Pancreatic Ductal Epithelium Research Articles

Population Dynamics of MafA-Positive Cells During Ontogeny of Human Pancreas

Diverse transcription factors influencing cell differentiation are often chosen as markers of β cell progenitors. One of these transcription factors is MafA, which according to data in literature activates the expression of insulin gene in β cells. Other data support the hypothesis that MafA is required solely for the regulation of insulin secretion by β cells of adult islets and is not involved in the development of islet cells. In this paper, we study the role of MafA through both examination of human pancreas ontogeny and comparison of these data with dynamic changes in the population of insulin-positive and glucagon-positive cells. The aim of this research was the immunohistochemical analysis of MafA, insulin, and glucagon expression during human pancreas ontogeny. The research was conducted on whole embryos and isolated human fetal organs, obtained as a result of either miscarriage or legal medical abortion with voluntary consent of patients, and also on autopsy material from infant and adult human pancreas. As a result of this study, it was found that, during the ontogeny of human pancreas, the first MafA-positive cells appear in the islets at 12.5 weeks of gestation, that is, later than both insulin-positive cells (11.5 weeks of gestation) and glucagon-positive cells (8.5 weeks of gestation). No MafA-positive cells were found in the epithelium of pancreatic ducts. The number of MafA-positive cells in the islets increases during pancreatic organogenesis. The results of our research do not allow us to regard MafA as a marker of undifferentiated progenitors of islet cells. Based on this, we believe that the results obtained in this study are a supplementary contribution to the hypothesis that relates MafA to the markers of adult β cells.

CD90 and CD24 Co-Expression Is Associated with Pancreatic Intraepithelial Neoplasias.

Thy-1 (CD90) has been shown to be a potential marker for several different types of cancer. However, reports on CD90 expression in pancreatic intraepithelial neoplasia (PanIN) lesions are still limited where PanINs are the most important precursor lesion of pancreatic ductal adenocarcinoma (PDAC). Herein, we investigate candidate markers for PanIN lesions by examining the distribution and trend of CD90 and CD24 expression as well as their co-expression in various stages of PanINs. Thirty cases of PanINs, which were confirmed histopathologically and clinically, were used to evaluate protein expression of CD90 and CD24 by immunofluoresence double staining. CD90 was found to be mainly expressed in stroma around lesion ducts while not observed in acini and islets in PanINs. CD90 also showed increased expression in PanIN III compared to PanIN I. CD24 was mainly present in the cytoplasm and membrane of pancreatic ductal epithelia, especially in the apical epithelium of the duct. CD24 had higher expression in PanIN III compared with PanIN II or PanIN I. CD90 was expressed around CD24 sites, but there was little overlap between cells that expressed each of these proteins. A correlation analysis showed that these two proteins have a moderate relationship with PanIN stages respectively. These results suggest that co-expression of CD90 and CD24 may have an important role in the development and progression of PanINs, which is also conducive to early detection and treatment of PDAC.

Tu1454 The Evaluation of Pancreatic Ductal Epithelium by Using the Novel High Frequency Small-Diameter Ultrasound Probe

Tu1454 The Evaluation of Pancreatic Ductal Epithelium by Using the Novel High Frequency Small-Diameter Ultrasound Probe

Using Quantitative Seroproteomics to Identify Antibody Biomarkers in Pancreatic Cancer.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Less than 6% of patients survive beyond the fifth year due to inadequate early diagnostics and ineffective treatment options. Our laboratory has developed an allogeneic, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting pancreatic cancer vaccine (GVAX) that has been tested in phase II clinical trials. Here, we employed a serum antibodies-based SILAC immunoprecipitation (SASI) approach to identify proteins that elicit an antibody response after vaccination. The SASI approach uses immunoprecipitation with patient-derived antibodies that is coupled to quantitative stable isotope-labeled amino acids in cell culture (SILAC). Using mass spectrometric analysis, we identified more than 150 different proteins that induce an antibody response after vaccination. The regulatory subunit 12A of protein phosphatase 1 (MYPT1 or PPP1R12A), regulatory subunit 8 of the 26S proteasome (PSMC5), and the transferrin receptor (TFRC) were shown to be pancreatic cancer-associated antigens recognized by postvaccination antibodies in the sera of patients with favorable disease-free survival after GVAX therapy. We further interrogated these proteins in over 80 GVAX-treated patients' pancreases and uniformly found a significant increase in the expression of MYPT1, PSMC5, and TFRC in neoplastic compared with non-neoplastic pancreatic ductal epithelium. We show that the novel SASI approach can identify antibody targets specifically expressed in patients with improved disease-free survival after cancer vaccine therapy. These targets need further validation to be considered as possible pancreatic cancer biomarkers.

