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Pancreatic stellate cells (PSCs), a pivotal component of the tumor microenvironment, contribute to tumor growth and metastasis. PSC-derived factors are essential for triggering the generation and maintenance of cancer stem cells (CSCs). However, the mechanisms by which paracrine signals regulate CSC-like properties such as glycolytic metabolism have not been fully elucidated. Here, we report that two pancreatic cancer cell lines, Panc-1 and MiaPaCa-2, reacted differently when treated with hepatocyte growth factor (HGF) secreted from PSCs. MiaPaCa-2 cells showed little response with regard to CSC-like properties after HGF treatment. We have shown that in Panc-1 cells by activating its cognate receptor c-MET, paracrine HGF resulted in YAP nuclear translocation and HIF-1α stabilization, thereby promoting the expression of CSC pluripotency markers NANOG, OCT-4 and SOX-2 and tumor sphere formation ability. Furthermore, HGF/c-MET/YAP/HIF-1α signaling enhanced the expression of Hexokinase 2 (HK2) and promoted glycolytic metabolism, which may facilitate CSC-like properties. Collectively, our study demonstrated that HGF/c-MET modulates tumor metabostemness by regulating YAP/HIF-1α and may hold promise as a potential therapeutic target against pancreatic cancer.

Background Hippo/YAP pathway is known to be important for development, growth and organogenesis, and dysregulation of this pathway leads to tumor progression. We and others find that YAP is up-regulated in pancreatic ductal adenocarcinoma (PDAC) and associated with worse prognosis of patients. Activated pancreatic stellate cells (PSCs) forming the components of microenvironment that enhance pancreatic cancer cells (PCs) invasiveness and malignance. However, the role and mechanism of YAP in PDAC tumor-stromal interaction is largely unknown. Methods The expression of YAP in Pancreatic cancer cell lines and PDAC samples was examined by Western blot and IHC. The biological role of YAP on cancer cell proliferation, epithelial-mesenchymal transition (EMT) and invasion were evaluated by MTT, Quantitative real-time PCR analysis, Western blot analysis and invasion assay. The effect of YAP on PSC activation was evaluated by PC-PSC co-culture conditions and xenograft PDAC mouse model. Results Firstly, knockdown of YAP inhibits PDAC cell proliferation and invasion in vitro. In addition, YAP modulates the PC and PSC interaction via reducing the production of connective tissue growth factor (CTGF) from PCs, inhibits paracrine-mediated PSC activation under PC-PSC co-culture conditions and in turn disrupts TGF-β1-mediated tumor-stromal interactions. Lastly, inhibiting YAP expression prevents tumor growth and suppresses desmoplastic reaction in vivo. Conclusions These results demonstrate that YAP contributes to the proliferation and invasion of PC and the activation of PSC via tumor-stromal interactions and that targeting YAP may be a promising therapeutic strategy for PDAC treatment. Electronic supplementary material The online version of this article (10.1186/s13046-018-0740-4) contains supplementary material, which is available to authorized users.

We examined the impact of crosstalk between the insulin receptor and G protein–coupled receptor (GPCR) signaling pathways on the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in the context of human pancreatic ductal adenocarcinoma (PDAC). Stimulation of PANC-1 or MiaPaCa-2 cells with insulin and neurotensin, a potent mitogenic combination of agonists for these cells, promoted striking YAP nuclear localization and decreased YAP phosphorylation at Ser127 and Ser397. Challenging PDAC cells with either insulin or neurotensin alone modestly induced the expression of YAP/TEAD–regulated genes, including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and CXCL5, whereas the combination of neurotensin and insulin induced a marked increase in the level of expression of these genes. In addition, siRNA-mediated knockdown of YAP/TAZ prevented the increase in the expression of these genes. A small-molecule inhibitor (A66), selective for the p110α subunit of PI3K, abrogated the increase in phosphatidylinositol 3,4,5-trisphosphate production and the expression of CTGF, CYR61, and CXCL5 induced by neurotensin and insulin. Furthermore, treatment of PDAC cells with protein kinase D (PKD) family inhibitors (CRT0066101 or kb NB 142-70) or with siRNAs targeting the PKD family prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels in response to insulin and neurotensin stimulation. Thus, PI3K and PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells. Implications: Inhibitors of PI3K or PKD disrupt crosstalk between insulin receptor and GPCR signaling systems by blocking YAP/TEAD–regulated gene expression in pancreatic cancer cells. Mol Cancer Res; 15(7); 929–41. ©2017 AACR.

Emerging evidence suggests that metformin, an activator of AMP-activated protein kinase (AMPK), may be useful in preventing and treating pancreatic ductal adenocarcinoma (PDAC). However, whether metformin has an effect on the stromal reaction of PDAC remains unknown. In this study, we first evaluated the expression of AMPK and phosphorylated-AMPK (P-AMPK) in normal and PDAC tissues, our data indicate that reduced P-AMPK expression is a frequent event in PDAC and correlated with poor prognosis and the dense stromal reaction. We then determined the efficacy of metformin on PDAC growth in vitro and in vivo. We reveal that metformin reduces the production of fibrogenic cytokines from pancreatic cancer cells (PCs) and inhibits paracrine-mediated pancreatic stellate cells (PSCs) activation under PCsPSCs co-culture conditions. By using a xenograft PDAC mouse model, we show that metformin intervention prevents tumor growth and enhances the antitumor effect of gemcitabine via suppression of desmoplastic reaction. Taken together, these results suggest that induction of AMPK activation by metformin represents a novel therapeutic approach for treating advanced PDAC through reducing the desmoplastic reaction in PDAC.

