Shin Hamada

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.

MiR-126 acts as a tumor suppressor in pancreatic cancer cells via the regulation of ADAM9.

The epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry of metastatic disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entire picture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targeting multiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductal adenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, and human pancreatic cancer cell line to elucidate essential miRNAs which regulate invasive growth of pancreatic cancer cells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reduced expression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to target ADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreatic cancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 3′ untranslated region assay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted in reduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first time that the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells. Mol Cancer Res; 10(1); 3–10. ©2011 AACR.

Fibrinogen induces cytokine and collagen production in pancreatic stellate cells

Objective: Fibroblasts in the area of fibrosis in chronic pancreatitis and of the desmoplastic reaction associated with pancreatic cancer are now recognised as activated pancreatic stellate cells (PSCs). Recent studies have shown strong expression of fibrinogen, the central protein in the haemostasis pathway, in the stromal tissues of pancreatic cancer and chronic pancreatitis, suggesting that PSCs are embedded in and exposed to abundant fibrinogen in these pathological settings. The effects of fibrinogen on cell functions in PSCs were examined here. Methods: PSCs were isolated from human pancreas tissues of patients undergoing operations for pancreatic cancer, and from rat pancreatic tissues. The effects of fibrinogen on key cell functions and activation of signalling pathways in PSCs were examined. Results: Fibrinogen induced the production of interleukin 6 (IL6), interleukin 8 (IL8), monocyte chemoattractant protein-1, vascular endothelial growth factor, angiopoietin-1 and type I collagen, but not proliferation or intercellular adhesion molecule-1 expression. Fibrinogen increased α-smooth muscle actin expression and induced the activation of nuclear factor-κB (NF-κB), Akt and three classes of mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK)). Fibrinogen-induced IL6 and IL8 production was inhibited by antibodies against αvβ3 and α5β1 integrins, suggesting that these integrins worked as counter receptors for fibrinogen in PSCs. In addition, fibrinogen-induced production of these cytokines was abolished by an inhibitor of NF-κB, and partially inhibited by inhibitors of ERK and p38 MAPK. Conclusion: Fibrinogen directly stimulated profibrogenic and proinflammatory functions in PSCs.

Diagnosis of autoimmune pancreatitis by EUS-FNA by using a 22-gauge needle based on the International Consensus Diagnostic Criteria

BACKGROUND It is controversial whether EUS-guided FNA by using 22-gauge (G) needles is useful for the diagnosis or evaluation of autoimmune pancreatitis (AIP). OBJECTIVE To evaluate the usefulness of EUS-FNA by 22-G needles for the histopathological diagnosis of AIP. DESIGN A retrospective study. SETTING Single academic center. PATIENTS A total of 273 patients, including 25 with AIP, underwent EUS-FNA and histological examinations. RESULTS EUS-FNA by using 22-G needles provided adequate tissue samples for histopathological evaluation because more than 10 high-power fields were available for evaluation in 20 of 25 patients (80%). The mean immunoglobulin G4-positive plasma cell count was 13.7/high-power field. Obliterative phlebitis was observed in 10 of 25 patients (40%). In the context of the International Consensus Diagnostic Criteria for AIP, 14 and 6 of 25 patients were judged to have level 1 (positive for 3 or 4 items) and level 2 (positive for 2 items) histological findings, respectively, meaning that 20 of 25 patients were suggested to have lymphoplasmacytic sclerosing pancreatitis based on the International Consensus Diagnostic Criteria. The diagnosis in 1 patient was type 2 AIP because a granulocytic epithelial lesion was identified in this patient. LIMITATIONS A retrospective study with a small number of patients. CONCLUSIONS The results of this study suggest that EUS-FNA by using 22-G needles provides tissue samples adequate for histopathological evaluation and greatly contributes to the histological diagnosis of AIP.

Nationwide epidemiological survey of acute pancreatitis in Japan.

Objectives A nationwide epidemiological survey of acute pancreatitis (AP) was performed to estimate the number of patients with AP in 2011 in Japan and to clarify the clinical features. Methods The first survey was performed by sending a questionnaire to randomly selected 4175 departments to determine the number of hospitalized patients with AP during 2011. The second survey was conducted by sending a questionnaire to departments in which hospitalized patients with AP were treated based on the first questionnaire. Evaluation of the AP severity was based on the revised severity scoring system of AP of the Japanese Ministry of Health, Labor and Welfare (2008). Results The estimated total number of patients with AP in 2011 was 63,080 (95% confidence interval, 57,678–68,484), with an overall prevalence rate of 49.4 per 100,000 population. The male-to-female ratio was 1.9. The mean age was 58.5 in male and 65.3 in female patients. Alcoholic AP was the most common in male and gallstone AP was the most common in female patients. The overall mortality of AP was 2.6%, and in severe AP, 10.1%. Conclusions The number of patients with AP is still increasing. The revised severity scoring system provided a more precise prediction of prognosis.

