Syndecan-2 promotes perineural invasion and cooperates with K-ras to induce an invasive pancreatic cancer cell phenotype.

BACKGROUND: We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells. METHODS: Syndecan-2 (SDC-2) expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. RESULTS: SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides) did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and--further downstream--phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. CONCLUSION: SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.

Glutamate increases pancreatic cancer cell invasion and migration via AMPA receptor activation and Kras-MAPK signaling.

Glutamate has been implicated in tumorigenesis through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR). However, the function of a glutamate-to-AMPAR signal in pancreatic ductal adenocarcinoma (PDAC) has remained elusive. We now show that glutamate-mediated AMPA receptor activation increases invasion and migration of pancreatic cancer cells via activation of the classical MAPK pathway. Glutamate levels were increased in pancreatic cancer accompanied by downregulation of GluR subunits 1, 2, and 4. In pancreatic cancer precursor lesions, pancreatic intraepithelial neoplasia (PanIN), GluR1 subunit levels were strikingly and step-wise increased but its expression was rare in PDAC. Pharmacological inhibition or RNAi-mediated suppression of GluR1 or GluR2 did not affect cancer cell growth but significantly decreased invasion. In a K-ras wildtype cell line, AMPA receptor activation enhanced K-ras activity and--further downstream--phosphorylation of p38 and of p44/42. Preemptive blockade of AMPA receptors in a mouse model of pancreatic cancer inhibited tumor cell settling. AMPA receptor activation thus not only activates MAPK signalling but also directly increases activity of K-ras. Glutamate might serve as a molecular switch that decreases the threshold of K-ras-induced oncogenic signalling and increases the chance of malignant transformation of pancreatic cancer precursor lesions.

ALCAM is associated with chemoresistance and tumor cell adhesion in pancreatic cancer.

AIMS: Cell-cell adhesion is a major factor in integrity of epithelia which is frequently disturbed in cancer leading to local invasion and distant metastasis. METHODS: To define expression and function of activated leukocyte cell adhesion molecule (ALCAM, CD166) in pancreatic cancer and in pancreatic neuroendocrine tumors (PNET), microarray analyses, RT-PCR, immunohistochemistry, RNAi, adhesion, migration, invasion, and chemoresistance assays were used. RESULTS: We demonstrate that expression of ALCAM is altered and its serum levels are increased in pancreatic ductal adenocarcinoma (PDAC). ALCAM was expressed on the membranes of islet cells in the normal pancreas whereas normal pancreatic ducts were ALCAM-negative. In PDAC, ALCAM expression was generally rare though in some tumors, membranous, or cytoplasmic ALCAM was found. PNET were mostly ALCAM-positive with a cytoplasmic staining pattern which was in contrast to the membrane expression observed in non-transformed islet cells. In vitro, ALCAM silencing using RNAi had no effects on growth or invasion of pancreatic cancer cells but reduced cell adhesion and induced chemoresistance. In neuroendocrine tumor cell lines, silencing of ALCAM decreased cell growth. CONCLUSIONS: We propose ALCAM as a novel serum biomarker in human pancreatic tumors which is associated with cell adhesion, growth and chemoresistance.

Cancer-stellate cell interactions perpetuate the hypoxia-fibrosis cycle in pancreatic ductal adenocarcinoma.

BACKGROUND AND AIMS: Although both cancer and stellate cells (PSCs) secrete proangiogenic factors, pancreatic cancer is a scirrhous and hypoxic tumor. The impact of cancer-PSCs interactions on angiogenesis was analyzed. METHODS: Expression of periostin, CD31, and alpha-smooth muscle actin was assessed by immunohistochemistry. Human PSCs and cancer cells were cultivated under normoxia and hypoxia alone, or in coculture, to analyze the changes in their angiogenic and fibrogenic attributes, using enzyme-linked immunosorbent assay, immunoblot, and quantitative polymerase chain reaction analyses and growth of cultured endothelial cells in vitro. RESULTS: On the invasive front of the activated stroma, PSCs deposited a periostin-rich matrix around the capillaries in the periacinar spaces. Compared with the normal pancreas, there was a significant reduction in the microvessel density in chronic pancreatitis (five-fold, P < .001) and pancreatic cancer (four-fold, P < .01) tissues. In vitro, hypoxia increased PSCs' activity and doubled the secretion of periostin, type I collagen, fibronectin, and vascular endothelial growth factor (VEGF). Cancer cells induced VEGF secretion of PSCs (390 +/- 60%, P < .001), whereas PSCs increased the endostatin production of cancer cells (210 +/- 14%, P < .001) by matrix metalloproteinase-dependent cleavage. In vitro, PSCs increased the endothelial cell growth, whereas cancer cells alone, or their coculture with PSCs, suppressed it. CONCLUSIONS: Although PSCs are the dominant producers of VEGF and increase endothelial cell growth in vitro, in the peritumoral stroma, they contribute to the fibrotic/hypoxic milieu through abnormal extracellular matrix deposition and by amplifying endostatin production of cancer cells.