The anti-oxidative transcription factor Nuclear factor E2 related factor-2 (Nrf2) counteracts TGF-β1 mediated growth inhibition of pancreatic ductal epithelial cells -Nrf2 as determinant of pro-tumorigenic functions of TGF-β1.

BackgroundNuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being essential in regulating cell homeostasis. Thus, acute induction of Nrf2 in epithelial cells exposed to inflammation confers protection from oxidative cell damage and mutagenesis supporting an anti-tumorigenic role for Nrf2. However, pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf2 activity conferring therapy resistance which points to a pro-tumorigenic role of Nrf2. A similar dichotomous role in tumorigenesis is described for the Transforming Growth Factor-beta 1 (TGF-β1). The present study therefore aimed at elucidating whether the switch of Nrf2 function towards a tumor promoting one relates to the modulation of TGF-β1 induced cell responses and whether this might occur early in PDAC development.MethodsIn situ analysis comprised immunohistochemical stainings of activated (phosphorylated) Nrf2 and Ki67 in pancreatic tissues containing normal ducts and pancreatic intraepithelial neoplasia (PanINs). In vitro, Nrf2 levels in benign (H6c7-pBp), premalignant (H6c7-kras) and malignant (Colo357) pancreatic ductal epithelial cells were modulated by Nrf2 specific siRNA or Nrf2 overexpression. Then, the effect of Nrf2 alone and in combination with TGF-β1 on cell growth and survival was investigated by cell counting, Ki67 staining and apoptosis assays. The underlying cell signaling was investigated by western blotting. Statistical analysis was performed by Shapiro-Wilk test for normal distribution. Parametric data were analyzed by one-way ANOVA, while non-parametric data were analyzed by Kruskal-Wallis one-way ANOVA on ranks.ResultsSignificantly elevated expression of activated Nrf2 and Ki67 could be detected in PanINs but not in normal pancreatic ductal epithelium. While the effect of Nrf2 on basal cell growth of H6c7-pBp, H6c7-kras and Colo357 cells was minor, it clearly attenuated the growth inhibiting effects of TGF-β1 in all cell lines. This enhanced Nrf2-mediated cell survival was predominantly based on an enhanced proliferative activity. Accordingly, expression of p21 expression along with expression of phospho-p38 and phospho-Smad3 was diminished whereas Erk-phosphorylation was enhanced under these conditions.ConclusionsOverall, our data demonstrate that Nrf2 being elevated in early precursor lesions counteracts the growth inhibiting function of TGF-β1 already in benign and premalignant pancreatic ductal epithelial cells. This could represent one fundamental mechanism underlying the functional switch of both- TGF-β1 and Nrf2 – which may manifest already in early stages of PDAC development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2191-7) contains supplementary material, which is available to authorized users.

Diagnostic value of IMP3 and mesothelin in differentiating pancreatic ductal adenocarcinoma from chronic pancreatitis

