Mouad Edderkaoui

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and is characterized by remarkable desmoplasia. The desmoplasia is composed of extracellular matrix (ECM) proteins, myofibroblastic pancreatic stellate cells, and immune cells associated with a multitude of cytokines, growth factors, and ECM metabolizing enzymes. The mechanisms of participation of this complex matrix process in carcinogenesis are only starting to be appreciated. Recent studies showed key roles for stellate cells in the production of ECM proteins as well as cytokines and growth factors that promote the growth of the cancer cells all present in the desmoplastic parts of PDAC. In addition, interactions of ECM proteins and desmoplastic secreted growth factors with the cancer cells of PDAC activate intracellular signals including reactive oxygen species that act to make the cancer cells resistant to dying. These findings suggest that the desmoplasia of PDAC is a key factor in regulating carcinogenesis of PDAC as well as responses to therapies. A better understanding of the biology of desmoplasia in the mechanism of PDAC will likely provide significant opportunities for better treatments for this devastating cancer.

Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways

BACKGROUND AND AIMS Pancreatic cancer is a very aggressive malignancy. Normal cells die through apoptosis when detached from extracellular matrix (ECM), but the role of ECM in cancer cell survival is poorly understood. Here, we determined the effects of ECM proteins on death responses and underlying signaling pathways in human pancreatic cancer cells. METHODS We measured apoptosis and necrosis, caspase activation, and mitochondrial dysfunction in MIA PaCa-2 and PANC-1 pancreatic carcinoma cells both detached and attached to ECM proteins. RESULTS Detachment of pancreatic cancer cells from ECM did not induce classic apoptosis, as it does in normal cells, but induced necrosis and apoptosis associated with secondary necrosis. It caused a pronounced mitochondrial depolarization and release of cytochrome c and Smac/DIABLO. However, as different from normal cells, cytochrome c release did not result in downstream caspase activation. Executioner caspases were activated in detached pancreatic cancer cells independent of cytochrome c. Laminin and fibronectin, but not collagen I, markedly increased pancreatic cancer cell survival by inhibiting both mitochondrial dysfunction (leading to inhibition of necrosis) and caspase activity (leading to decreased apoptotic DNA fragmentation). CONCLUSIONS ECM proteins greatly protect pancreatic cancer cells from death by mechanisms different from those operating in normal cells. The results suggest ECM proteins and their receptors as potential targets for treatment of pancreatic cancer.

The Phosphatase PHLPP1 Regulates Akt2, Promotes Pancreatic Cancer Cell Death, and Inhibits Tumor Formation

BACKGROUND & AIMS The kinase Akt mediates resistance of pancreatic cancer (PaCa) cells to death and is constitutively active (phosphorylated) in cancer cells. Whereas the kinases that activate Akt are well characterized, less is known about phosphatases that dephosporylate and thereby inactivate it. We investigated regulation of Akt activity and cell death by the phosphatases PHLPP1 and PHLPP2 in PaCa cells, mouse models of PaCa, and human pancreatic ductal adenocarcinoma (PDAC). METHODS We measured the effects of PHLPP overexpression or knockdown with small interfering RNAs on Akt activation and cell death. We examined regulation of PHLPPs by growth factors and reactive oxygen species, as well as associations between PHLPPs and tumorigenesis. RESULTS PHLPP overexpression inactivated Akt, whereas PHLPP knockdown increased phosphorylation of Akt in PaCa cells. Levels of PHLPPs were greatly reduced in human PDAC and in mouse genetic and xenograft models of PaCa. PHLPP activities in PaCa cells were down-regulated by growth factors and Nox4 reduced nicotinamide adenine dinucleotide phosphate oxidase. PHLPP1 selectively dephosphorylated Akt2, whereas PHLPP2 selectively dephosphorylated Akt1. Akt2, but not Akt1, was up-regulated in PDAC, and Akt2 levels correlated with mortality. Consistent with these results, high levels of PHLPP1, which dephosphorylates Akt2 (but not PHLPP2, which dephosphorylates Akt1), correlated with longer survival times of patients with PDAC. In mice, xenograft tumors derived from PaCa cells that overexpress PHLPP1 (but not PHLPP2) had inactivated Akt, greater extent of apoptosis, and smaller size. CONCLUSIONS PHLPP1 has tumor suppressive activity and might represent a therapeutic or diagnostic tool for PDAC.

NADPH oxidase activation in pancreatic cancer cells is mediated through Akt-dependent up-regulation of p22phox.