We examined the impact of statins on Yes-associated Protein (YAP) localization, phosphorylation and transcriptional activity in human and mouse pancreatic ductal adenocarcinoma (PDAC) cells. Exposure of sparse cultures of PANC-1 and MiaPaCa-2 cells to cerivastatin or simvastatin induced a striking re-localization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEAD-regulated genes Connective Tissue Growth Factor (CTGF) and Cysteine-rich angiogenic inducer 61 (CYR61). Statins also prevented YAP nuclear import and expression of CTGF and CYR61 stimulated by the mitogenic combination of insulin and neurotensin in dense culture of these PDAC cells. Cerivastatin, simvastatin, atorvastatin and fluvastatin also inhibited colony formation by PANC-1 and MiaPaCa-2 cells in a dose-dependent manner. In contrast, the hydrophilic statin pravastatin did not exert any inhibitory effect even at a high concentration (10 μM). Mechanistically, cerivastatin did not alter the phosphorylation of YAP at Ser(127) in either PANC-1 or MiaPaCa-2 cells incubated without or with neurotensin and insulin but blunted the assembly of actin stress fiber in these cells. We extended these findings with human PDAC cells using primary KC and KPC cells, (expressing KrasG12D or both KrasG12D and mutant p53, respectively) isolated from KC or KPC mice. Using cultures of these murine cells, we show that lipophilic statins induced striking YAP translocation from the nucleus to the cytoplasm, inhibited the expression of Ctgf, Cyr61 and Birc5 and profoundly inhibited colony formation of these cells. Administration of simvastatin to KC mice subjected to diet-induced obesity prevented early pancreatic acini depletion and PanIN formation. Collectively, our results show that lipophilic statins restrain YAP activity and proliferation in pancreatic cancer cell models in vitro and attenuates early lesions leading to PDAC in vivo.

Background: Recent studies have suggested that 85% of pancreatic cancer patients accompanied with impaired glucose tolerance or even Diabetes Mellitus (DM) and the invasive and migratory abilities of pancreatic cancer could be enhanced by high glucose. This study aimed to investigate whether Hypoxia- Inducible Factor-1α (HIF-1α) mediates hyperglycemia-induced pancreatic cancer glycolysis. Methods: The cellular glycolytic activity was assessed by determining lactate production, glucose uptake and lactate dehydrogenase enzymatic activity. Pancreatic cancer cells (BxPC-3 cells) were transfected with short hairpin RNA targeting the HIF-1α. Results: Hyperglycemia promotes pancreatic cancer glycolysis. Lactate dehydrogenase A (LDHA) activity and hexokinase 2 (HK2), platelet-type of phosphofructokinase (PFKP) expression were significantly upregulated under hyperglycemic conditions. HIF-1α knockdown prominently down-regulated the activity of LDHA and the expression of HK2, PFKP and decreased lactate production in BxPC-3 cells. Under hypoxia condition, hyperglycemia induced pancreatic glycolysis by mechanisms that are both dependent on HIF-1α and independent of it. Conclusion: The accumulation of HIF-1α induced by hyperglycemia increases LDHA activity and HK2, PFKP expression, thereby promoting pancreatic glycolysis to facilitate cancer progression.

Cancer stem cells (CSCs), which are found in various types of human cancer, including pancreatic cancer, possess elevated metastatic potential, lead to tumor recurrence and cause chemoradiotherapy resistance. Alterations in cellular bioenergetics through the regulation of 5' adenosine monophosphate‑activated protein kinase (AMPK) signaling may be a prerequisite to stemness. Betulinic acid (BA) is a well‑known bioactive compound with antiretroviral and anti‑inflammatory potential, which has been reported to exert anticancer effects on various types of cancer, including pancreatic cancer. The present study aimed to investigate whether BA could inhibit pancreatic CSCs via regulation of AMPK signaling. The proliferation of pancreatic cancer cells was examined by MTT and colony formation assays. The migratory and invasive abilities of pancreatic cancer cells were assessed using wound‑scratch and Transwell invasion assays. In addition, the expression levels of candidate genes were measured by reverse transcription‑quantitative polymerase chain reaction and western blotting. The results revealed that BA inhibited the proliferation and tumorsphere formation of pancreatic cancer cells, suppressed epithelial‑mesenchymal transition (EMT), migration and invasion, and reduced the expression of three pluripotency factors [SRY‑box 2 (Sox2), octamer‑binding protein 4 (Oct4) and Nanog]. Furthermore, immunohistochemical analysis confirmed that there was a significant inverse association between the expression levels of phosphorylated (P)‑AMPK and Sox2 in pancreatic cancer, and it was revealed that BA may activate AMPK signaling. Notably, knockdown of AMPK reversed the suppressive effects of BA on EMT and stemness of pancreatic cancer cells. In addition, BA reversed the effects of gemcitabine on stemness and enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Collectively, these results indicated that BA may effectively inhibit pluripotency factor expression (Sox2, Oct4 and Nanog), EMT and the stem‑like phenotype of pancreatic cancer cells via activating AMPK signaling. Therefore, BA may be considered an attractive therapeutic candidate and an effective inhibitor of the stem‑like phenotype in pancreatic cancer cells. Further investigation into the development of BA as an anticancer drug is warranted.

