NADPH oxidase 1 in chronic pancreatitis-activated pancreatic stellate cells facilitates the progression of pancreatic cancer.

Patients suffering from chronic pancreatitis (CP) have a higher risk of pancreatic ductal adenocarcinoma (PDAC) compared to the general population. For instance, the presence of an activated pancreatic stellate cell (PaSC)-rich stroma in CP has facilitated the progression of non-invasive pancreatic intraepithelial neoplasia (PanIN) lesions to invasive PDAC. We have previously found that in a mouse model of CP, NADPH oxidase 1 (Nox1) in activated PaSCs forms fibrotic tissue and up-regulates both matrix metalloproteinase (MMP) 9 and the transcription factor Twist1. Yet, the role and mechanism of Nox1 in activated PaSCs from mice with CP (CP-activated PaSCs) in the progression of PDAC is unknown. For that, we tested the ability of Nox1 in CP-activated PaSCs to facilitate the growth of pancreatic cancer cells, and the mechanisms involved in these effects by identifying proteins in the secretome of CP-activated PaSCs whose production were Nox1-dependent. We found that, in vitro, Nox1 evoked a pro-invasive and cancer-promoting phenotype in CP-activated PaSCs via Twist1/MMP-9 expression, causing changes in the extracellular matrix composition. In vivo, Nox1 in CP-activated PaSCs facilitated tumor growth and stromal expansion. Using mass spectrometry, we identified proteins protecting from endoplasmic reticulum, oxidative and metabolic stresses in the secretome of CP-activated PaSCs whose production was Nox1-dependent, including peroxiredoxins (Prdx1 and Prdx4), and thioredoxin reductase 1. In conclusion, inhibiting the Nox1 signaling in activated PaSCs from patients with CP at early stages can reduce the reorganization of extracellular matrix, and the protection of neoplastic cells from cellular stresses, ameliorating the progression of PDAC.

NADPH oxidase 1 mediates caerulein-induced pancreatic fibrosis in chronic pancreatitis.

Inflammatory disorders of the pancreas are divided into acute (AP) and chronic (CP) forms. Both states of pancreatitis are a result of pro-inflammatory mediators, including reactive oxygen species (ROS). One of the sources of ROS is NADPH oxidase (Nox). The rodent genome encodes Nox1-4, Duox1 and Duox2. Our purpose was to assess the extent to which Nox enzymes contribute to the pathogenesis of both AP and CP using Nox-deficient mice. Using RT-PCR, Nox1 was found in both isolated mouse pancreatic acini and pancreatic stellate cells (PaSCs). Subsequently, mice with genetically deleted Nox1 were further studied and showed that the histo-morphologic characteristics of caerulein-induced CP, but not caerulein-induced AP, was ameliorated in Nox1 KO mice. We also found that the lack of Nox1 impaired caerulein-induced ROS generation in PaSCs. Using Western blotting, we found that AKT mediates the fibrotic effect of Nox1 in a mouse model of CP. We also found a decrease in phospho-ERK and p38MAPK levels in Nox1 KO mice with CP, but not with AP. Both CP-induced TGF-ß up-regulation and NF-ĸB activation were impaired in pancreas from Nox1 KO mice. Western blotting indicated increases in proteins involved in fibrosis and acinar-to-ductal metaplasia in WT mice with CP. No change in those proteins were observed in Nox1 KO mice. The lack of Nox1 lowered mRNA levels of CP-induced matrix metalloproteinase MMP-9 and E-cadherin repressor Twist in PaSCs. CONCLUSION: Nox1-derived ROS in PaSCs mediate the fibrotic process of CP by activating the downstream redox-sensitive signaling pathways AKT and NF-ĸB, up-regulating MMP-9 and Twist, and producing α-smooth muscle actin and collagen I and III.

Contrasting roles of H3K4me3 and H3K9me3 in regulation of apoptosis and gemcitabine resistance in human pancreatic cancer cells.

