Promoted Pancreatic Ductal Adenocarcinoma Cell Research Articles

Phosphoglycerate Mutase 1 (PGAM1) Promotes Pancreatic Ductal Adenocarcinoma (PDAC) Metastasis by Acting as a Novel Downstream Target of the PI3K/Akt/mTOR Pathway.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors known, with an overall 5-year survival rate of less than 6% due to early local invasion and distant metastasis. Exploring suitable therapeutic targets associated with invasion and metastasis is required for improving the prognosis of PDAC. In this study, we investigated the role of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) in PDAC. PGAM1 expression was examined in tissue samples of 54 PDAC patients using immunohistochemistry, and the correlation between clinicopathological expression and PGAM1 expression was determined. A survival curve was generated using the Kaplan–Meier method. After silencing PGAM1 by siRNA in pancreatic cancer cell lines Aspc-1 and Panc-1, the changes in proliferation, migration, and invasion, and signal pathways were determined. In this study, the expression of PGAM1 was found positively related to poor differentiation, metastasis, advanced clinical stage, and poor survival rate. Silencing PGAM1 decreased the proliferation of Aspc-1 and Panc-1 cells with an S phase arrest, but without influencing cell apoptosis. Migration and invasion also decreased significantly, independent of proliferation. PGAM1 was also found to promote EMT of PDAC cell lines by regulating the Wnt/β-catenin pathway. PGAM1 itself was modulated by the PI3K/Akt/mTOR pathway as a novel downstream target and has a positive mutual regulation with HIF-1α. This study indicates that PGAM1 is closely associated with clinical metastasis and poor prognosis of PDAC. PGAM1 is considered as a potential therapeutic target in PDAC metastasis.

The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF–ERK signaling

Myc-associated zinc-finger protein (MAZ) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of action of MAZ in PDAC progression is largely unknown. Here, we present evidence that MAZ mRNA expression and protein levels are increased in human PDAC cell lines, tissue samples, a subcutaneous tumor xenograft in a nude mouse model, and spontaneous cancer in the genetically engineered PDAC mouse model. We also found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis indicated that MAZ depletion in PDAC cells inhibits invasive phenotypes such as the epithelial-to-mesenchymal transition, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we detected no direct effects of MAZ on the expression of K-Ras mutants, but MAZ increased the activity of CRAF-ERK signaling, a downstream signaling target of K-Ras. The MAZ-induced activation of CRAF-ERK signaling was mediated via p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades and promoted PDAC cell invasiveness. Moreover, we found that the matricellular oncoprotein cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) regulates MAZ expression via Notch-1-sonic hedgehog signaling in PDAC cells. We propose that Cyr61/CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells not through direct K-Ras activation but instead through the activation of CRAF-ERK signaling. Collectively, these results highlight key molecular players in PDAC invasiveness and may help inform therapeutic strategies to improve clinical management and outcomes of PDAC.

MiRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1

Background/Aims: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. Methods: We used miRCURY^TM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. Results: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722–0.936 and 0.749–0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. Conclusion: MiR-1290 may act as an oncogene by directly targeting the 3’-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions

Tissue architecture contributes to pancreatic ductal adenocarcinoma (PDAC) phenotypes. Cancer cells within PDAC form gland-like structures embedded in a collagen-rich meshwork where nutrients and oxygen are scarce. Altered metabolism is needed for tumour cells to survive in this environment, but the metabolic modifications that allow PDAC cells to endure these conditions are incompletely understood. Here we demonstrate that collagen serves as a proline reservoir for PDAC cells to use as a nutrient source when other fuels are limited. We show PDAC cells are able to take up collagen fragments, which can promote PDAC cell survival under nutrient limited conditions, and that collagen-derived proline contributes to PDAC cell metabolism. Finally, we show that proline oxidase (PRODH1) is required for PDAC cell proliferation in vitro and in vivo. Collectively, our results indicate that PDAC extracellular matrix represents a nutrient reservoir for tumour cells highlighting the metabolic flexibility of this cancer.