We recently showed that Nox4 NADPH oxidase is highly expressed in pancreatic ductal adenocarcinoma and that it is activated by growth factors and plays a pro-survival, anti-apoptotic role. Here we investigate the mechanisms through which insulin-like growth factor I and serum (FBS) activate NADPH oxidase in pancreatic cancer (PaCa) cells. We show that in PaCa cells, NADPH oxidase is composed of Nox4 and p22phox catalytic subunits, which are both required for NADPH oxidase activity. Insulin-like growth factor I and FBS activate NADPH oxidase through transcriptional up-regulation of p22phox. This involves activation of the transcription factor NF-κB mediated by Akt kinase. Up-regulation of p22phox by the growth factors results in increased Nox4-p22phox complex formation and activation of NADPH oxidase. This mechanism is different from that for receptor-induced activation of phagocytic NADPH oxidase, which is mediated by phosphorylation of its regulatory subunits. Up-regulation of p22phox represents a novel pro-survival mechanism through which growth factors and Akt inhibit apoptosis in PaCa cells.

Protein kinase D1 mediates NF-κB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

Ethanol augments PDGF-induced NADPH oxidase activity and proliferation in rat pancreatic stellate cells.

Background/Aims: Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation. Methods:PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA. Results: mRNA for NADPH oxidase components Nox1, gp91phox, Nox4, p22phox, p47phox and p67phox and protein for NADPH oxidase subunits gp91phox, p22phox, p47phox and p67phox are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47phox. Conclusions: Ethanol causes proliferation of stellate cells by augmenting the activation of the cell’s NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.

Insulin-like Growth Factor-I Receptor Mediates the Prosurvival Effect of Fibronectin *

We recently showed that extracellular matrix (ECM) proteins, which are abundant in desmoplastic pancreatic tumor, are as potent as growth factors in inhibiting apoptosis in pancreatic cancer (PaCa) cells. Here we show that fibronectin, a major ECM component, engages insulin-like growth factor-I receptor (IGF-IR) to inhibit PaCa cell death. We found that fibronectin-induced protection from apoptosis is fully mediated by IGF-IR and is independent of IGF-I. Pharmacologic and molecular inhibitions of IGF-IR stimulated apoptosis and prevented the prosurvival effect of fibronectin in PaCa cells. Our data indicate that fibronectin protects from apoptosis through trans-activation of IGF-IR. We showed that fibronectin stimulated complex formation between its receptor β3 integrin and protein-tyrosine phosphatase SHP-2. This process of complex formation, in turn, prevents SHP-2 from dephosphorylating IGF-IR resulting in sustained phosphorylation of IGF-IR and leading to the downstream activation of Akt kinase, up-regulation of antiapoptotic BclxL, and inhibition of apoptosis. Among ECM proteins tested only fibronectin and laminin but not vitronectin and collagen I stimulated trans-activation of IGF-IR. Interaction of fibronectin with β3 but not β1 integrin receptors mediates the survival pathway. In contrast, fibronectin-induced adhesion is mediated through β1 integrin receptor and is IGF-IR-independent. Thus, our results indicate that the prosurvival effect of fibronectin in PaCa cells is mediated by trans-activation of IGF-IR induced by the β3 integrin receptor. The data suggest IGF-IR as a key target for prevention of the prosurvival effects of ECM proteins and growth factors in pancreatic cancer.

Ellagic Acid and Embelin Affect Key Cellular Components of Pancreatic Adenocarcinoma, Cancer, and Stellate Cells

Ellagic acid is a polyphenolic phytochemical present in many fruits and nuts with anticancer properties demonstrated in experimental tumor studies. Embelin is a benzoquinone phytochemical isolated from the Japanese herb Ardisiae Japonicae and has been shown to induce apoptosis in cancer cells. We found that ellagic acid and embelin each dose-dependently increased apoptosis and inhibited proliferation in human pancreatic cancer cells, MIA PaCa-2 and HPAF-II cells, and in pancreatic stellate cells, which are progenitors of pancreatic cancer desmoplasia. In each of these cell types, combinations of ellagic acid and embelin at low micromolar concentrations (0.5–3 μM) induced synergistic increases in apoptosis and decreases in proliferation. Ellagic acid decreased NF-κB transcriptional activity, whereas embelin decreased STAT-3 phosphorylation and protein expression of its downstream target survivin in cancer cells. In vivo dietary ellagic acid alone or in combination with embelin decreased tumor size and tumor cellularity in a subcutaneous xenograft mouse model of pancreatic cancer. These results show that ellagic acid and embelin interact with divergent intracellular signaling pathways resulting in augmentation of apoptosis and inhibition of proliferation at low micromolar concentrations for the key cellular components of pancreatic adenocarcinoma.

The burning question: why is smoking a risk factor for pancreatic cancer?

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease. The prognosis is poor; less than 5% of those diagnosed are still alive five years after diagnosis, and complete remission is still rare. Tobacco smoking is a major risk factor of pancreatic cancer. However, the mechanism(s) through which it causes the disease remains unknown. Accumulating evidence indicates that carcinogenic compounds in cigarette smoke stimulate pancreatic cancer progression through induction of inflammation and fibrosis which act in concert with genetic factors leading to the inhibition of cell death and stimulation of proliferation resulting in the promotion of the PDAC.

Determination of Rottlerin, a Natural Protein Kinases C Inhibitor, in Pancreatic Cancer Cells and Mouse Xenografts by RP-HPLC Method

Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer.