DPEP1 inhibits tumor cell invasiveness, enhances chemosensitivity and predicts clinical outcome in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. To identify biologically relevant genes with prognostic and therapeutic significance in PDAC, we first performed the microarray gene-expression profiling in 45 matching pairs of tumor and adjacent non-tumor tissues from resected PDAC cases. We identified 36 genes that were associated with patient outcome and also differentially expressed in tumors as compared with adjacent non-tumor tissues in microarray analysis. Further evaluation in an independent validation cohort (N = 27) confirmed that DPEP1 (dipeptidase 1) expression was decreased (T: N ratio ∼0.1, P<0.01) in tumors as compared with non-tumor tissues. DPEP1 gene expression was negatively correlated with histological grade (Spearman correlation coefficient = −0.35, P = 0.004). Lower expression of DPEP1 in tumors was associated with poor survival (Kaplan Meier log rank) in both test cohort (P = 0.035) and validation cohort (P = 0.016). DPEP1 expression was independently associated with cancer-specific mortality when adjusted for tumor stage and resection margin status in both univariate (hazard ratio = 0.43, 95%CI = 0.24–0.76, P = 0.004) and multivariate analyses (hazard ratio = 0.51, 95%CI = 0.27–0.94, P = 0.032). We further demonstrated that overexpression of DPEP1 suppressed tumor cells invasiveness and increased sensitivity to chemotherapeutic agent Gemcitabine. Our data also showed that growth factor EGF treatment decreased DPEP1 expression and MEK1/2 inhibitor AZD6244 increased DPEP1 expression in vitro, indicating a potential mechanism for DPEP1 gene regulation. Therefore, we provide evidence that DPEP1 plays a role in pancreatic cancer aggressiveness and predicts outcome in patients with resected PDAC. In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments.     

Analysis of dynamic molecular networks for pancreatic ductal adenocarcinoma progression

Background

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid tumors. The rapid progression of PDAC results in an advanced stage of patients when diagnosed. However, the dynamic molecular mechanism underlying PDAC progression remains far from clear.

Methods

The microarray GSE62165 containing PDAC staging samples was obtained from Gene Expression Omnibus and the differentially expressed genes (DEGs) between normal tissue and PDAC of different stages were profiled using R software, respectively. The software program Short Time-series Expression Miner was applied to cluster, compare, and visualize gene expression differences between PDAC stages. Then, function annotation and pathway enrichment of DEGs were conducted by Database for Annotation Visualization and Integrated Discovery. Further, the Cytoscape plugin DyNetViewer was applied to construct the dynamic protein–protein interaction networks and to analyze different topological variation of nodes and clusters over time. The phosphosite markers of stage-specific protein kinases were predicted by PhosphoSitePlus database. Moreover, survival analysis of candidate genes and pathways was performed by Kaplan–Meier plotter. Finally, candidate genes were validated by immunohistochemistry in PDAC tissues.

Results

Compared with normal tissues, the total DEGs number for each PDAC stage were 994 (stage I), 967 (stage IIa), 965 (stage IIb), 1027 (stage III), 925 (stage IV), respectively. The stage-course gene expression analysis showed that 30 distinct expressional models were clustered. Kyoto Encyclopedia of Genes and Genomes analysis indicated that the up-regulated DEGs were commonly enriched in five fundamental pathways throughout five stages, including pathways in cancer, small cell lung cancer, ECM-receptor interaction, amoebiasis, focal adhesion. Except for amoebiasis, these pathways were associated with poor PDAC overall survival. Meanwhile, LAMA3, LAMB3, LAMC2, COL4A1 and FN1 were commonly shared by these five pathways and were unfavorable factors for prognosis. Furthermore, by constructing the stage-course dynamic protein interaction network, 45 functional molecular modules and 19 nodes were identified as featured regulators for all PDAC stages, among which the collagen family and integrins were considered as two main regulators for facilitating aggressive progression. Additionally, the clinical relevance analysis suggested that the stage IV featured nodes MLF1IP and ITGB4 were significantly correlated with shorter overall survival. Moreover, 15 stage-specific protein kinases were identified from the dynamic network and CHEK1 was particularly activated at stage IV. Experimental validation showed that MLF1IP, LAMA3 and LAMB3 were progressively increased from tumor initiation to progression.

Conclusions

Our study provided a view for a better understanding of the dynamic landscape of molecular interaction networks during PDAC progression and offered potential targets for therapeutic intervention.

     

Caveolin-1 overexpression correlates with tumour progression markers in pancreatic ductal adenocarcinoma

The assessment of caveolin-1 (Cav-1) as a marker of tumor aggressiveness in pancreatic ductal adenocarcinoma (PDAC). In this study, we examined the expression of Cav-1 in 34 human PDAC tissue samples and the associated peritumoral tissues by immunohistochemistry and western blot. Additionally, we correlated Cav-1 expression with other tissue (Ki-67, p53) and serum (CA 19-9) tumor markers. In the tumor-derived tissue, both tumor cells and blood vessels expressed Cav-1. In contrast, in peritumoral tissue, Cav-1 expression was confined mainly to blood vessels and was only occasionally expressed in ductal or parenchymal cells. Western blot analysis confirmed the overexpression of Cav-1 in pancreatic tumors compared with peritumoral tissue. Cav-1 expression in tumor tissues was correlated with both the Ki-67 LI (r = 0.95, P < 0.0001) and p53 expression (χ2 = 9.91, P < 0.005). Overexpression of Cav-1 was associated with tumor size, grade and stage and Cav-1 expression in tumors was correlated with an increased serum level of CA 19-9 (r = 0.795, P < 0.001). Based on the results of this study, the inclusion of Cav-1 in a putative panel of biomarkers predicting pancreatic cancer aggressiveness is warranted.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.     

SIRT1 and circadian gene expression in pancreatic ductal adenocarcinoma: Effect of starvation

Pancreatic cancer (PC), the fourth leading cause of cancer-related deaths, is characterized by high aggressiveness and resistance to chemotherapy. Pancreatic carcinogenesis is kept going by derangement of essential cell processes, such as proliferation, apoptosis, metabolism and autophagy, characterized by rhythmic variations with 24-h periodicity driven by the biological clock. We assessed the expression of the circadian genes ARNLT, ARNLT2, CLOCK, PER1, PER2, PER3, CRY1, CRY2 and the starvation-activated histone/protein deacetylase SIRT1 in 34 matched tumor and non-tumor tissue specimens of PC patients, and evaluated in PC derived cell lines if the modulation of SIRT1 expression through starvation could influence the temporal pattern of expression of the circadian genes. We found a significant down-regulation of ARNLT (p?=?0.015), CRY1 (p?=?0.013), CRY2 (p?=?0.001), PER1 (p?<?0.0001), PER2 (p?<?0.001), PER3 (p?=?0.001) and SIRT1 (p?=?0.017) in PC specimens. PER3 and CRY2 expression levels were lower in patients with jaundice at diagnosis (?<?0.05). Having adjusted for age, adjuvant therapy and tumor stage, we evidenced that patients with higher PER2 and lower SIRT1 expression levels showed lower mortality (p?=?0.028). Levels and temporal patterns of expression of many circadian genes and SIRT1 significantly changed upon serum starvation in vitro, with differences among four different PC cell lines examined (BXPC3, CFPAC, MIA-PaCa-2 and PANC-1). Serum deprivation induced changes of the overall mean level of the wave and amplitude, lengthened or shortened the cycle time and phase-advanced or phase-delayed the rhythmic oscillation depending on the gene and the PC cell line examined. In conclusion, a severe deregulation of expression of SIRT1 and circadian genes was evidenced in the cancer specimens of PC patients, and starvation influenced gene expression in PC cell lines, suggesting that the altered interplay between SIRT1 and the core circadian proteins could represent a crucial player in the process of pancreatic carcinogenesis.     

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.     

Stroma and pancreatic ductal adenocarcinoma: An interaction loop

Pancreatic ductal adenocarcinom a (PDA) has two exceptional features. First, it is a highly lethal disease, with a median survival of less than 6months and a 5-year survival rate less than 5%. Second, PDA tumor cells are surrounded by an extensive stroma, which accounts for up to 90% of the tumor volume. It is well recognized that stromal microenvironment can accelerate malignant transformation, tumor growth and progression. More importantly, the interaction loop between PDA and its stroma greatly contributes to tumor growth and progression. We propose that the extensive stroma of PDA is closely linked to its poor prognosis. An improved understanding of the mechanisms that contribute to pancreatic tumor growth and progression is therefore urgently needed. Targeting the stroma may thus provide novel prevention, earlier detection and therapeutic options to this deadly malignancy. Accordingly, in this review, we will summarize the mechanism of PDA stroma formation, the role of the stroma in tumor progression and therapy resistance and the potential of stroma-targeted therapeutics strategies.     