Small Nucleolar Noncoding RNA SNORA23, Up-Regulated in Human Pancreatic Ductal Adenocarcinoma, Regulates Expression of Spectrin Repeat-Containing Nuclear Envelope 2 to Promote Growth and Metastasis of Xenograft Tumors in Mice

Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes, and specific snoRNAsare dysregulated in some cancer cells. We investigated dysregulation of snoRNAs in pancreatic ductal adenocarcinoma (PDAC) cells. We investigated snoRNA expression in PDAC cell lines by complementary DNA microarray and quantitative reverse transcription polymerase chain reaction. In PDAC (n= 133), intraductal papillary mucinous neoplasm (n=16), mucinous cystic neoplasm-associated PDAC (n= 1), and non-tumor pancreas (n= 8) and liver (n= 3) tissues from subjects who underwent surgical resection, levels of snoRNA were measured by quantitative reverse transcription polymerase chain reaction and compared with clinicopathologic parameters and survival times determined by Kaplan-Meier analysis. To examine snoRNA function, PDAC cells were transfected with snoRNA-antisense oligonucleotides flanked with amido-bridged nucleic acids, or snoRNA-expression plasmids, and analyzed in proliferation, colony formation, spheroid formation, and invasion assays. To identify snoRNA-related factors, cells were analyzed by gene expression and proteomic profiling and immunoblot assays. Mice were given intrasplenic injections of MIA PaCa2- or Suit2-HLMC cells; tumor-bearing nude mice were then given 3 weekly injections of an antisense oligonucleotides against SNORA23, a H/ACA-box type snoRNA, and tumor growth and metastasis to liver, blood, and pancreas were analyzed. Levels of SNORA23 increased and accumulated at the nucleolus in highly metastatic MIA PaCa2- or Suit2-HLMC cells compared with their parental cells. We detected SNORA23 in human PDACspecimens but not in non-tumor pancreatic tissue. PDAC level of SNORA23 correlated with invasion grade and correlated inversely with disease-free survival time of patients. Expression of SNORA23 in PDAC cells increased their invasive activity and colony formation, and spheroid formation was inhibited by SNORA23 knockdown. In gene expression and proteomic profile analyses, we found SNORA23 to increase expression of spectrin repeat-containing nuclear envelope 2 (SYNE2) messenger RNA and protein. Knockdown of SYNE2 in PDAC cells reduced their invasive activities and anchor-independent survival. Administration of SNORA23 antisense oligonucleotides to mice slowed growth of xenograft tumors, tumor expression of SYNE2, tumor cell dissemination, and metastasis to liver. We found expression of the snoRNA SNORA23, which mediates sequence-specific pseudouridylation of ribosomal RNAs, to be increased in human PDAC tissues compared with non-tumor tissues, and levels to correlate with tumor invasion grade and patient survival time. SNORA23 increases expression of SYNE2, possibly through modulation of ribosome biogenesis, to promote PDAC cell survival and invasion, and growth and metastasis of xenograft tumors in mice.

CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells.

BackgroundCoiled-Coil Domain Containing 88A (CCDC88A) was identified as a substrate of the serine/threonine kinase Akt that is capable of binding to the actin cytoskeleton. The aim of this study was to investigate the potential role of CCDC88A in the migration and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was performed to determine whether high CCDC88A expression in human PDAC tissues is correlated with poor prognosis. Immunoprecipitation, immunoblotting and immunocytochemistry were performed to determine the intracellular distribution of CCDC88A, and its association with the serine/threonine kinase Akt and actin-filaments in PDAC cells. Phosphoprotein array analysis was performed to determine CCDC88A-associated intracellular signaling pathways. Finally, immunofluorescence analyses and Matrigel invasion assays were performed to examine the effects of CCDC88A on the formation of cell protrusions and PDAC cell invasion.ResultsExpression of CCDC88A in PDAC tissue was significantly correlated with overall survival. CCDC88A was co-localized with peripheral actin structures in cell protrusions of migrating PDAC cells. Knockdown of CCDC88A inhibited the migration and invasiveness of PDAC cells through a decrease in cell protrusions. Although CCDC88A has been previously reported to be a binding partner and substrate of Akt, the level of active Akt was not associated with the translocation of CCDC88A towards cell protrusions. CCDC88A-dependent promotion of cell migration and invasiveness was not modulated by Akt signaling. Knockdown of CCDC88A decreased phosphorylated Src and ERK1/2 and increased phosphorylated AMPK1 in PDAC cells. Knockdown of AMPK1 inhibited the migration and invasiveness of PDAC cells. The combined data suggest that CCDC88A may be a useful marker for predicting the outcome of patients with PDAC and that CCDC88A can promote PDAC cell migration and invasion through a signaling pathway that involves phosphorylation of Src and ERK1/2 and/or dephosphorylation of AMPK1.ConclusionsCCDC88A was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and increased the migration and invasiveness of PDAC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0466-0) contains supplementary material, which is available to authorized users.