Cannabinoids reduce markers of inflammation and fibrosis in pancreatic stellate cells.

BACKGROUND: While cannabinoids have been shown to ameliorate liver fibrosis, their effects in chronic pancreatitis and on pancreatic stellate cells (PSC) are unknown. METHODOLOGY/PRINCIPAL FINDINGS: The activity of the endocannabinoid system was evaluated in human chronic pancreatitis (CP) tissues. In vitro, effects of blockade and activation of cannabinoid receptors on pancreatic stellate cells were characterized. In CP, cannabinoid receptors were detected predominantly in areas with inflammatory changes, stellate cells and nerves. Levels of endocannabinoids were decreased compared with normal pancreas. Cannabinoid-receptor-1 antagonism effectuated a small PSC phenotype and a trend toward increased invasiveness. Activation of cannabinoid receptors, however, induced de-activation of PSC and dose-dependently inhibited growth and decreased IL-6 and MCP-1 secretion as well as fibronectin, collagen1 and alphaSMA levels. De-activation of PSC was partially reversible using a combination of cannabinoid-receptor-1 and -2 antagonists. Concomitantly, cannabinoid receptor activation specifically decreased invasiveness of PSC, MMP-2 secretion and led to changes in PSC phenotype accompanied by a reduction of intracellular stress fibres. CONCLUSIONS/SIGNIFICANCE: Augmentation of the endocannabinoid system via exogenously administered cannabinoid receptor agonists specifically induces a functionally and metabolically quiescent pancreatic stellate cell phenotype and may thus constitute an option to treat inflammation and fibrosis in chronic pancreatitis.

Cannabinoids in pancreatic cancer: correlation with survival and pain.

Cannabinoids exert antiproliferative properties in a variety of malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). In our study, we quantitatively evaluated the immunoreactivity for cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptors as well as for the endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGLL). Furthermore, quantitative real-time RT-PCR for CB1, CB2, FAAH and MGLL in normal pancreas and pancreatic cancer tissues was performed. Levels of endocannabinoids were determined by liquid chromatography/mass spectrometry. Immunoreactivity scores and QRT-PCR expression levels were correlated with the clinico-pathological (TNM, survival, pain) status of the patients. Evaluation of endocannabinoid levels revealed that these remained unchanged in PDAC compared to the normal pancreas. Patients with high CB1 receptor levels in enlarged nerves in PDAC had a lower combined pain score (intensity, frequency, duration; p = 0.012). There was a significant relationship between low CB1 receptor immunoreactivity or mRNA expression levels (p = 0.0011 and p = 0.026, respectively), or high FAAH and MGLL cancer cell immunoreactivity (p = 0.036 and p = 0.017, respectively) and longer survival of PDAC patients. These results are underlined by a significant correlation of high pain scores and increased survival (p = 0.0343). CB2 receptor immunoreactivity, CB2 receptor, FAAH and MGLL mRNA expression levels did not correlate with survival. Therefore, changes in the levels of endocannabinoid metabolizing enzymes and cannabinoid receptors on pancreatic cancer cells may affect prognosis and pain status of PDAC patients.

Cannabinoids ameliorate pain and reduce disease pathology in cerulein-induced acute pancreatitis.

BACKGROUND & AIMS: The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed. METHODS: We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects. RESULTS: Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis. CONCLUSIONS: In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.