a reductionin the ability of cells to migrate and invade towards HGF. Furthermore, the activity of thesmall GTPase Rac1, which was necessary for HGF-stimulated chemotaxis, decreased whenα6β4 integrin expression was reduced. We discover that the expression of the Rac-specificnucleotide exchange factor, Tiam1 was upregulated in cells overexpressing the integrin α6β4and required for the elevated Rac1 activity in these cells. We conclude that the integrinα6β4 promotes the migratory and invasive phenotype of pancreatic carcinoma cells throughthe Tiam1-Rac1 pathway in part through the upregulation of Tiam1.

M2023

Expression of Cathepsin E Reverses EMT in Pancreatic Cancer Cells In Vitroand Expression Correlates with Increased Patient Survival in HumanPancreatic AdenocarcinomaTobias Grote, Baoan Ji, Vijaya Ramachandran, Thiruvengadam Arumugam, HuaminWang, Craig D. Logsdon

Introduction: Based on mRNA expression profiling cathepsin E (CTSE), an aspartic protease,was found to be highly expressed in human pancreatic adenocarcinoma. The aim of thisstudy was to verify CTSE expression in human tissue and to investigate its functionalrelevance in pancreatic cancer. Methods: CTSE mRNA expression was quantified by QRT-PCR in normal pancreatic tissue, chronic pancreatitis and pancreatic adenocarcinoma (n=5). A tissue microarray with normal and cancer tissue (n=62) was analyzed for CTSE byimmunocytochemistry (IHC). The results were correlated with clinical data. Tissue from atransgenic K-Ras mouse model was stained for CTSE by IHC. CTSE protein expression inhuman pancreatic cancer cell lines (MOH, HPAC, BxPC3, MPanc96, Panc1, MiaPaca2 andHPDE as a control) was analyzed by western blot. MiaPaca2 cells were infected with a CTSE/GFP lentiviral construct or with GFP alone and effects on proliferation (MTS) and migration(Boydon chamber) were analyzed and E-cadherin and vimentin protein levels were deter-mined by western blot. Results: CTSE mRNA was highly expressed in pancreatic cancer(116-fold vs. normal and 22-fold vs. chronic pancreatitis) as determined by QRT-PCR. Atprotein level, 39/62 (63%) of the tumor tissues showed staining for CTSE, which correlated