Paracrine IL-6 signaling mediates the effects of pancreatic stellate cells on epithelial-mesenchymal transition via Stat3/Nrf2 pathway in pancreatic cancer cells

BackgroundWe previously showed that pancreatic stellate cells (PSC) secreted interleukin (IL)-6 and promoted pancreatic ductal adenocarcinoma (PDAC) cell proliferation via nuclear factor erythroid 2 (Nrf2)-mediated metabolic reprogramming. Epithelial-mesenchymal transition (EMT) is a key process for the metastatic cascade. To study the mechanism of PDAC progression to metastasis, we investigated the role of PSC-secreted IL-6 in activating EMT and the involvement of Nrf2 in this process. MethodsGene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion. ResultsPSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6. ConclusionsIL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway. General significanceTargeting Stat3/Nrf2 pathway activated by PSC-secreted IL-6 may provide a novel therapeutic option to improve the prognosis of PDAC.

Vav3 is linked to poor prognosis of pancreatic cancers and promotes the motility and invasiveness of pancreatic cancer cells

Background/objectivesThe aim of this study was to investigate the role of the guanine nucleotide exchange factor Vav3 in the motility and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells. MethodsImmunohistochemistry was used to determine whether high Vav3 expression in human PDAC tissues is correlated with poor prognosis. Immunocytochemistry was used to determine the association and intracellular distribution of Vav3, Rac1 and Akt in PDAC cells. Phosphoprotein array analysis was performed to determine the Vav3-associated intracellular signaling pathways. Immunocytochemistry and Matrigel invasion assays were used to examine the effects of Vav3 on the formation of cell protrusions and PDAC cell invasion. ResultsExpression of Vav3 in PDAC tissue was significantly correlated with overall survival. Vav3 was localized in cell protrusions of migrating PDAC cells. Knockdown of Vav3 inhibited the motility and invasiveness of PDAC cells through a decrease in cell protrusions. The levels of active Rac1 or active Akt were not associated with the concentration of Vav3 in cell protrusions. The Vav3-dependent promotion of motility and invasiveness was not modulated by Rac1 or Akt. Additionally, knockdown of Vav3 increased phosphorylated WNK1 in PDAC cells, and knockdown of WNK1 inhibited the motility and invasiveness. This study suggests that Vav3 can be a useful marker for predicting the outcome of patients with PDAC and that Vav3 can promote PDAC cell motility and invasion through association with dephosphorylation of WNK1. ConclusionsVav3 was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and thereby increased the motility and invasiveness of PDAC cells.

S100 Calcium-Binding Protein A6 Promotes Epithelial-Mesenchymal Transition through β-Catenin in Pancreatic Cancer Cell Line

The pathogenesis of pancreatic ductal adenocarcinoma (PDAC) remains poorly understood. S100 calcium-binding protein A6 (S100A6) has been associated with PDAC; however, the effect of S100A6 on PDAC migration and invasion has not yet been explored. In this study, Panc-1 cells were transfected with a plasmid to induce overexpression of S100A6, and β-catenin was knocked down using a specific short hairpin RNA (shRNA). The wound-healing and Transwell assays demonstrated that S100A6 promoted PDAC cell migration and invasion. Furthermore, β-catenin shRNA inhibited the migration and invasion of PDAC cells. We confirmed that S100A6 induces PDAC cell migration and invasion via activation of β-catenin in vitro. Assessment of mRNA and protein levels revealed that S100A6 induces increased expression of β-catenin, N-cadherin and vimentin, and decreased expression of E-cadherin in PDAC cells. β-catenin shRNA also altered the expression of epithelial-mesenchymal transition (EMT)-related markers in PDAC cells. Specifically, expression of E-cadherin was increased, whereas expression of N-cadherin and vimentin was decreased. Finally, we demonstrated that S100A6 alters the expression of EMT-related markers via β-catenin activation. In conclusion, S100A6 induces EMT and promotes cell migration and invasion in a β-catenin-dependent manner. S100A6 may therefore represent a novel potential therapeutic target for the treatment of pancreatic cancer.