BackgroundThe discrimination between pancreatic ductal adenocarcinoma (PDA) and chronic pancreatitis may be confusing at both clinical and radiologic levels. So, the search for biomarkers able to distinguish both clinical conditions is of great interest. AimThis study was undertaken to assess the value of insulin-like growth factor II mRNA binding protein 3 (IMP3) and mesothelin to differentiate PDA from non-neoplastic/reactive pancreatic duct epithelium. MethodsImmunohistochemical staining for IMP3 and mesothelin was performed on 40 formalin-fixed, paraffin-embedded tissue sections of PDA, 20 biopsies of chronic pancreatitis and 10 normal pancreatic tissue obtained from tumor-free surgical margins. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy were calculated. ResultsIMP3 immunoreactivity was observed in 34 of 40 (85%) cases of PDA. The staining reaction was moderate to strong in 30 (75%) cases and diffuse in 26 (65%) cases. Eighteen of 20 (90%) biopsies of chronic pancreatitis were negative for IMP3, while the other two cases (10%) showed weak and focal IMP3 immunoreactivity. On the other hand, mesothelin demonstrated positive immunoreactivity in 30 of 40 (75%) cases of PDA. The staining reaction was moderate to strong in 24 (60%) cases and diffuse in 22 (55%) cases. Sixteen of 20 (80%) biopsies of chronic pancreatitis were negative for mesothelin, while weak and focal mesothelin staining was detected in the other 4 cases. All normal pancreatic tissues were negative for IMP3 and mesothelin expression. IMP3 showed higher sensitivity (85%) and specificity (90%) than mesothelin (75% and 80%, respectively). ConclusionsOur results showed that IMP3 immunostaining has a higher sensitivity and specificity than mesothelin for the diagnosis of PDA. IMP3 and mesothelin may be useful markers in distinguishing neoplastic from reactive lesions of the pancreas in instances where this is impossible by morphology alone in surgical pathology practice.

Effect of All-Trans Retinoic Acid on the Pancreas of Streptozotocin-Induced Diabetic Rat.

All-trans Retinoic acid (atRA) is instructive for the development of endocrine pancreas and is an integral component of β-cell induction protocols. We showed that atRA induces glucose-responsive endocrine transdifferentiation of pleomorphic pancreatic ductal adenocarcinoma cells in vitro. This study aimed to detect the role of atRA in improving the histological changes of the pancreas in diabetic rats. Forty young male Wistar rats were used and divided into three groups. Group I: normal vehicle control (N = 5). Group II: streptozotocin-induced diabetic rats (N = 20) were followed up at 0.0, 1, 2, and 4 weeks. Group III: streptozotocin-induced diabetic rats (N = 15) treated with atRA (2.5 mg/kg/day), were followed up at 1, 2, and 4 weeks. Specimens from the pancreas were processed for light, electron microscopy and pancreatic insulin mRNA expression. Blood samples were assayed for the levels of glucose, insulin, and total peroxides. In the atRA-treated group, the number of the islets and the islet area significantly increased. Strong insulin-immunoreactive endocrine-like cells were observed nearby the pancreatic acini and the interlobular ducts. Interestingly, insulin-positive cells seemed to arise from pancreatic acinar and ductal epithelium. Ultrastructurally, ß-cells, acinar, and ductal cells restored their normal appearance. Pancreatic insulin mRNA and blood indices were almost normalized. AtRA improved the histological changes of the pancreas and the blood indices in diabetic rats.

TRPM8 Ion Channels as Potential Cancer Biomarker and Target in Pancreatic Cancer.

This article provides a review and discussion of the transient receptor potential melastatin-subfamily member 8 (TRPM8) ion channel as a potential biomarker and target in cancer. TRPM8 is a Ca(2+)-permeable channel that plays a major physiological role in cellular sensation and transduction of cold temperature. TRPM8 is aberrantly expressed in a variety of solid tumors including pancreatic cancer. In pancreatic adenocarcinoma cell lines and tissues, TRPM8 is overexpressed as compared to normal pancreatic ductal epithelia. Analysis of anti-TRPM8 immunoreactivity in pancreatic adenocarcinoma indicates positive correlation of TRPM8 expression with tumor size and stages. The biological roles of TRPM8 in pancreatic cancer cells have been revealed from studies using RNA interference-mediated silencing of TRPM8. The experimental data show that TRPM8 channels are required for sustaining proliferation and cell cycle progression, preventing replicative senescence, and promoting cell invasion. Evidence to date implicates a contributory role of TRPM8 channels in the pathogenesis of pancreatic neoplasms and other tumors. Research focus on the mechanisms that underlie TRPM8-mediated roles in tumor growth and metastasis may help establish a novel link of physicochemical changes with pancreatic carcinogenesis. Translational and clinical investigation to exploit TRPM8 as a molecular biomarker and therapeutic target is expected to make a positive impact on precision medicine in pancreatic cancer and other malignant diseases.

