The Matricellular Protein CCN1/Cyr61 Is a Critical Regulator of Sonic Hedgehog in Pancreatic Carcinogenesis

CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvβ3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance.     

Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer

Modulation of KRAS activity by upstream signals has revealed a promising new approach for pancreatic cancer therapy; however, it is not clear whether microRNA-associated KRAS axis is involved in the carcinogenesis of pancreatic cancer. Here, we identified miR-193b as a tumor-suppressive miRNA in pancreatic ductal adenocarcinoma (PDAC). Expression analyses revealed that miR-193b was downregulated in (10/11) PDAC specimens and cell lines. Moreover, we found that miR-193b functioned as a cell-cycle brake in PDAC cells by inducing G1-phase arrest and reducing the fraction of cells in S phase, thereby leading to dampened cell proliferation. miR-193b also modulated the malignant transformation phenotype of PDAC cells by suppressing anchorage-independent growth. Mechanistically, KRAS was verified as a direct effector of miR-193b, through which the AKT and ERK pathways were modulated and cell growth of PDAC cells was suppressed. Taken together, our findings indicate that miR-193b-mediated deregulation of the KRAS axis is involved in pancreatic carcinogenesis, and suggest that miR-193b could be a potentially effective target for PDAC therapy.     

Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.     

ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancer

ADAM8 belongs to a family of transmembrane proteins implicated in cell-cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy.     

Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

Background

Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.

Methodology/Principal Findings

Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.

Conclusions/Significance

Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer.     

Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding ras and activating ras signaling

Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.     

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.     

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.     

Hypoxia-Inducible Factor-1α Regulates Chemotactic Migration of Pancreatic Ductal Adenocarcinoma Cells through Directly Transactivating the CX3CR1 Gene

CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC) cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF)-1α expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1α bound to the hypoxia-response element (HRE; 5'-A/GCGTG-3') of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1α significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1α may regulate cancer cell migration through CX3CR1. The HIF-1α/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC.     

Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.     

Increased B Cell-Activating Factor Promotes Tumor Invasion and Metastasis in Human Pancreatic Cancer

B cell-activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) superfamily. It has been reported that BAFF is elevated in patients with autoimmune pancreatitis and contributes to the malignant potential of blood cancers and solid tumors. In this study, clinical evidence of increased BAFF levels in patients with pancreatic ductal adenocarcinoma (PDAC) was obtained, and the roles and mechanisms of BAFF in PDAC were clarified in human tissues of PDAC and from in vitro data of PDAC cell lines. Serum levels of BAFF in patients with PDAC were significantly higher than in healthy subjects (p = 0.0121). Patients with UICC stage IV PDAC (T1-4, N0-1, M1) had significantly higher levels of serum BAFF compared to patients with PDAC (p = 0.0182). BAFF was remarkably expressed in infiltrating B lymphocytes surrounding pancreatic cancer in human pancreatic tissues, suggesting that BAFF may play a role in progression of pancreatic cancer. PDAC cell lines were cultured with human recombinant BAFF, and morphology and gene expression were analyzed; pancreatic cancer cells changed to a fibroblast-like morphology, and showed altered gene expression of E-cadherin, vimentin and Snail. These BAFF-induced changes reflect enhanced cell motility and invasion. BAFF-R-overexpressing cell clones confirmed the association between these BAFF-induced changes and epithelial-mesenchymal transition (EMT)-related genes. BAFF was elevated in patients with metastatic advanced PDAC and induced alterations in PDAC cells via regulation of EMT-related genes. Elucidation of the precise role and mechanism of control of BAFF may lead to new therapeutic approaches with the aim of improving pancreatic cancer survival.     

MiR-371-5p Facilitates Pancreatic Cancer Cell Proliferation and Decreases Patient Survival

Background

microRNAs (miRNAs) play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.

Methods

The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC) and their adjacent normal pancreatic tissues (ANPT) or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). Protein expression was analyzed by Western blot.

Results

The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1) downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.

Conclusions

our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.     

LncRNA PWAR6 regulates proliferation and migration by epigenetically silencing YAP1 in tumorigenesis of pancreatic ductal adenocarcinoma

Long non-coding RNAs (lncRNAs) are a novel class of regulators in multiple cancer biological processes. However, the functions of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. In this study, we identified PWAR6 as a frequently down-regulated lncRNA in PDAC samples as well as a panel of pancreatic cancer cell lines. Down-regulated PWAR6 was associated with multiple clinical outcomes, including advanced tumour stage, distant metastasis, and overall survival of PDAC patients. In our cell-based assays, ectopic expression of PWAR6 dramatically repressed PDAC cells proliferation, invasion and migration, accelerated apoptosis, and induced cell cycle arrest at G0/G1 phase. In contrast, depletion of PWAR6 mediated by siRNA exhibited opposite effects on PDAC cell behaviours. In vivo study further validated the anti-tumour role of PWAR6 in PDAC. By taking advantage of available online sources, we also identified YAP1 as a potential PWAR6 target gene. Negative correlation between YAP1 and PWAR6 expressions were observed in both online database and our PDAC samples. Notably, rescue experiments further indicated that YAP1 is an important downstream effector involved in PWAR6-mediated functions. Mechanistically, PWAR6 could bind to methyltransferase EZH2, a core component of Polycomb Repressive Complex 2 (PRC2) in regulating gene expression, and scaffold EZH2 to the promoter region of YAP1, resulting in epigenetic repression of YAP1. In conclusion, our data manifest the vital roles of PWAR6 in PDAC tumorigenesis and underscore the potential of PWAR6 as a promising target for PDAC diagnosis and therapy.     

MiR-200a Suppresses the Proliferation and Metastasis in Pancreatic Ductal Adenocarcinoma through Downregulation of DEK Gene

MiR-200a has been reported to be able to suppress the epithelial-mesenchymal transition process in pancreatic cancer stem cells, suggesting that miR-200a could suppress the metastasis of pancreatic ductal adenocarcinoma (PDAC). However, its role in proliferation and metastasis of PDAC and the underlying mechanism by which miR-200a works in PDAC have not been elucidated. In our study, we for the first time identified that DEK gene is a direct downstream target of miR-200a. It was found that overexpression of miR-200a decreased DEK expression, suppressing the proliferation, migration, and invasion of PDAC cells. Meanwhile, knockdown of miR-200a can increase DEK level, promoting the proliferation, migration, and invasion of PDAC cells. Our study demonstrated that miR-200a suppresses the metastasis in pancreatic PDAC through downregulation of DEK, suggesting that miR-200a may be used as a novel potential marker in prediction of metastasis of PDAC.     

Death of a tumor: targeting CCN in pancreatic cancer

The matricellular protein CCN2 (connective tissue growth factor, CTGF) has been previously implicated in tumorigenesis. In pancreatic cancer cells, CCN2 expression occurs downstream of ras/MEK/ERK. Direct evidence that CCN2 mediates tumor progression in pancreatic cancer has been lacking. An exciting recent report by Bennewith et al. (Cancer Res 69:775–784, 2009) has used shRNA knockdown of CCN2 to illustrate that CCN2 contributes to growth of pancreatic tumor cells, both in vitro and in vivo. This report briefly summarizes these findings.