The long non-coding RNA HOTTIP promotes progression and gemcitabine resistance by regulating HOXA13 in pancreatic cancer.

BackgroundThe human genome encodes many long non-coding RNAs (lncRNAs). However, their biological functions, molecular mechanisms, and the prognostic value associated with pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. Here, we identify a fundamental role for the lncRNA HOXA transcript at the distal tip (HOTTIP) in the progression and chemoresistance of PDAC.MethodsHigh-throughput microarrays were performed to detect the expression profiles of lncRNAs and messenger RNAs in eight human PDAC tissues and four pancreatic tissues. Quantitative real-time PCR was used to determine the levels of HOTTIP and HOXA13 transcripts in PDAC cell lines and 90 PDAC samples from patients. HPDE6 cells (immortalized human pancreatic ductal epithelial cells) and corresponding adjacent non-neoplastic tissues were used as controls, respectively. The functions of HOTTIP and HOXA13 in cell proliferation, invasion, and epithelial-mesenchymal transition were evaluated by targeted knockdown in vitro. CCK-8 assays, colony formation assays, and xenografts in nude mice were used to investigate whether targeted silencing of HOTTIP could sensitize pancreatic cancer cells to gemcitabine. Immunohistochemistry was performed to investigate the relationship between HOXA13 expression and patient outcome.ResultsMicroarray analyses revealed that HOTTIP was one of the most significantly upregulated lncRNAs in PDAC tissues compared with pancreatic tissues. Quantitative PCR further verified that HOTTIP levels were increased in PDAC cell lines and patient samples compared with controls. Functionally, HOTTIP silencing resulted in proliferation arrest by altering cell-cycle progression, and impaired cell invasion by inhibiting epithelial-mesenchymal transition in pancreatic cancer. Additionally, inhibition of HOTTIP potentiated the antitumor effects of gemcitabine in vitro and in vivo. Furthermore, knockdown of HOXA13 by RNA interference (siHOXA13) revealed that HOTTIP promoted PDAC cell proliferation, invasion, and chemoresistance, at least partly through regulating HOXA13. Immunohistochemistry results revealed that higher HOXA13 expression was correlated with lymph node metastasis, poor histological differentiation, and decreased overall survival in PDAC patients.ConclusionsAs a crucial tumor promoter, HOTTIP promotes cell proliferation, invasion, and chemoresistance by modulating HOXA13. Therefore, the HOTTIP/HOXA13 axis is a potential therapeutic target and molecular biomarker for PDAC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0442-z) contains supplementary material, which is available to authorized users.

Kindlin-2 induced by TGF-β signaling promotes pancreatic ductal adenocarcinoma progression through downregulation of transcriptional factor HOXB9

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with no effective therapeutics. Invasion and metastasis are the major characteristics of PDAC. However, mechanisms underlying PDAC invasion and metastasis are elusive. In this report, we found that Kindlin-2 is a target protein of transforming growth factor β (TGF-β) signaling and is upregulated by TGF-β1 in PDAC cells. TGF-β1-upregulated Kindlin-2 promotes PDAC cell growth, migration and invasion, whereas Kindlin-2 upregulates transforming growth factor receptor I (TβRI), a key component of TGF-β signaling. Thereby Kindlin-2 and TGF-β signaling constitute a positive feedback loop. Mechanistically, Kindlin-2 promotes PDAC progression by downregulation of HOXB9 and E-cadherin. For clinical relevance, enhanced expression of Kindlin-2 predicts a poor overall survival for PDAC patients. Gene expression levels of Kindlin-2, TGF-β, TβRI and HOXB9 are all correlated with the overall survival of PDAC patients in an Oncomine dataset. Taken together, our findings demonstrated that TGF-β1-induced Kindlin-2 expression promotes PDAC progression by downregulation of HOXB9 and E-cadherin.

Peroxiredoxin 1 promotes pancreatic cancer cell invasion by modulating p38 MAPK activity.

ObjectiveThe aim of this study was to investigate the role of peroxiredoxin 1 (Prdx1) in the invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was used to determine overexpression of Prdx1 in human PDAC tissues. Immunoprecipitation and immunocytochemistry were used to determine the interaction and intracellular distribution of Prdx1 and a member of the mitogen-activated protein kinase (MAPK) family protein, p38 MAPK, in PDAC cells. Finally, immunocytochemistry and Matrigel invasion assay were used to examine the effects of Prdx1 and p38 MAPK on the formation of cell protrusions and PDAC cell invasion.ResultsPrdx1 is overexpressed in human PDAC tissues. Peroxiredoxin 1 interacts with active forms of p38 MAPK, and complexes of Prdx1 and phosphorylated p38 MAPK localize at the leading edges of migrating PDAC cells. Suppression of Prdx1 decreases active p38 MAPK localized in cell protrusions and inhibits the invasiveness of PDAC cells. Consequently, suppression of Prdx1 inhibits membrane ruffling and protrusions. The p38 MAPK inhibitor SB203580 also decreases the formation of membrane protrusions and inhibits invasiveness.ConclusionsPrdx1 associates with the formation of membrane protrusions through modulation of the activity of p38 MAPK, which in turn promotes PDAC cell invasion.

MiR-940 Inhibited Pancreatic Ductal Adenocarcinoma Growth by Targeting MyD88

Background: Pancreatic ductal adenocarcinoma (PDAC) is an almost universally lethal disease. Deregulation or dysfunction of miRNAs contribute to cancer development. The role of miR-940 in PDAC remains unclear. Methods: The level of miR-940 in PDAC tissues and cell lines was measured by qRT-PCR. MiR-940 was over-expressed by miRNAs mimics transfection and reduced by miRNAs antisense oligonucleotides (ASO) transfection. Cell proliferation was analyzed by MTT assay and cell apoptosis was evaluated by FACS analysis. Targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Myeloid differentiation primary response gene (88) (MyD88) protein level was assayed by immunohistochemistry and Western blot analysis. Results: Low miR-940 level and high MyD88 protein level in PDAC tissues were both correlated with low survival rate. Up-regulation of miR-940 inhibited PDAC cell lines growth while down-regulation induced cell growth. The 3' UTR of MyD88 was targeted by miR-940. Conclusions: Low level of miR-940 and high level of MyD88 in PDAC promoted PDAC cells growth which might be related to the low survival rate of PDAC patients. MiR-940 exerted its effect by targeting MyD88.

MiR-429 Determines Poor Outcome and Inhibits Pancreatic Ductal Adenocarcinoma Growth by Targeting TBK1

Background: Pancreatic ductal adenocarcinoma (PDAC) ranks fourth on the list of cancer-related causes of death and its prognosis has not improved significantly over the past decades. Deregulation or dysfunction of miRNAs contribute to cancer development. Previous data indicates that miR-429 is involved in the pathogenesis of PDAC. However, the role of miR-429 in PDAC remained unknown. Methods: MiR-429 levels in sample tissues of 78 patients and in PANC1 and SW1990 cell lines were quantified by real-time PCR. MiR-429 expression was modulated using specific pre- and anti-miRNAs and cell growth was assayed by MTT analysis. Bioinformatics prediction of the miR-429 putative target genes was performed and luciferase assays confirmed TBK1 as a direct target gene. TBK1 levels in PDAC tissues were analyzed by immunohistochemistry. Results: MiR-429 was remarkably decreased in PDAC tissues and cell lines. Lower miR-429 expression in PDAC tissues significantly correlated with shorter survival of PDAC patients. Overexpression of miR-429 inhibited PDAC cell lines growth in vitro and vice versa. TBK1 was found to be the direct target gene of miR-429. Higher TBK1 protein level in PDAC tissues correlated with shorter survival of PDAC patients. Overexpression of TBK1 partly restored cell proliferation. Conclusions: Low level of miR-429 and high level of TBK1 in PDAC promoted PDAC cells growth which might be related to the low survival rate of PDAC patients. MiR-429 play its role in PDAC by targeting TBK1.