Abdominal pain is a critical clinical symptom in pancreatic cancer (PC) that affects the quality of life for PC patients. However, the pathogenesis of PC pain is largely unknown. In this study, we show that PC pain is initiated by the sonic hedgehog (sHH) signaling pathway in pancreatic stellate cells (PSCs), which is activated by sHH secreted from PC cells, and then, neurotrophic factors derived from PSCs mediate the pain. The different culture systems were established in vitro, and the expression of sHH pathway molecules, neurotrophic factors, TRPV1, and pain factors were examined. Capsaicin-evoked TRPV1 currents in dorsal root ganglion (DRG) neurons were examined by the patch-clamp technique. Pain-related behavior was observed in an orthotopic tumor model. sHH and PSCs increased the expression and secretion of TRPV1, SP, and CGRP by inducing NGF and BDNF in a co-culture system, also increasing TRPV1 current. But, suppressing sHH pathway or NGF reduced the expression of TRPV1, SP, and CGRP. In vivo, PSCs and PC cells that expressed high levels of sHH could enhance pain behavior. Furthermore, the blockade of NGF or TRPV1 significantly attenuated the pain response to mechanical stimulation compared with the control. Our results demonstrate that sHH signaling pathway is involved in PC pain, and PSCs play an essential role in the process greatly by inducing NGF.

Cigarette smoking is a risk factor for pancreatic cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, we show that smoking, HIF-1α expression and β2-adrenogenic receptor (β2-AR) expression are negatively correlated with the overall survival of pancreatic cancer patients. Moreover, HIF-1α expression and β2-AR expression are positively correlated with smoking status, different histological differentiation and among the tumor node metastasis (TNM) stages in pancreatic cancer patients. NNK increases HIF-1α expression in pancreatic cancer in vitro and in vivo. Furthermore, knockdown of HIF-1α and ICI118, 551 (a β2-AR selective antagonist) abrogates NNK-induced pancreatic cancer proliferation and invasion in vitro and inhibits NNK-induced pancreatic cancer growth in vivo. However, using CoCl2 (a HIF-1α stabilizing agent which decreases HIF-1α degradation under normoxia conditions) reverses ICI118, 551 induced effects under NNK exposure. Thus, our data indicate that β2-AR signaling regulates NNK-induced pancreatic cancer progression via upregulation of HIF-1α. Taken together, β2-AR signaling and HIF-1α may represent promising therapeutic targets for preventing smoking induced pancreatic cancer progression.

Diabetes mellitus and pancreatic cancer are intimately related, as approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes. In this study, we evaluate the underlying mechanism by which hyperglycemia modulates the invasive potential of cancer cells and contributes to their enhanced metastatic behavior. Here we show that hyperglycemia increases the hydrogen peroxide (H2O2) concentration through up-regulation of manganese superoxide dismutase (SOD2) expression, which further activates the ERK and p38 MAPK pathways, as well as the transcription factors NF-κB and AP-1, in a time-dependent manner. The invasion of pancreatic cancer cells resulting from the activation of the H2O2/MAPK axis under high glucose conditions is effectively inhibited by PD 98059 (ERK inhibitor), SB 203580 (p38 MAPK inhibitor), polyethylene glycol-conjugated catalase (PEG-CAT), or the siRNA specific to SOD2. In addition, streptozotocin-treated diabetic nude mice exhibit a stronger tumor invasive ability in renal capsule xenografts which could be suppressed by PEG-CAT treatment. Furthermore, the integrated optical density (IOD) of SOD2 and uPA stainings is higher in the tumor tissues of pancreatic cancer patients with diabetes compared with pancreatic cancer patients with euglycemia. Taken together, our results demonstrate that hyperglycemia enhances cell invasive ability through the SOD2/H2O2/MAPK axis in human pancreatic cancer. Thus, SOD2/H2O2/MAPK axis may represent a promising therapeutic target for pancreatic cancer patients combined with diabetes mellitus.

Glucose intolerance and frank diabetes mellitus (DM) can increase the risk of cancer death for pancreatic cancer (PanCa). However, the mechanism by which these factors influence cancer deaths is not clear. In this study, we established a model system to mimic the pancreatic tumor microenvironment in patients with DM to examine the biological behavior of PanCa cells and nerves in cell culture and in animals. Our in vitro studies demonstrated that hyperglycemia promoted the proliferation and invasion of PanCa cell lines and upregulated the expression of nerve growth factor in these cells. Also, the migration of Schwann cells (SCs) was inhibited by hyperglycemia and neurites exerted pathological regeneration. Furthermore, the interaction between the PanCa cells and nerves was enhanced in the tumor microenvironment. We further showed that hyperglycemia promoted the perineural invasion (PNI) of PanCa in vivo. These data suggest that DM worsens the prognosis of PanCa because of aggravated PNI. Thus, our study illustrates a novel mechanism by which hyperglycemia decreases survival in patients with PanCa.

// Qinhong Xu 1 ,* , Pei Li 1 ,2,* , Xin Chen 1 , Liang Zong 1 , Zhengdong Jiang 1 , Ligang Nan 1 , Jianjun Lei 1 , Wanxing Duan 1 , Dong Zhang 1 , Xuqi Li 3 , Huanchen Sha 1 , Zheng Wu 1 Qingyong Ma 1 and Zheng Wang 1 1 Department of Hepatobiliary Surgery, First Affiliated Hospital Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi, China 2 Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China 3 Department of General Surgery, First Affiliated Hospital Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi, China * These authors have contributed equally to this work Correspondence to: Zheng Wang, email: // Keywords : miR-221/222, pancreatic cancer, matrix metalloproteinases Received : December 20, 2014 Accepted : March 03, 2015 Published : March 29, 2015 Abstract MicroRNAs are involved in the initiation and progression of pancreatic cancer. In this study, we showed that miR-221/222 is overexpressed in pancreatic cancer. MiR-221/222 overexpression significantly promoted pancreatic cancer cell proliferation and invasion while inhibiting apoptosis. The expression of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 was increased in miR-221/222 mimic-transfected pancreatic cancer cells. Validation experiments identified TIMP-2 as a direct target of miR-221/222. These data indicate that overexpressed miR-221/222 may play an oncogenic role in pancreatic cancer by inducing the expression of MMP-2 and MMP-9, thus leading to cancer cell invasion.