Ro60/SSA levels are increased and promote the progression of pancreatic ductal adenocarcinoma

Ro60/SSA is a vital auto antigen that is targeted in Sjogren's syndrome and systemic lupus erythematosus (SLE). However, its role in solid cancers has rarely been reported. The present study investigated the expression and function of Ro60/SSA in the development of pancreatic ductal adenocarcinoma (PDAC) both in vitro and in vivo. Immunohistochemistry was used to examine the expression of Ro60/SSA in PDAC and normal pancreatic tissues by using tissue microarray chips. The results showed that Ro60/SSA expression was increased in PDAC tissues compared with normal pancreatic tissues. Knockdown of Ro60/SSA by siRNA transfection significantly decreased cell proliferation and invasion in vitro. Furthermore, knockdown of Ro60/SSA inhibited the growth of subcutaneous tumors in vivo. Taken together, the current study provides evidence of new function of Ro60/SSA in the development of cancer. It facilitates pancreatic cancer proliferation, migration and invasion. Therefore, it may represent a novel molecular target for the management of pancreatic cancer.     

MUC1 enhances tumor progression and contributes toward immunosuppression in a mouse model of spontaneous pancreatic adenocarcinoma

MUC1, a membrane tethered mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic adenocarcinoma. However, the role of MUC1 in pancreatic cancer has been elusive, partly due to the lack of an appropriate model. We report the characterization of a novel mouse model that expresses human MUC1 as a self molecule (PDA.MUC1 mice). Pancreatic tumors arise in an appropriate MUC1-tolerant background within an immune-competent host. Significant enhancement in the development of pancreatic intraepithelial preneoplastic lesions and progression to adenocarcinoma is observed in PDA.MUC1 mice, possibly due to increased proliferation. Tumors from PDA.MUC1 mice express higher levels of cyclooxygenase-2 and IDO compared with PDA mice lacking MUC1, especially during early stages of tumor development. The increased proinflammatory milieu correlates with an increased percentage of regulatory T cells and myeloid suppressor cells in the pancreatic tumor and tumor draining lymph nodes. Data shows that during pancreatic cancer progression, MUC1-mediated mechanisms enhance the onset and progression of the disease, which in turn regulate the immune responses. Thus, the mouse model is ideally suited for testing novel chemopreventive and therapeutic strategies against pancreatic cancer.     

Increased SPON1 promotes pancreatic ductal adenocarcinoma progression by enhancing IL‐6 trans‐signalling

ObjectivesThis study investigated the specific molecular mechanism and the roles of extracellular matrix protein Spondin 1 (SPON1) in the development of pancreatic ductal adenocarcinoma (PDAC).Materials and MethodsThe expression pattern and clinical relevance of SPON1 was determined in GEO, Ren Ji and TCGA datasets, further validated by immunohistochemical staining and Kaplan‐Meier analysis. Loss and gain of function experiments were employed to investigate the cellular function of SPON1 in vitro. Gene set enrichment analysis, luciferase assay, immunofluorescence and Western blot and immunoprecipitation were applied to reveal the underlying molecular mechanisms. Subcutaneous xenograft model was used to test the role of SPON1 in tumour growth and maintenance in vivo.ResultsSPON1 is significantly upregulated in PDAC tumour tissues and correlated with progression of PDAC. Loss and gain of function experiments showed that SPON1 promotes the growth and colony formation ability of pancreatic cancer cells. Combining bioinformatics assays and experimental signalling evidences, we found that SPON1 can enhance the IL‐6/JAK/STAT3 signalling. Mechanistically, SPON1 exerts its oncogenic roles in pancreatic cancer by maintaining IL‐6R trans‐signalling through stabilizing the interaction of soluble IL‐6R (sIL‐6R) and glycoprotein‐130 (gp130) in PDAC cells. Furthermore, SPON1 depletion greatly reduced the tumour burden, exerted positive effect with gemcitabine, prolonging PDAC mice overall survival.ConclusionsOur data indicate that SPON1 expression is dramatically increased in PDAC and that SPON1 promotes tumorigenicity by activating the sIL‐6R/gp130/STAT3 axis. Collectively, our current work suggests SPON1 may be a potential therapy target for PDAC patient.

Extracellular matrix protein spondin 1 is significantly upregulated in PDAC tumour cell, which exerts its oncogenic roles in pancreatic cancer by maintaining IL6R trans‐signalling through stabilizing the interaction of sIL6R and GP130 in PDAC cell, resulting in STAT3 signalling activating and tumour cell growth.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Overexpression and elevated serum levels of phosphoglycerate kinase 1 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy with a very low 5-year survival rate. Currently, there are no valid markers for early detection and targets for therapy. Here, we used 2-DE to analyze the protein profiles of eight PDAC specimens and paired adjacent nontumor tissues. MS was used to identify 15 protein spots that were found to be overexpressed in PDAC tissues versus adjacent controls. One of them was identified as phosphoglycerate kinase (PGK) 1, a secretable glycolytic enzyme known to participate in angiogenesis. Immunohistochemical analysis of 63 PDAC specimens revealed moderate to strong expression of PGK1 in >70% of the tumors. Further Western blotting analysis of cells from tumor and adjacent nontumor tissues obtained by laser capture microdissection confirmed the enhanced expression of PGK1 in tumor cells. Furthermore, the serum levels of PGK1 were significantly higher in PDAC patients (n = 21) than in the control group (n = 25) (p < 0.005), as determined by ELISA. These observations indicate that protein profile analysis using a combination of 2-DE and MS provides an effective strategy for identifying biomarkers that may have diagnostic potential for PDAC, and identify PGK1 as a potential biomarker and/or therapeutic target for PDAC.     