MiR-365 induces gemcitabine resistance in pancreatic cancer cells by targeting the adaptor protein SHC1 and pro-apoptotic regulator BAX

The poor prognosis of invasive ductal adenocarcinoma of the pancreas is mainly due to its resistance against therapeutic agents. The molecular mechanism by which morbidity enhances cell survival has been extensively studied, but radical improvements in the therapeutic strategy have not yet been achieved. Recent reports have indicated the substantial contribution of miRNA in multiple cell functions by comprehensively targeting clusters of genes. We identified several miRNAs highly expressed in invasive ductal adenocarcinoma in our previous study, and clarified their contribution to the epithelial-mesenchymal transition. Among the differentially expressed miRNAs, miR-365 was highly expressed in invasive ductal adenocarcinoma, whose functional role has not been reported. In the current study, we found that miR-365 induced gemcitabine resistance in pancreatic cancer cells. MiR-365 directly targeted adaptor protein Src Homology 2 Domain Containing 1 (SHC1) and apoptosis-promoting protein BAX. The siRNA-based knockdown of SHC1 and BAX increased gemcitabine resistance, indicating the miR-365/SHC1/BAX axis influences the survival of pancreatic cancer cells. In addition, miR-365 up-regulated cancer-promoting molecules such as Inhibitor of DNA binding 2 and S100P, suggesting the existence of cross-talk with other cancer-promoting signals. MiR-365 could exert orchestrated effects on pancreatic cancer cell survival.

miR-197 induces epithelial-mesenchymal transition in pancreatic cancer cells by targeting p120 catenin.

Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR‐126 is down‐regulated in IDA compared with IPMA or IPMC, and miR‐126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain‐containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA, and IPMC, we newly identified miR‐197 as an up‐regulated miRNA specifically in IDA. Expression of miR‐197 in pancreatic cancer cells resulted in the induction of epithelial–mesenchymal transition (EMT) along with the down‐regulation of p120 catenin which is a putative target of miR‐197. Direct interaction between miR‐197 and p120 catenin mRNA sequence was confirmed by 3′UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR‐197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR‐197 in the regulation of p120 catenin. This miR‐197/p120 catenin axis could be a novel therapeutic target. J. Cell. Physiol. 228: 1255–1263, 2013.

Circulating miR-483-3p and miR-21 is highly expressed in plasma of pancreatic cancer

Several recent studies have revealed that microRNAs (miRNAs) have a role in carcinogenesis and cancer development, and that it is stably detectable in plasma/serum. The aim of this study was to test whether miR-483-3p as well as miR-21 could be plasma biomarkers for PDAC. The plasma samples were obtained from three groups including 32 pancreatic ductal adenocarcinoma (PDAC) patients, 12 patients with intraductal papillary mucinous neoplasm (IPMN) patients and 30 healthy controls (HC). We evaluated the plasma miR-483-3p and miR-21 expression level by quantitative RT-PCR. We compared the differences in the plasma level of these miRNAs among the three groups, and investigated the relevance of their plasma expression level to the clinical factors in PDAC. The expressions of miR-483-3p and miR-21 were detected in all examined plasma samples. The plasma expression levels of these miRNAs were significantly higher in PDAC compared to HC (P<0.01). The plasma miR-483-3p expression was significantly higher in PDAC patients than IPMN patients (P<0.05). The plasma miR-21 level was associated with advanced stage (P<0.05), metastasis to lymph node and liver (P<0.01), and shorter survival (P<0.01) of the PDAC patients. Together, these findings suggest that measurement of the plasma miR-483-3p level is useful for discriminating PDAC from IPMN, and that the plasma miR-21 level predicts outcome of PDAC patients.

miR-210 regulates the interaction between pancreatic cancer cells and stellate cells

There is accumulating evidence that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression at the post-transcriptional level. This study aimed to clarify the role of miRNAs in the interaction between PSCs and pancreatic cancer cells. Pancreatic cancer cells were mono-cultured or indirectly co-cultured with PSCs. miRNAs were prepared, and Agilents miRNA microarray containing probes for 904 human miRNAs was used to identify differentially expressed miRNAs. miR-210 was identified as an upregulated miRNA by co-culture with PSCs. Conditioned media of PSCs activated ERK and Akt, but not hypoxia-inducible factor-1α pathway. PSCs-induced miR-210 upregulation was inhibited by inhibitors of ERK and PI3K/Akt pathways. Inhibition of miR-210 expression decreased migration, decreased the expression of vimentin and snai-1, and increased the membrane-associated expression of β-catenin in Panc-1 cells co-cultured with PSCs. In conclusion, our results suggest a novel role of miR-210 in the interaction between PSCs and pancreatic cancer cells.

The sixth nationwide epidemiological survey of chronic pancreatitis in Japan

OBJECTIVES A nationwide survey was conducted to clarify the epidemiological features of patients with chronic pancreatitis (CP) in Japan. METHODS Two sequential surveys were conducted. In the first survey, both the prevalence and incidence of CP in Japan in 2007 were estimated by a questionnaire, which was mailed to 3027 randomly chosen Japanese facilities. In the second survey, the second questionnaire was then mailed to 1110 facilities selected by the first survey to clarify the clinicoepidemiological features of the patients. RESULTS The estimated annual prevalence of CP was 36.9 per 100,000; 53.2 in males and 21.2 in females. The estimated annual incidence was 11.9 per 100,000. The prevalence and the incidence of CP gradually increased in Japan as compared to former surveys. The sex ratio (male/female) of definitive and probable CP patients was 4.5, with a mean age of 59.4 years; 59.2 years in males and 60.2 years in females. Alcoholic (69.7%) was most the common and idiopathic (21.0%) was the second most common cause of CP. The proportion of alcoholic CP increased as compared to the 55.5% found in 1994. The clinical features of overall Japanese patients with CP were: abdominal pain (60.6%), malabsorbtion (12.2%), diabetes mellitus (39.7%) and pancreatolithiasis (75.7%). Alcoholic patients were characterized by high morbidity as compared to nonalcoholic patients: abdominal pain (alcoholic 65.0% vs nonalcoholic 53.0%, p < 0.0001), diabetes mellitus (44.8% vs 31.4%, p < 0.0001) and pancreatolithiasis (84.0% vs 60.8%, p < 0.0001). CONCLUSION The prevalence and the incidence of CP, especially alcoholic CP, have been increasing in Japan.