// Jiguang Ma 1,* , Wanxing Duan 1,* , Suxia Han 1 , Jianjun Lei 2 , Qinhong Xu 2 , Xin Chen 2 , Zhengdong Jiang 2 , Ligang Nan 2 , Jiahui Li 2 , Ke Chen 2 , Liang Han 2 Zheng Wang 2 , Xuqi Li 3 , Erxi Wu 4 , Xiongwei Huo 3 1 Department of Oncology, First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 2 Department of Hepatobiliary Surgery, First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 3 Department of General Surgery, First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 4 Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, USA * These authors have contributed equally to this work Correspondence to: Suxia Han, email: // Xiongwei Huo, email: // Keywords : lipogenesis, cancer metabolism, ginkgolic acid (GA), AMP-activated protein kinase (AMPK), pancreatic cancer Received : January 26, 2015 Accepted : February 28, 2015 Published : March 26, 2015 Abstract Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration, and invasion ability and increased apoptosis of cancer cells. In addition, GA inhibited the de novo lipogenesis of cancer cells through inducing activation of AMP-activated protein kinase (AMPK) signaling and downregulated the expression of key enzymes (e.g. acetyl-CoA carboxylase [ACC], fatty acid synthase [FASN]) involved in lipogenesis. Moreover, the in vivo experiment showed that GA reduced the expression of the key enzymes involved in lipogenesis and restrained the tumor growth. Taken together, our results suggest that GA may serve as a new candidate against tumor growth of pancreatic cancer partially through targeting pathway driving lipogenesis.

Background Pancreatic cancer is a lethal disease in part because of its potential for aggressive invasion and metastasis. Lipoxin A4 (LXA4) is one of the metabolites that is derived from arachidonic acid and that is catalyzed by 15-lipoxygenase (15-LOX), and it has recently been reported to exhibit anti-cancer effects. However, the role of LXA4 in pancreatic cancer remains to be elucidated. Methods Pancreatic cell lines were treated with vehicle or LXA4, and the invasive capacity was then assessed by Transwell assays. The expression of epithelial and mesenchymal markers was determined by western blotting and immunofluorescence. Anti-TGF-β1 neutralizing antibody and exogenous recombinant human TGF-β1 (rhTGF-β1) were used to study the effect of LXA4 on the TGF-β signaling. A liver metastasis model was applied to investigate the effect of LXA4 in vivo. The correlation between the Lipoxin effect score (LES) and the clinical-pathological features of pancreatic cancer was also analyzed. Results We found that in patients with pancreatic cancer, low LES was correlated with aggressive metastatic potential. The LXA4 activity, which was mediated by the LXA4 receptor FPRL1, could significantly suppress invasion capacity and mesenchymal phenotypes. The expression and autocrine signaling pathway activity of TGF-β1 were also downregulated by LXA4. In the liver metastasis model in nude mice, the stable analog of LXA4, BML-111, could inhibit the metastasis of pancreatic cancer cells. Conclusion Our results demonstrated that LXA4 could reverse mesenchymal phenotypes, which attenuated invasion and metastasis via the inhibition of autocrine TGF-β1 signaling in pancreatic cancer, which may provide a new strategy to prevent the metastasis of pancreatic cancer.

Perineural invasion (PNI) is considered as an alternative route for the metastatic spread of pancreatic cancer cells; however, the molecular changes leading to PNI are still poorly understood. In this study, we show that the CXCL12/CXCR4 axis plays a pivotal role in the neurotropism of pancreatic cancer cells to local peripheral nerves. Immunohistochemical staining results revealed that CXCR4 elevation correlated with PNI in 78 pancreatic cancer samples. Both in vitro and in vivo PNI models were applied to investigate the function of the CXCL12/CXCR4 signaling in PNI progression and pathogenesis. The results showed that the activation of the CXCL12/CXCR4 axis significantly increased pancreatic cancer cells invasion and promoted the outgrowth of the dorsal root ganglia. CXCL12 derived from the peripheral nerves stimulated the invasion and chemotactic migration of CXCR4-positive cancer cells in a paracrine manner, eventually leading to PNI. In vivo analyses revealed that the abrogation of the activated signaling inhibited tumor growth and invasion of the sciatic nerve toward the spinal cord. These data indicate that the CXCL12/CXCR4 axis may be a novel therapeutic target to prevent the perineural dissemination of pancreatic cancer.

Purpose: Pancreatic cancer is characterized by stromal desmoplasia and perineural invasion (PNI). We sought to explore the contribution of pancreatic stellate cells (PSC) activated by paracrine Sonic Hedgehog (SHH) in pancreatic cancer PNI and progression. Experimental Design: In this study, the expression dynamics of SHH were examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of carcinomatous and nonneoplastic pancreatic tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of PSCs activated by paracrine SHH signaling in pancreatic cancer PNI and progression. Results: We show that SHH overexpression in tumor cells is involved in PNI in pancreatic cancer and is an important marker of biologic activity of pancreatic cancer. Moreover, the overexpression of SHH in tumor cells activates the hedgehog pathway in PSCs in the stroma instead of activating tumor cells. These activated PSCs are essential for the promotion of pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies in an in vitro three-dimensional model of nerve invasion in cancer. Furthermore, the coimplantation of PSCs activated by paracrine SHH induced tumor cell invasion of the trunk and nerve dysfunction along sciatic nerves and also promoted orthotropic xenograft tumor growth, metastasis, and PNI in in vivo models. Conclusions: These results establish that stromal PSCs activated by SHH paracrine signaling in pancreatic cancer cells secrete high levels of PNI-associated molecules to promote PNI in pancreatic cancer. Clin Cancer Res; 20(16); 4326–38. ©2014 AACR.