H19 promotes pancreatic cancer metastasis by derepressing let-7's suppression on its target HMGA2-mediated EMT.

The long noncoding RNA (lncRNA) H19 has been recently characterized as an oncogenic lncRNA in some tumors. However, the role of H19 in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we found that not only the levels of H19 was overexpressed in PDAC compared with adjacent normal tissues, but also H19 expression was upregulated remarkably in primary tumors which subsequently metastasized, compared to those did not metastasis. Subsequently, the efficacy of knockdown of H19 by H19-small interfering RNA (siRNA) was evaluated in vitro, and we found that downregulation of H19 impaired PDAC cell invasion and migration. We further demonstrated that H19 promoted PDAC cell invasion and migration at least partially by increasing HMGA2-mediated epithelial-mesenchymal transition (EMT) through antagonizing let-7. This study suggests an important role of H19 in regulating metastasis of PDAC and provides some clues for elucidating the lncRNA-miRNA functional network in cancer.

Hypoxia-Inducible Factor-1 Promotes Pancreatic Ductal Adenocarcinoma Invasion and Metastasis by Activating Transcription of the Actin-Bundling Protein Fascin

Because of the early onset of local invasion and distant metastasis, pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor, with a 5-year survival rate of less than 5%. In this study, we investigated the role of fascin, a prometastasis actin-bundling protein, in PDAC progression, invasion, and the molecular mechanisms underlying fascin overexpression in PDAC. Our data showed that the expression levels of fascin were higher in cancer tissues than in normal tissues, and fascin overexpression correlated with the PDAC differentiation and prognosis. Fascin overexpression promoted PDAC cell migration and invasion by elevating matrix metalloproteinase-2 (MMP-2) expression. Fascin regulated MMP-2 expression through protein kinase C and extracellular signal-regulated kinase. Importantly, our data showed that hypoxia induced fascin overexpression in PDAC cells by promoting the binding of hypoxia-inducible factor-1 (HIF-1) to a hypoxia response element on the fascin promoter and transactivating fascin mRNA transcription. Intriguingly, HIF-1α expression levels in PDAC patient specimens significantly correlated with fascin expression. Moreover, immunohistochemistry staining of consecutive sections demonstrated colocalization between HIF-1α and fascin in PDAC specimens, suggesting that hypoxia and HIF-1α were responsible for fascin overexpression in PDAC. When ectopically expressed, fascin was able to rescue PDAC cell invasion after HIF-1α knockdown. Our results demonstrated that fascin is a direct target gene of HIF-1. Our data suggested that the hypoxic tumor microenvironment in PDAC might promote invasion and metastasis by inducing fascin overexpression, and fascin might be targeted to block PDAC progression.

CypA, a Gene Downstream of HIF-1α, Promotes the Development of PDAC

Hypoxia-inducible factor-1α (HIF-1α) is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC). In response to tumor hypoxia, cyclophilin A (CypA) is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE) in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.

KERR-MCGEE SANCTIONS DEEPWATER GAS DEVELOPMENT

We previously showed that pancreatic stellate cells (PSC) secreted interleukin (IL)-6 and promoted pancreatic ductal adenocarcinoma (PDAC) cell proliferation via nuclear factor erythroid 2 (Nrf2)-mediated metabolic reprogramming. Epithelial-mesenchymal transition (EMT) is a key process for the metastatic cascade. To study the mechanism of PDAC progression to metastasis, we investigated the role of PSC-secreted IL-6 in activating EMT and the involvement of Nrf2 in this process.Gene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion.PSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6.IL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway.Targeting Stat3/Nrf2 pathway activated by PSC-secreted IL-6 may provide a novel therapeutic option to improve the prognosis of PDAC.