BACKGROUND: Pancreas ductal adenocarcinoma (PDAC) has the most dismal prognosis among all human cancers since it is highly resistant to chemotherapy, radiotherapy and immunotherapy. The anticipated consequence of all therapies is induction of tumor apoptosis. The highly resistance nature of PDACs to all therapies suggests that the intrinsic tumor cell factors, likely the deregulated apoptosis pathway, are key mechanisms underlying PDAC non-response to these therapies, rather than the therapeutic agents themselves. The aim of this study is to test the hypothesis that epigenetic dysregulation of apoptosis mediators underlies PDAC resistance to gemcitabine, the standard chemotherapy for human PDAC. METHODS: PDAC cells were analyzed for apoptosis sensitivity in the presence of a selective epigenetic inhibitor. The epigenetic regulation of apoptosis regulators was determined by Western Blotting and quantitative PCR. The specific epigenetic modification of apoptosis regulator promoter chromatin was determined by chromatin immunoprecipitation in PDAC cells. RESULTS: Inhibition of histone methyltransferase (HMTase) by a selective HMTase inhibitor, verticillin A, significantly increased human PDAC cell sensitivity to gemcitabine-induced growth suppression. Verticillin A treatment decreased FLIP, Mcl-1, Bcl-x and increased Bak, Bax and Bim protein level in the tumor cells, resulting in activation of caspases, elevated cytochrome C release and increased apoptosis as determined by upregulated PARP cleavage in tumor cells. Analysis of human PDAC specimens indicated that the expression levels of anti-apoptotic mediators Bcl-x, Mcl-1, and FLIP were significantly higher, whereas the expression levels of pro-apoptotic mediators Bim, Bak and Bax were dramatically lower in human PDAC tissues as compared to normal pancreas. Verticillin A downregulated H3K4me3 levels at the BCL2L1, CFLAR and MCL-1 promoter to decrease Bcl-x, FLIP and Mcl-1 expression level, and inhibited H3K9me3 levels at the BAK1, BAX and BCL2L11 promoter to upregulate Bak, Bax and Bim expression level. CONCLUSION: We determined that PDAC cells use H3K4me3 to activate Bcl-x, FLIP and Mcl-1, and H3K9me3 to silence Bak, Bax and Bim to acquire an apoptosis-resistant phenotype. Therefore, selective inhibition of H3K4me3 and H3K9me3 is potentially an effective approach to overcome PDAC cells resistance to gemcitabine.

The MLL1-H3K4me3 Axis-Mediated PD-L1 Expression and Pancreatic Cancer Immune Evasion.

BACKGROUND: Pancreatic cancer is one of the cancers where anti-PD-L1/PD-1 immunotherapy has been unsuccessful. What confers pancreatic cancer resistance to checkpoint immunotherapy is unknown. The aim of this study is to elucidate the underlying mechanism of PD-L1 expression regulation in the context of pancreatic cancer immune evasion. METHODS: Pancreatic cancer mouse models and human specimens were used to determine PD-L1 and PD-1 expression and cancer immune evasion. Histone methyltransferase inhibitors, RNAi, and overexpression were used to elucidate the underlying molecular mechanism of PD-L1 expression regulation. All statistical tests were two-sided. RESULTS: PD-L1 is expressed in 60% to 90% of tumor cells in human pancreatic carcinomas and in nine of 10 human pancreatic cancer cell lines. PD-1 is expressed in 51.2% to 52.1% of pancreatic tumor-infiltrating cytotoxic T lymphocytes (CTLs). Tumors grow statistically significantly faster in FasL-deficient mice than in wild-type mice (P = .03-.001) and when CTLs are neutralized (P = .03-<.001). H3K4 trimethylation (H3K4me3) is enriched in the cd274 promoter in pancreatic tumor cells. MLL1 directly binds to the cd274 promoter to catalyze H3K4me3 to activate PD-L1 transcription in tumor cells. Inhibition or silencing of MLL1 decreases the H3K4me3 level in the cd274 promoter and PD-L1 expression in tumor cells. Accordingly, inhibition of MLL1 in combination with anti-PD-L1 or anti-PD-1 antibody immunotherapy effectively suppresses pancreatic tumor growth in a FasL- and CTL-dependent manner. CONCLUSIONS: The Fas-FasL/CTLs and the MLL1-H3K4me3-PD-L1 axis play contrasting roles in pancreatic cancer immune surveillance and evasion. Targeting the MLL1-H3K4me3 axis is an effective approach to enhance the efficacy of checkpoint immunotherapy against pancreatic cancer.

Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

Adenylyl cyclase 6 mediates the action of cyclic AMP-dependent secretagogues in mouse pancreatic exocrine cells via protein kinase A pathway activation.

Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation.

Rap1 activation plays a regulatory role in pancreatic amylase secretion.

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.

C-type natriuretic peptide stimulates pancreatic exocrine secretion in the rat: role of vagal afferent and efferent pathways.