Recent advances indicating a key role of microenvironment for tumor progression, we investigated the role of PSCs and hypoxia in pancreatic cancer aggressiveness, and examined the potential protective effect of α-mangostin on hypoxia-driven pancreatic cancer progression. Our data indicate that hypoxic PSCs exploit their oxidative stress due to hypoxia to secrete soluble factors favouring pancreatic cancer invasion. α-mangostin suppresses hypoxia-induced PSC activation and pancreatic cancer cell invasion through the inhibition of HIF-1α stabilization and GLI1 expression. Increased generation of hypoxic ROS is responsible for HIF-1α stabilization and GLI1 upregulation. Therefore, α-mangostin may be beneficial in preventing hypoxia-induced pancreatic cancer progression.

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. MicroRNAs control gene expression by negatively regulating protein-coding mRNAs. Several reports demonstrated that miR-106a is up-regulated in gastric and colorectal cancers and promotes tumor progression. In contrast, in glioma miR-106a plays the role of a tumor suppressor gene rather than an oncogene. Here we demonstrate that a high level of miR-106a expression is present in pancreatic cancer. Furthermore, our investigation shows that miR-106a has an oncogenic role in pancreatic tumorigenesis by promoting cancer cell proliferation, epithelial–mesenchymal transition and invasion by targeting tissue inhibitors of metalloproteinase 2 (TIMP-2). MiR-106a could be a critical therapeutic target in pancreatic cancer.

Indometacin, an inhibitor of cyclooxygenase-2 (COX-2), has been shown to exert anticancer effects in a variety of cancers. However, the effect and mechanism of indometacin on high glucose (HG)-induced proliferation and invasion of pancreatic cancer (PC) cells remain unclear. Multiple lines of evidence suggest that a large portion of pancreatic cancer (PC) patients suffer from either diabetes or HG which contributing PC progression. In this study, we report that indometacin down-regulated HG-induced proliferation and invasion via up-regulating E-cadherin but not COX-2 in PC cells. Additionally, the E-cadherin transcriptional repressors, Snail and Slug, were also involved in the process. Furthermore, the proliferation and invasion of PC cells, incubated in HG medium and treated with indometacin were significantly increased when E-cadherin was knocked down (Si-E-cad). Moreover, the protein levels of MMP-2, MMP-9, and VEGF were increased in PC cells transfected with Si-E-cad. Finally, the activation of the PI3K/AKT/GSK-3β signaling pathway was demonstrated to be involved in indometacin reversing HG-induced cell proliferation and invasion in PC cells. In conclusion, these results suggest that indometacin plays a key role in down-regulating HG-induced proliferation and invasion in PC cells. Our findings indicate that indometacin could be used as a novel therapeutic strategy to treat PC patients who simultaneously suffer from diabetes or HG.

In our previous study, we found that blockade of SDF-1/CXCR4 signaling inhibits pancreatic cancer cell migration and invasion in vitro. However, the mechanism governing the downstream regulation of SDF-1/CXCR4-mediated invasion remains unclear. Here we report the role of SDF-1/CXCR4 in pancreatic cancer and the possible mechanism of SDF-1/CXCR4-mediated pancreatic cancer invasion. We show that there is a cross-talk between SDF-1/CXCR4 axis and non-canonical Hedgehog (Hh) pathway in pancreatic cancer. Furthermore, our data demonstrate that the ligand of CXCR4, SDF-1 induces CXCR4-positive pancreatic cancer invasion, epithelial-mesenchymal transition (EMT) process and activates the non-canonical Hh pathway. Moreover, we also demonstrate that the invasion of a pancreatic cancer and EMT resulting from the activation of SDF-1/CXCR4 axis is effectively inhibited by Smoothened (SMO) inhibitor cyclopamine and siRNA specific to Gli-1. Collectively, these data demonstrate that SDF-1/CXCR4 modulates the non-canonical Hh pathway by increasing the transcription of SMO in a ligand-independent manner. Taken together, SDF-1/CXCR4 axis may represent a promising therapeutic target to prevent pancreatic cancer progression.

Perineural invasion (PNI) is the initial infiltration of tumor cells into the retroperitoneal nerve plexus and along the nerves. It precludes curative resection, is thought to be the major cause of local recurrence following resection, and is a special metastatic route in pancreatic cancer. Glial cell line-derived neurotrophic factor (GDNF) was recently recognized as a key player in the PNI process. This review covers the most recently published studies on the role of GDNF in pancreatic cancer. We introduce the players in PNI, summarize the distribution of GDNF and its receptors in pancreatic cancer, and discuss the effects and underlying mechanism of GDNF in the PNI process. Finally, we also review some potential inhibitors for GDNF-targeted therapy.

// Jianjun Lei 1, * , Zheng Wu 1, * , Zhengdong Jiang 1 , Jiahui Li 1 , Liang Zong 1 , Xin Chen 1 , Wanxing Duan 1 , Qinhong Xu 1 , Lun Zhang 1 , Liang Han 1 , Qingyong Ma 1 , Zheng Wang 1 , Dong Zhang 1 1 Department of Hepatobiliary and Pancreas Surgery, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi’an 710061, Shaanxi Province, China * These authors contributed equally to this work Correspondence to: Zheng Wang, email: zheng.wang11@mail.xjtu.edu.cn Dong Zhang, email: pavilion1021@hotmail.com Keywords: tumor specific cytotoxic T lymphocytes, immunotherapy, pancreatic carcinoma Received: January 24, 2016 Accepted: September 24, 2016 Published: November 19, 2016 ABSTRACT Pancreatic cancer represents one of the most lethal human cancers. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTLs) have recently been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral particles containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to SW1990 cells. We found that the novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL inhibited pancreatic cancer cell growth and angiogenesis in vitro and in vivo . Furthermore, Ad5/35-TRAIL transduced CTL induced significant apoptosis in pancreatic carcinoma cell lines and upregulated IFN-gamma (IFN-γ) secretion of CTLs. Importantly, Ad5/35-TRAIL transduced CTLs had no inhibitory effect on normal cells. Thus, the novel CTLs may be safe and feasible for the development of gene therapy approaches to pancreatic carcinoma.