Acinar to ductal cell trans-differentiation: A prelude to dysplasia and pancreatic ductal adenocarcinoma

Pancreatic cancer (PC) is the deadliest neoplastic epithelial malignancies and is projected to be the second leading cause of cancer-related mortality by 2024. Five years overall survival being ~10%, mortality and incidence rates are disturbing. Acinar to ductal cell metaplasia (ADM) encompasses cellular reprogramming and phenotypic switch-over, making it a cardinal event in tumor initiation. Differential cues and varied regulatory factors drive synchronous functions of metaplastic cell populations leading to multiple cell fates and physiological outcomes. ADM is a precursor for developing early pre-neoplastic lesions further progressing into PC due to oncogenic signaling. Hence delineating molecular events guiding tumor initiation may provide cues for regenerative medicine and precision onco-medicine. Therefore, understanding PC pathogenesis and early diagnosis are crucial. We hereby provide a timely overview of the current progress in this direction and future perspectives we foresee unfolding in the best interest of patient well-being and better clinical management of PC.     

Microvascular density in chronic pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma represent two pathologic phenomena with marked production of connective tissue stroma containing numerous small blood vessels. The aim of this study was to characterise quantitatively the vascular supply of pancreatic adenocarcinoma and fragments of the periductal tissue collected from patients with chronic pancreatitis. The study material included 18 cases of pancreatitis and 22 cases of pancreatic ductal adenocarcinoma. Microvessels were marked using monoclonal anti-CD34 antibodies. The number of blood vessels in the fibrous stroma was significantly higher in the chronic pancreatitis samples compared to the pancreatic carcinoma group (mean vessel count 298 and 194 vessel/mm2; median 251 and 187 vessel/mm2 respectively; p<0.01). Distributions of the vascular diameter in both studied groups were very similar. The obtained results suggest that the development of vascular network accompanying chronic pancreatitis is more effective in some parts of pancreas compared to angiogenic intensity in pancreatic adenocarcinoma.     

Prognostic significance of microRNA-141 expression and its tumor suppressor function in human pancreatic ductal adenocarcinoma

Increasing evidence shows that dysregulation of microRNAs is correlated with tumor development. This study was performed to determine the expression of miR-141 and investigate its clinical significance in pancreatic ductal adenocarcinoma (PDAC). Taqman quantitative RT-PCR was used to detect miR-141 expressions in 94 PDAC tissues and 16 nontumorous pancreatic tissues. Correlations between miR-141 expression and clinicopathologic features and prognosis of patients were statistically analyzed. The effects of miR-141 expression on growth and apoptosis of PDAC cell line (PANC-1) were determined by MTT, colony formation, and flow cytometry assays. Potential target genes were identified by luciferase reporter and Western blot assays. The expression level of miR-141 in PDAC tissues was significantly lower than that in corresponding nontumorous tissues. Downregulation of miR-141 correlated with poorer pT and pN status, advanced clinical stage, and lymphatic invasion. Also, low miR-141 expression in PDAC tissues was significantly correlated with shorter overall survival, and multivariate analysis showed that miR-141 was an independent prognostic factor for PDAC patients. Further, functional researches suggested that miR-141 inhibits growth and colony formation, and enhances caspase-3-dependent apoptosis in PANC-1 cells by targeting Yes-associated protein-1 (YAP1). Therefore, miR-141 is an independent prognostic factor for PDAC patients, and functions as a tumor suppressor gene by targeting YAP1.     

Non-coding RNAs in pancreatic ductal adenocarcinoma: New approaches for better diagnosis and therapy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate less than 8%, which has remained unchanged over the last 50 years. Early detection is particularly difficult due to the lack of disease-specific symptoms and a reliable biomarker. Multimodality treatment including chemotherapy, radiotherapy (used sparingly) and surgery has become the standard of care for patients with PDAC. Carbohydrate antigen 19–9 (CA 19–9) is the most common diagnostic biomarker; however, it is not specific enough especially for asymptomatic patients. Non-coding RNAs are often deregulated in human malignancies and shown to be involved in cancer-related mechanisms such as cell growth, differentiation, and cell death. Several micro, long non-coding and circular RNAs have been reported to date which are involved in PDAC. Aim of this review is to discuss the roles and functions of non-coding RNAs in diagnosis and treatments of PDAC.     

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.