The Crosstalk between Nrf2 and TGF-β1 in the Epithelial-Mesenchymal Transition of Pancreatic Duct Epithelial Cells.

Nrf2 and TGF-β1 both affect tumorigenesis in a dual fashion, either by preventing carcinogen induced carcinogenesis and suppressing tumor growth, respectively, or by conferring cytoprotection and invasiveness to tumor cells during malignant transformation. Given the involvement of Nrf2 and TGF-β1 in the adaptation of epithelial cells to persistent inflammatory stress, e.g. of the pancreatic duct epithelium during chronic pancreatitis, a crosstalk between Nrf2 and TGF-β1 can be envisaged. By using premalignant human pancreatic duct cells (HPDE) and the pancreatic ductal adenocarcinoma cell line Colo357, we could show that Nrf2 and TGF-β1 independently but additively conferred an invasive phenotype to HPDE cells, whereas acting synergistically in Colo357 cells. This was accompanied by differential regulation of EMT markers like vimentin, Slug, L1CAM and E-cadherin. Nrf2 activation suppressed E-cadherin expression through an as yet unidentified ARE related site in the E-cadherin promoter, attenuated TGF-β1 induced Smad2/3-activity and enhanced JNK-signaling. In Colo357 cells, TGF-β1 itself was capable of inducing Nrf2 whereas in HPDE cells TGF-β1 per-se did not affect Nrf2 activity, but enhanced Nrf2 induction by tBHQ. In Colo357, but not in HPDE cells, the effects of TGF-β1 on invasion were sensitive to Nrf2 knock-down. In both cell lines, E-cadherin re-expression inhibited the proinvasive effect of Nrf2. Thus, the increased invasion of both cell lines relates to the Nrf2-dependent downregulation of E-cadherin expression. In line, immunohistochemistry analysis of human pancreatic intraepithelial neoplasias in pancreatic tissues from chronic pancreatitis patients revealed strong Nrf2 activity already in premalignant epithelial duct cells, accompanied by partial loss of E-cadherin expression. Our findings indicate that Nrf2 and TGF-β1 both contribute to malignant transformation through distinct EMT related mechanisms accounting for an invasive phenotype. Provided a crosstalk between both pathways, Nrf2 and TGF-β1 mutually promote their tumorigenic potential, a condition manifesting already at an early stage during inflammation induced carcinogenesis of the pancreas.

Abstract A10: In search of Kras resistance genes: Whole transcriptome analysis identifies critical pathways mediating resistance and sensitivity to oncogenic Kras