Nrf2 (NF-E2-related factor 2) pathway and autophagy both can respond to oxidative stress to promote cancer cells to survive in the tumor microenvironment. We, therefore, explored the relevance between Nrf2 pathway and autophagy in pancreatic cancer cells upon stimulation of reactive oxygen species (ROS). Pancreatic cancer cells were cultured under controlled ROS stressing condition or basal condition. Different inhibitors were used to prevent autophagy at particular stages. Nrf2 siRNA was used to inhibit Nrf2 pathway activation. Ad-mRFP-GFP-LC3 infection was used to monitor autophagic flux. The result shows that a small amount of exogenous hydrogen peroxide (H2O2) can significantly improve the level of intracellular ROS. Moreover, our findings indicate that ROS promotes the activation of both Nrf2 pathway and autophagy in pancreatic cancer cells. Moreover, our data demonstrate that suppression of autophagic activity at particular stages results in an increased promotion of Nrf2 pathway activation upon ROS stimulation. Furthermore, we found that silencing of Nrf2 promotes autophagy upon ROS stimulation. In addition, Nrf2 interference effectively promotes autophagic flux upon ROS stimulation. In summary, our findings suggest that Nrf2 pathway and autophagy have a negative interaction with each other upon ROS stimulation.

Lipoxin A4(LXA4), an endogenous arachidonic acid metabolite, was previously considered an anti-inflammatory lipid mediator. But it also has the potential to inhibit cancer progression. To explore the therapeutic effect of LXA4in pancreatic cancer, we used Panc-1 cells to investigate the mechanism by which LXA4can attenuate pancreatic cancer cell invasion. Our data showed that LXA4significantly inhibited both cell invasion and the expression of matrix metalloproteinase- (MMP-) 9 and MMP-2. Further experiments implied that LXA4decreased the levels of intracellular reactive oxygen species (ROS) and the activity of the extracellular signal regulated kinases (ERK) pathway to achieve similar outcome to ROS scavenger N-acetyl-L-cysteine (NAC). However, a decreased level of intracellular ROS was not observed in cells treated with the specific ERK pathway inhibitor FR180204. The blocking of either intracellular ROS or ERK pathway caused the downregulation of MMP-9 and MMP-2 expression. Furthermore, tests revealed that LXA4inhibited MMP-9 and MMP-2 at the mRNA, protein, and functional levels. Finally, LXA4dramatically limited the invasion of CoCl2-mimic hypoxic cells and abrogated intracellular ROS levels, ERK activity, and MMPs expression. These results suggest that LXA4attenuates cell invasion in pancreatic cancer by suppressing the ROS/ERK/MMPs pathway, which may be beneficial for preventing the invasion of pancreatic cancer.

Pancreatic cancer (PCa), which is now the fourth most frequent cause of cancer-related death, has a median survival of less than 6 months and a 5-year survival rate of6%. The hallmarks of this cancer include poor outcome, short survival duration, and resistance to therapy. The poor prognosis of PCa is related to its local recurrence, lymph node and liver metastases, and peritoneal dissemination. Recent studies have indicated that resveratrol has cancer-chemopreventive and anticancer activities. In this short review we summarize the chemopreventive and treatment effects of resveratrol in PCa, as follows: resveratrol inhibits the proliferation of pancreatic cancer cells; induces apoptosis and cell cycle arrest; inhibits metastasis and invasion of PCa cells; inhibits the proliferation and viability of PCa stem cells; enhances the chemoradiosensitization of PCa cells; and can affect diabetes mellitus in addition to PCa. On the basis of these data, resveratrol may be considered a potential anticancer agent for the treatment of PCa.

Our previous study revealed that superoxide dismutase (SOD)-dependent production of reactive oxygen species (ROS) was able to increase the invasive ability of pancreatic cancer cells. However, the underlying mechanisms by which SOD enhances metastasis are still not fully elucidated. As epithelial-mesenchymal transition (EMT) is a key player in tumor metastasis, the aim of this study was to evaluate whether SOD affects EMT in pancreatic cancer cells and the related mechanism. Human pancreatic cancer cells BxPC-3 and Panc-1 were utilized to examine the level of hydrogen peroxide (H2O) in the absence or presence of SOD and catalase (CAT). The activation of phospho-ERK and phospho-NF-κB were measured by western blot analysis. Wound healing assay and transwell invasion assay were used to detect the migratory and invasive potential of cancer cells. The EMT-related factors, E-cadherin, N-cadherin and vimentin were detected by QT-PCR and western blot analysis. The results of present study showed that SOD not only increased cell migration and invasion in pancreatic cancer, but also mediated the expression of EMT-related factors and cell morphology. In addition, the levels of phospho-ERK and phospho-NF-κB were induced by SOD which could be counter-balanced by both CAT treatment and PD 98059 (an ERK inhibitor). Taken together, these data indicate that SOD promotes the invasive and migratory activity of pancreatic cancer. Blocking the H2O2/ERK/NF-κB axis might be a novel strategy for the treatment of this severe malignancy.

Approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes because high glucose levels can contribute to oxidative stress which promotes tumor development. As one of the reactive oxygen species (ROS)-regulating factors, thioredoxin-interacting protein (TXNIP), is involved in the maintenance of thioredoxin (TRX)-mediated redox regulation. In this study, we demonstrated that high glucose levels increased the expression of TXNIP in time- and concentration-dependent manners and modulated the activity of TRX and ROS production in pancreatic cancer cells, BxPC-3 and Panc-1. We also found that glucose activated both p38 MAPK and ERK pathways and inhibitors of these pathways impaired the TXNIP/TRX/ROS axis. Knockdown of TXNIP restored TRX activity and decreased ROS production under high glucose conditions. Moreover, we observed that the integrated optical density (IOD) of TXNIP staining as well as the protein and mRNA expression levels of TXNIP were higher in the tumor tissues of pancreatic cancer patients with diabetes. Taken together, these results indicate that hyperglycemia-induced TXNIP expression is involved in diabetes-mediated oxidative stress in pancreatic cancer via p38 MAPK and ERK pathways.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the excessive deposition of extracellular matrix (ECM), which is thought to contribute to this tumor's malignant behavior. However, the detailed mechanism and the contribution of excessive deposition of ECM in PDAC progression remain unclear. A better understanding of the mechanism involved in this process is essential for the design of new effective therapies. In this study, we demonstrated that pancreatic cancer cells exhibited increased proliferation and decreased apoptosis in response to type I collagen. In addition, PDAC cells exposed to type I collagen lost the expression of E-cadherin and increased expression of mesenchymal markers, including N-cadherin and vimentin. This epithelial- mesenchymal transition (EMT) was correlated with enhanced cell migration and invasiveness. Knockdown of β1-integrin abolished the effects induced by type I collagen, and further investigation revealed that type I collagen activates β1-integrin (marked by phosphorylation of β1 integrin downstream effectors, focal adhesion kinase [FAK], AKT, and ERK) accompanied by markedly up-regulation of Gli-1, a component of the Hedgehog (HH) pathway. Knockdown of Gli-1 reversed the effects of type I collagen on PDAC invasion and EMT. These results suggest that there is cross-talk between the β1-integrin signaling pathway and the HH pathway in pancreatic cancer and that activation of the HH pathway plays a key role in the type I collagen-induced effects on pancreatic cancer.

Background Hypoxia plays a vital role in cancer epithelial to mesenchymal transition (EMT) and invasion. However, it is not quite clear how hypoxia may contribute to these events. Here we investigate the role of Hedgehog (Hh) signaling in hypoxia induced pancreatic cancer EMT and invasion. Methods Pancreatic cancer cells were cultured under controlled hypoxia conditions (3% O2) or normoxic conditions. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation. The effect of hypoxia and Hh signaling on cancer cell EMT and invasion were evaluated by Quantitative real-time PCR analysis, Western blot analysis and invasion assay. Results Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in pancreatic cancer cells. Moreover, our data demonstrate hypoxia induces EMT process as well as invasion, and activates the non-canonical Hh pathway without affecting sonic hedgehog homolog (SHH) expression. Moreover, these effects are reversible upon HIF-1α siRNA interference with unchanged SHH and patched1 (PTCH1) level. Furthermore, our data demonstrate that hypoxia induced invasion and EMT process are effectively inhibited by Smoothened (SMO) antagonist cyclopamine and GLI1 siRNA. In addition, GLI1 interference inhibited EMT progress with significantly suppressed vimentin expression, whereas inhibition of SMO through cyclopamine could not reduce vimentin level. This data indicate that hypoxia could trigger other factors (such as TGF-β, KRAS or RTK) bypassing SMO to activate GLI1 directly. Conclusions Our findings suggest that Hh signaling modulates hypoxia induced pancreatic cancer EMT and invasion in a ligand-independent manner. Thus, Hh signaling may represent a promising therapeutic target for preventing pancreatic cancer progression.

Resveratrol (trans-3,4',5-trihydroxystilbene), a natural polyphenolic compound detected in grapes, berries, and peanuts, possesses a wide spectrum of pharmacological properties, including anti-tumor metastasis activities. However, the underlying mechanisms through which resveratrol inhibits the metastasis of pancreatic cancer are still not fully elucidated. As epithelial-to-mesenchymal transition (EMT) is a key player for metastasis in tumor, the aim of this study is to determine whether resveratrol affects EMT in pancreatic cancer cells and the related mechanism. The results showed that resveratrol not only inhibited cell proliferation, migration, and invasion in a dose-dependent manner, but also mediated the expression of EMT-related genes (E-cadherin, N-cadherin, vimentin, MMP-2, and MMP-9) which are important for cancer cellular motility, invasiveness and metastasis during tumorigenesis. In addition, the levels of phospho-Akt and phospho- NF-κB in BxPC-3 and Panc-1 cells were reduced by both resveratrol and LY294002 (a PI3-K inhibitor). Furthermore, transforming growth factor-β (TGF-β)-induced alterations in cell morphology that are characteristic of EMT as well as increased cell invasive ability could also be reversed by resveratrol. Taken together, these data indicate that resveratrol suppresses pancreatic cancer migration and invasion through the inhibition of the PI-3K/Akt/NF-κB signaling pathway. This study suggests that resveratrol may be a potential anticancer agent for pancreatic cancer.

