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Mazarico J.M.,Epithelial Carcinogenesis Group | Lobo V.J.S.-A.,Epithelial Carcinogenesis Group | Favicchio R.,Imperial College London | Greenhalf W.,NIHR Liverpool Pancreas Biomedical Research Unit | And 7 more authors.
Molecular Cancer Therapeutics | Year: 2016

Choline kinase a (CHKa) plays a crucial role in the regulation of membrane phospholipid synthesis and has oncogenic properties in vitro. We have analyzed the expression of CHKa in cell lines derived from pancreatic ductal adenocarcinoma (PDAC) and have found increased CHKa expression, associated with differentiation. CHKa protein expression was directly correlated with sensitivity to MN58b, a CHKa inhibitor that reduced cell growth through the induction of apoptosis. Accordingly, CHKa knockdown led to reduced drug sensitivity. In addition, we found that gemcitabine-resistant PDAC cells displayed enhanced sensitivity to CHKa inhibition and, in vitro, MN58b had additive or synergistic effects with gemcitabine, 5-fluorouracil, and oxaliplatin, three active drugs in the treatment of PDAC. Using tissue microarrays, CHKa was found to be overexpressed in 90% of pancreatic tumors. While cytoplasmic CHKa did not relate to survival, nuclear CHKa distribution was observed in 43% of samples and was associated with longer survival, especially among patients with well/moderately differentiated tumors. To identify the mechanisms involved in resistance to CHKa inhibitors, we cultured IMIM-PC-2 cells with increasingly higher concentrations of MN58b and isolated a subline with a 30-fold higher IC50 RNA-Seq analysis identified upregulation of ABCB1 and ABCB4 multidrug resistance transporters, and functional studies confirmed that their upregulation is the main mechanism involved in resistance. Overall, our findings support the notion that CHKa inhibition merits further attention as a therapeutic option in patients with PDAC and that expression levels may predict response. © 2016 AACR. Source

Brandi J.,University of Verona | Pozza E.D.,University of Verona | Dando I.,University of Verona | Biondani G.,University of Verona | And 9 more authors.
Journal of Proteomics | Year: 2016

Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9.Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. Biological significance: The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72 kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9.We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered. © 2016 Elsevier B.V. Source

Radon T.P.,Queen Mary, University of London | Massat N.J.,Queen Mary, University of London | Jones R.,Bioworks Inc. | Alrawashdeh W.,Queen Mary, University of London | And 12 more authors.
Clinical Cancer Research | Year: 2015

Purpose: Noninvasive biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are currently not available. Here, weaimedto identify a set of urine proteins able to distinguish patients with early-stage PDAC from healthy individuals. Experimental design: Proteomes of 18 urine samples from healthy controls, chronic pancreatitis, and patients with PDAC (six/group) were assayed using GeLC/MS/MS analysis. The selected biomarkers were subsequently validated with ELISA assays using multiple logistic regression applied to a training dataset in a multicenter cohort comprising 488 urine samples. Results: LYVE-1, REG1A, and TFF1 were selected as candidate biomarkers. When comparing PDAC (n = 192) with healthy (n = 87) urine specimens, the resulting areas under the receiver-operating characteristic curves (AUC) of the panel were 0.89 [95% confidence interval (CI), 0.84-0.94] in the training (70% of the data) and 0.92 (95% CI, 0.86-0.98) in the validation (30% of the data) datasets. When comparing PDAC stage I-II (n = 71) with healthy urine specimens, the panel achieved AUCs of 0.90 (95% CI, 0.84-0.96) and 0.93 (95% CI, 0.84-1.00) in the training and validation datasets, respectively. In PDAC stage I-II and healthy samples with matching plasma CA19.9, the panel achieved a higher AUC of 0.97 (95% CI, 0.94-0.99) than CA19.9 (AUC = 0.88; 95% CI, 0.81-0.95, P = 0.005). Adding plasma CA19.9 to the panel increased the AUC from 0.97 (95% CI, 0.94-0.99) to 0.99 (95% CI, 0.97-1.00, P = 0.04), but did not improve the comparison of stage I-IIA PDAC (n = 17) with healthy urine. Conclusions: We have established a novel, three-protein biomarker panel that is able to detect patients with early-stage pancreatic cancer in urine specimens. © 2015 American Association for Cancer Research. Source

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