Abstract In human tumorigenesis, mutational oncogene activation is postulated to occur in either single cells or small numbers of cells, followed by clonal expansion and tumor initiation. In the field of pancreatic cancer research, transgenic animal models mimicking the human disease have contributed to major advancements in our understanding of pancreatic cancer biology. These models have allowed the effects of oncogenic Kras (KrasG12D) activation to be evaluated in different pancreatic compartments using Cre/Lox technology. When oncogenic Kras is expressed under the control of endogenous Kras regulatory elements and selectively activated in embryonic pancreatic epithelium using either Pdx-1:Cre, Ptf1a:Cre or Nestin:Cre driver lines, the KrasG12D allele induces murine pancreatic intraepithelial neoplastic lesions (mPanIN) at as early as two weeks of age. In adult pancreas, selective activation of oncogenic Kras in the acinar cell compartment using either tamoxifen-regulated Elastase (Ela):CreERT2, Mist1:CreERT2 or Ptf1:CreER driver lines also leads to effective mPanIN formation, supporting the possibility that adult acinar cells may serve as the cell type of origin for “ductal” neoplasia. In comparison, activation of oncogenic Kras in Sox9-expressing ductal epithelium leads to only low frequency mPanIN formation (Kopp et al, 2012), indicating that Sox9-expressing duct cells may be more resistant to transformation by oncogenic Kras. We have thoroughly interrogated the ability of the pancreatic ductal epithelium to respond to oncogenic Kras using the HNF1β:CreERT2 transgenic mouse. Our unpublished data support the findings of Kopp et al., as activation of Kras fails to induce mPanIN up to six months after the induction of recombination. Furthermore, combined KrasG12D activation and loss of the potent tumor suppressor p53 also fails to induce mPanIN formation from the HNF1β ductal compartment. In order to better understand the differential responsiveness of adult acinar and ductal cells to oncogenic Kras, we have FACS sorted acinar cells from PanIN-forming Mist1:CreERT2;LsL-Kras;mTmG mice and duct cells from non-PanIN forming HNF1β:CreERT2;LsL-Kras;mTmG mice at 0, 1 and 3 weeks following tamoxifen administration, and prior to the onset of any morphologic change. Following FACS-based cell isolation, we have performed whole genome transcriptional profiling to identify candidate genes mediating differential responsiveness to oncogenic Kras. This approach has identified a panel of candidate genes mediating Kras resistance in ductal cells, and a corresponding panel of candidate genes mediating Kras sensitivity in acinar cells. Genes potentially mediating Kras resistance in the ductal compartment include Plkha1, Slc9a9 and Pdcd10, while the loss of Gmnn and Med6 in the acinar cells appears to be associated with cell type-specific sensitivity to oncogenic Kras. Functional interrogation of identified genes is currently underway. Citation Format: Jennifer M. Bailey, Janivette Alsina, Florencia McAllister, Audrey Hendley, Kelly Lafaro, Anzer Habibulla, Luigi Marchionni, Anirban Maitra, Steven Leach. In search of Kras resistance genes: Whole transcriptome analysis identifies critical pathways mediating resistance and sensitivity to oncogenic Kras. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A10.

Trefoil Factor Family 1 (TFF1) Inhibit the Invasion of Tumor Cells of the Pancreas

ObjectiveThe tumor suppressive role of TFF has been recently reported in gastric tumorigenesis. Here we aimed to clarify the role of TFF1 as tumor suppressor in pancreatic tumorigenesis.MethodsBy immunohistochemistry, the expression pattern of TFF1 was analyzed in surgically resected specimen of PDAC (Pancreatic Ductal Adenocarcinoma; n=14), invasive IPMN (Intraductal Papillary Mucinous Neoplasms: n=12) and non‐invasive IPMN (n=10). Human pancreatic cancer cell line was treated by TFF1‐siRNA to evaluate the role of TFF1 in vitro.ResultsTFF1 expression is not found in normal pancreatic ductal epithelium. Pancreatic Intraductal Neoplasm (PanIN) has high expression of TFF1, suggesting that up‐regulation of TFF1 is the early event of pancreatic tumorigenesis. While TFF1 is also expressed in the intraductal compartment of PDAC and IPMN, it is frequently lost in the invasive compartment (50% (7/14) in PDAC and 58.3% (7/12) in invasive IPMN). The expressions of TFF1 in human pancreatic cancer cell lines were evaluated and Panc1 was found to have TFF1 mRNA. Suppression of TFF1 in Panc1 cells by siRNA does not change the proliferative rate of Panc1 cells; however, it was found to induce the higher invasive ability (55.2% of invasive area in control, 94.9% in TFF1‐siRNA; p<0.01) by Boyden chamber assay.ConclusionWhile acquisition of TFF1 seems to be the early event of pancreatic tumorigenesis, loss of TFF1 may result in the invasive transformation of pancreatic tumor.

Comparison of neutrophil infiltration between type 1 and type 2 autoimmune pancreatitis