Inflammation has become a research hotspot in solid tumours and has been confirmed as a key factor in tumour development through the interactions of inflammatory mediators with gene expression, cell proliferation, and apoptosis. Pancreatic cancer (PC) is one of the most aggressive and deadliest forms of gastrointestinal cancer. A large case-control study found that aspirin, an anti-inflammatory drug, was associated with a decreased risk of PC. Moreover, aspirin has been shown to have inhibitory effects on PC in both in vitro and in vivo studies. However, the clinical data analysis has not been similarly promising. Results from genetic and pharmacological studies suggest that the anti-tumour effects of aspirin are mediated, at least in part, through the inhibition of COXs. Furtermore, other results suggest that the chemopreventive and therapeutic effects of aspirin are also mediated through COX-independent mechanisms. The COX-dependent and COX-independent mechanisms will be described in this review. In addition, we will discuss future research directions on the risks and benefits of the use of aspirin to treat PC and the potential cellular/molecular.

Pancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the underlying mechanism is not clear. This study aimed to determine the relationship between Substance P (SP) and pancreatic cancer perineural invasion (PNI) as well as the mechanism of SP mediating pancreatic cancer PNI, which causes pain in patients with pancreatic cancer. Human pancreatic cancer cells and newborn dorsal root ganglions (DRG) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzed using MTT assay and Transwell Matrigel invasion assay, respectively. Alterations in the neurotropism of pancreatic cancer cells were assessed by coculture system, which mimics the interaction of tumor/neuron in vivo. SP is not only widely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3 cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines examined. SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells, and NK-1R antagonists inhibit these effects. Furthermore, SP promotes neurite outgrowth and the migration of pancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the coculture model. Our results suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, and blocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer. Mol Cancer Res; 11(3); 294–302. ©2012 AACR.

Perineural invasion (PNI) is one of the most important causes of local recurrence and poor survival in pancreatic cancer. However, the exact mechanism of PNI is still not clear. In this study, we sought to identify the reciprocal signaling interactions between sympathetic nerves and pancreatic cancer cells and the underlying mechanisms. We used mouse dorsal root ganglia and pancreatic cancer cells cocultured in vitro, cellular and molecular biology, and animal models to evaluate the function of the sympathetic neurotransmitter norepinephrine (NE) in PNI progression and pathogenesis. NE promoted PNI of pancreatic cancer cells and increased levels of phosphorylated STAT3 in a concentration-dependent manner. NE-mediated activation of STAT3 was inhibited by blocking β-adrenergic receptors (AR) and by blocking protein kinase A, but not through blocking α-AR. Blocking STAT3 could inhibit NE-induced NGF, MMP2, and MMP9 expression and attenuate the migratory, invasive ability and PNI of pancreatic cancer cells. Furthermore, PNI of pancreatic cancer cells was blocked by treatment with a STAT3 phosphorylation inhibitor in vivo. These studies show that NE plays a critical role in pancreatic cancer PNI development and progression through the β-AR/PKA/STAT3 signaling pathway. Reciprocal signaling interactions between the sympathetic nerves and pancreatic cancer cells critically contribute to pancreatic cancer PNI pathogenesis. Inhibition of the activity of sympathetic nerves or STAT3 may be potential strategies for pancreatic cancer PNI therapy. Mol Cancer Ther; 12(3); 264–73. ©2012 AACR.

Pancreatic cancer is one of the most fatal human malignancies. Though a relatively rare malignancy, it remains one of the deadliest tumors, with an extremely high mortality rate. The prognosis of patients with pancreatic cancer remains poor; only patients with small tumors and complete resection have a chance of a complete cure. Pancreatic cancer responds poorly to conventional therapies, including chemotherapy and irradiation. Tumor-specific targeted therapy is a relatively recent addition to the arsenal of anti-cancer therapies. It is important to find novel targets to distinguish tumor cells from their normal counterparts in therapeutic approaches. In the past few decades, studies have revealed the molecular mechanisms of pancreatic tumorigenesis, growth, invasion and metastasis. The proteins that participate in the pathophysiological processes of pancreatic cancer might be potential targets for therapy. This review describes the main players in perineural invasion, hypoxia and desmoplasia and the molecular mechanisms of these pathophysiological processes.

Multiple lines of evidence suggest that a large portion of pancreatic cancer patients suffer from either hyperglycemia or diabetes, both of which are characterized by high blood glucose level. However, the underlying biological mechanism of this phenomenon is largely unknown. In the present study, we demonstrated that the proliferative ability of two human pancreatic cancer cell lines, BxPC-3 and Panc-1, was upregulated by high glucose in a concentration-dependent manner. Furthermore, the promoting effect of high glucose levels on EGF transcription and secretion but not its receptors in these PC cell lines was detected by using an EGF-neutralizing antibody and RT-PCR. In addition, the EGFR transactivation is induced by high glucose levels in concentration- and time-dependent manners in PC cells in the presence of the EGF-neutralizing antibody. These results suggest that high glucose promotes pancreatic cancer cell proliferation via the induction of EGF expression and transactivation of EGFR. Our findings may provide new insight on the links between high glucose level and PC in terms of the molecular mechanism and reveal a novel therapeutic strategy for PC patients who simultaneously suffer from either diabetes or hyperglycemia.

Recent studies have demonstrated that the interaction between the cancer and the stroma, play a key role in the development of pancreatic cancer. The desmoplasia, which consists of fibroblasts, pancreatic stellate cells, lymphatic and vascular endothelial cells, immune cells, pathologic increased nerves, and the extracellular matrix (ECM), creates a complex tumor microenvironment that promotes pancreatic cancer development, invasion, metastasis, and resistance to chemotherapy. Thus, the potential approach for targeting the components of this desmoplastic reaction or the pancreatic tumor microenvironment might represent a novel therapeutic approach to advanced pancreatic carcinoma. Novel therapies that target on the pancreatic tumor microenvironment should become one of the more effective treatments for pancreatic cancer.