Kazuhiro Kikuta

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.

Pancreatic stellate cells enhance stem cell-like phenotypes 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. Recent studies have identified that a portion of cancer cells, called cancer stem cells, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the stemness of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

Nationwide epidemiological survey of autoimmune pancreatitis in Japan.

Objectives To clarify the clinicoepidemiological features of autoimmune pancreatitis (AIP) in Japan, the nationwide survey was conducted. Methods Patients with AIP who had visited the selected hospitals in 2007 were surveyed. Autoimmune pancreatitis was diagnosed according to the Japanese clinical diagnostic criteria 2006. The study consisted of 2-stage surveys: the number of patients with AIP was estimated by the first questionnaire and their clinical features were assessed by the second questionnaire. Results The estimated total number of AIP patients in 2007 was 2790 (95% confidence interval, 2540–3040), with an overall prevalence rate of 2.2 per 100,000 populations. The number of patients, who were newly diagnosed as AIP, was estimated to be 1120 (95% confidence interval, 1000–1240), with an annual incidence rate of 0.9 per 100,000 populations. Sex ratio (male to female) was 3.7, and the mean (SD) age was 63.0 (11.4) years. Among the 546 patients whose clinical information was obtained, 87.6% of the patients presented high serum immunoglobulin G4 levels (≥135 mg/dL), and 83% received steroid therapy. Conclusions The data represent the current clinical features of AIP in Japan. From the results, most AIP patients in Japan can be categorized to type 1 AIP according to the recent classification of AIP. Abbreviations AIP - autoimmune pancreatitis CI - confidence interval IgG - immunoglobulin G MPD - main pancreatic duct

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

BACKGROUNDnIt is controversial whether EUS-guided FNA by using 22-gauge (G) needles is useful for the diagnosis or evaluation of autoimmune pancreatitis (AIP).nnnOBJECTIVEnTo evaluate the usefulness of EUS-FNA by 22-G needles for the histopathological diagnosis of AIP.nnnDESIGNnA retrospective study.nnnSETTINGnSingle academic center.nnnPATIENTSnA total of 273 patients, including 25 with AIP, underwent EUS-FNA and histological examinations.nnnRESULTSnEUS-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.nnnLIMITATIONSnA retrospective study with a small number of patients.nnnCONCLUSIONSnThe 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.

Nuclear expression of interleukin-33 in pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis in chronic pancreatitis and pancreatic cancer. Recent studies have suggested a role of IL-33, a newly identified IL-1 family member, in fibrosis. We here examined the expression of IL-33 and the IL-33-mediated regulation of cell functions in PSCs. PSCs were isolated from human and rat pancreas tissues. The expression of IL-33 was examined by Western blotting, PCR, ELISA, and immunostaining. The roles of IL-33 in the regulation of PSC functions were examined by using recombinant IL-33 and small interfering RNA. Activated PSCs expressed IL-33 in the nucleus, and the expression was increased by IL-1β, TNF-α, PDGF-BB, and IFN-γ, but not TGF-β1. Nuclear IL-33 expression was also observed in the pancreatic acinar and ductal cells. IL-1β induced IL-33 expression mainly through the activation of NF-κB and ERK pathways and partially through that of p38 MAP kinase, whereas PDGF-BB induced IL-33 expression mainly through the activation of ERK pathway. PSCs expressed soluble ST2, ST2L, and IL-1RAcP, but the expression level of ST2L was relatively low. Recombinant IL-33 did not stimulate key cell functions of PSCs. Decreased IL-33 expression by small interfering RNA resulted in decreased proliferation in response to PDGF-BB. In conclusion, activated PSCs expressed IL-33 in the nucleus. IL-33 might regulate the PDGF-induced proliferation in PSCs.

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-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.

The angiotensin II type I receptor blocker olmesartan inhibits the growth of pancreatic cancer by targeting stellate cell activities in mice

Abstract There is accumulating evidence that pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, promote the progression of pancreatic cancer. The interactions between PSCs and pancreatic cancer have attracted substantial attention as a novel therapeutic target for the treatment of pancreatic cancer. We examined here the effects of olmesartan, an angiotensin II type I receptor blocker, on pancreatic cancer-associated fibrosis using a subcutaneous tumor model developed by co-injection of pancreatic cancer cells with PSCs in nude mice. Co-injection of pancreatic cancer cells AsPC-1 with PSCs increased the size of tumors compared with AsPC-1 cells alone. Olmesartan administrated at 10 mg/kg in drinking water inhibited the growth of subcutaneous tumors derived from the co-injection, but not those derived from mono-injection. This effect was accompanied by decreased expression of α-smooth muscle actin (a marker of activated PSCs) and collagen deposition. The inhibitory effect of olmesartan was also observed even if it was administrated after significant development of subcutaneous tumors. In addition, olmesartan decreased cell growth and type I collagen production in PSCs in vitro. These results suggest that olmesartan inhibited the growth of tumors by targeting stellate cell activities, and that olmesartan might be useful as an anti-fibrosis therapy in pancreatic cancer.

The seventh nationwide epidemiological survey for chronic pancreatitis in Japan: Clinical significance of smoking habit in Japanese patients

OBJECTIVESnA nationwide survey was conducted to clarify the epidemiological features of patients with chronic pancreatitis (CP) in Japan.nnnMETHODSnIn the first survey, both the prevalence and the incidence of CP in 2011 were estimated. In the second survey, the clinicoepidemiological features of the patients were clarified by mailed questionnaires. Patients were diagnosed by the Japanese diagnostic criteria for chronic pancreatitis 2009.nnnRESULTSnThe estimated annual prevalence and incidence of CP in 2011 were 52.4/100,000 and 14.0/100,000, respectively. The sex ratio (male/female) of patients was 4.6, with a mean age of 62.3 years. Alcoholic (67.5%) was the most common and idiopathic (20.0%) was the second most common cause of CP. Comorbidity with diabetes mellitus (DM) and pancreatic calcifications (PC) occurred more frequent in ever smokers independently of their drinking status. Among patients without drinking habit, the incidences of DM and PC were significantly higher in ever smokers than in never smokers. The multiple logistic regression analysis revealed smoking was an independent factor of DM and PC in CP patients: DM, Odds ratio (OR) 1.644, 95% confidence interval (CI) 1.202 to 2.247 (P = 0.002): PC, OR 2.010, 95% CI 1.458 to 2.773 (P < 0.001). On the other hand, smoking was not identified as an independent factor for the appearance of abdominal pain by this analysis.nnnCONCLUSIONnThe prevalence of Japanese patients with CP has been increasing. Smoking was identified as an independent factor related to DM and PC in Japanese CP patients.