We previously reported that C-type natriuretic peptide (CNP) increases amylase release in isolated pancreatic acini through natriuretic peptide receptor C activation and enhances pancreatic exocrine secretion via vagal pathways when applied to the brain. In the present study we sought to establish whether CNP was involved in the peripheral regulation of pancreatic secretion. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation and bile diversion into the duodenum. CNP dose-dependently enhanced pancreatic flow, chloride and protein excretion but did not modify bicarbonate output. A selective natriuretic peptide receptor C agonist enhanced pancreatic flow and mimicked CNP-evoked protein output but failed to modify chloride secretion. Truncal vagotomy, perivagal application of capsaicin and hexamethonium reduced CNP-evoked pancreatic flow and abolished chloride excretion but did not affect protein output. Furthermore, pre-treatment with atropine reduced both CNP-stimulated pancreatic flow and chloride excretion but failed to modify protein excretion. Partial muscarinic blockade of CNP-evoked chloride output suggested that mediators other than acetylcholine were involved. However, CNP response was unaltered by cholecystokinin and vasoactive intestinal peptide receptor blockade or by nitric oxide synthase inhibition. In conclusion, CNP-stimulated pancreatic flow through the activation of the natriuretic peptide receptor C and the vago-vagal reflex but it increased protein output only by natriuretic peptide receptor C activation and chloride excretion by vago-vagal reflexes. Present results suggest that CNP may play a role as a local regulator of the exocrine pancreas.

Atrial natriuretic factor negatively modulates secretin intracellular signaling in the exocrine pancreas.

We previously reported that atrial natriuretic factor (ANF) stimulates pancreatic secretion through NPR-C receptors coupled to PLC and potentiates secretin response without affecting cAMP levels. In the present study we sought to establish the intracellular signaling mechanism underlying the interaction between both peptides. In isolated pancreatic acini 100 nM ANF abolished cAMP accumulation evoked by any dose of secretin. Lower doses of ANF (1 fM, 1 pM, 1 and 10 nM) dose dependently reduced EC50 secretin-evoked cAMP. Although ANF failed to affect cAMP stimulated by amthamine (selective H2 agonist) or isoproterenol (beta-adrenergic agonist), it abolished VIP-induced cAMP formation. ANF inhibitory effect was prevented by U-73122 (PLC inhibitor) and GF-109203X (PKC inhibitor) but unaltered by PKG and nitric oxide synthase inhibition, supporting that the PLC/PKC pathway mediated the effect. ANF response was mimicked by cANP (4-23 amide) and abolished by pertussis toxin, strongly supporting NPR-C receptor activation. In vivo studies showed that ANF at 0.5 microg x kg(-1) x h(-1) enhanced secretion stimulated by 1 U x kg(-1) x h(-1) secretin but at 1 and 2 microg x kg(-1) x h(-1) it abolished secretin response. However, ANF at such doses failed to modify the secretion evoked by carbachol or CCK. Present results show that ANF negatively modulated secretin secretory response and intracellular signaling through the activation of NPR-C receptors coupled to the PLC/PKC pathway. Furthermore, the finding that ANF also inhibited VIP-evoked cAMP supports a selective modulation of class II G-protein coupled receptors by ANF. Present findings suggest that ANF may play a protective role by reducing secretin response to avoid overstimulation.

C-type natriuretic peptide applied to the brain enhances exocrine pancreatic secretion through a vagal pathway.

C-type natriuretic peptide (CNP) is the major natriuretic peptide in the brain and its mRNA has been reported in the central nervous system, which supports local synthesis and its role as a neuromodulator. The aim of the present work was to study the effect of centrally applied CNP on pancreatic secretion. Rats were fitted with a lateral cerebroventricular cannula one-week before secretion studies. The central administration of CNP dose-dependently enhanced pancreatic fluid and protein output. CNP response was diminished by atropine and hexamethonium, but it was abolished by vagotomy. Neither adrenergic antagonists nor the administration of (D-p-Cl-Phe(6),Leu(17))-vasoactive intestinal peptide (VIP antagonist) or N(omega) Nitro-L arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor) affected CNP response. The effect induced by CNP was mimicked by 8-Br-cGMP but not by c-ANP-(4-23) amide (selective agonist of the natriuretic peptide receptor C). Furthermore, CNP interacted with cholecystokinin (CCK) and secretin in the brain to modify pancreatic secretion. Present findings show that centrally applied CNP enhanced pancreatic secretion through a vagal pathway and suggest that CNP response is mediated by the activation of natriuretic peptide guanylyl cyclase coupled receptors in the brain.