BackgroundCharacteristics of type 2 autoimmune pancreatitis (AIP) is granulocyte epithelial lesions, called idiopathic duct-centric pancreatitis (IDCP). To clarify pathogenesis of IDCP, we investigated mechanism of neutrophil infiltration in type 1 AIP, called lymphoplasmacytic sclerosing pancreatitis (LPSP) and IDCP. MethodThis study was performed on resected pancreata from patients with alcoholic chronic pancreatitis (ACP, n = 10), LPSP (n = 10) and IDCP (n = 12). The number of neutrophils around the pancreatic ducts was counted. The expression of neutrophils chemoattractants granulocyte chemotactic protein-2 (GCP-2) and interleukin-8 (IL-8) in the pancreatic duct epithelia was examined using immunohistochemistry. The cell staining intensity is scored as negative (0), weak (1), moderate (2) or strong (3). ResultsThe median number of neutrophils around the interlobular pancreatic ducts was significantly higher in IDCP (15.16; interquartile range [IQR]: 9.74–18.41) than in ACP (2.66; IQR: 1.33–4.33) (P < 0.05) and LPSP (3.16; IQR: 2.74–4.57) (P < 0.01). There was no significant difference in the median number of neutrophils around the intralobular pancreatic ducts among ACP (1.16; IQR: 0.33–3.41), LPSP (3.16; IQR: 0.74–5.5) and IDCP (3.00; IQR: 1.08–7.91). The median score of GCP-2 in the interlobular pancreatic duct epithelia was significantly higher in IDCP (1.5; IQR: 0.25–2) than in ACP (0; IQR: 0–0.75) (P < 0.05) and LPSP (0; IQR: 0–0.75) (P < 0.05). There was no significant difference in the median score of IL-8 in the interlobular pancreatic duct epithelia among ACP (0; IQR: 0–0.75), LPSP (1; IQR: 0–1.75) and IDCP (0.5; IQR: 0–1). ConclusionsSignificantly increased neutrophil infiltration around the interlobular pancreatic duct in IDCP may depend on GCP-2.

Intraductal papillary mucinous neoplasms of the pancreas (IPMNs): epidemiology, diagnosis and future aspects.

Intraductal papillary mucinous neoplasms of the pancreas (IPMNs) are potentially malignant intraductal epithelial neoplasms which consist of columnar, mucin-containing cells and arise from the epithelium of the main pancreatic duct or its branches. IPMNs as well as pancreatic intraepithelial neoplasias (PanINs) and mucinous cystic neoplasms represent noninvasive precursors of invasive ductal adenocarcinoma of the pancreas. The diagnosis of IPMNs includes radiographic (CT scanning, MRI, MRCP) and endoscopic evaluation (ERCP, EUS), PET, as well as serum tumor markers and molecular markers. The Sendai Consensus Guidelines help guide surgical resection for patients with IPMN. The follow-up of these patients, as well as of those who do not undergo surgical resection, is of great importance, since patients with IPMN appear to be at risk for other malignancies. Herein, the authors summarize the data presented at the 2013 ASCO Gastrointestinal Cancers Symposium regarding incidence and clinicopathological characteristics of IPMN (Abstracts #324, #187 and #179).

Gradual telomere shortening and increasing chromosomal instability among PanIN grades and normal ductal epithelia with and without cancer in the pancreas.

A large body of evidence supports a key role for telomere dysfunction in carcinogenesis due to the induction of chromosomal instability. To study telomere shortening in precancerous pancreatic lesions, we measured telomere lengths using quantitative fluorescence in situ hybridization in the normal pancreatic duct epithelium, pancreatic intraepithelial neoplasias (PanINs), and cancers. The materials employed included surgically resected pancreatic specimens without cancer (n = 33) and with invasive ductal carcinoma (n = 36), as well as control autopsy cases (n = 150). In comparison with normal ducts, telomere length was decreased in PanIN-1, −2 and −3 and cancer. Furthermore, telomeres were shorter in cancer than in PanIN-1 and −2. Telomere length in cancer was not associated with histological type, lesion location, or cancer stage. PanINs with or without cancer showed similar telomere lengths. The incidences of atypical mitosis and anaphase bridges, which are morphological characteristics of chromosomal instability, were negatively correlated with telomere length. The telomeres in normal duct epithelium became shorter with aging, and those in PanINs or cancers were shorter than in age-matched controls, suggesting that telomere shortening occurs even when histological changes are absent. Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium might be associated with carcinogenesis of the pancreas. Determination of telomere length in pancreatic ductal lesions may be valuable for accurate detection and risk assessment of pancreatic cancer.

SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium.

Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.

ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca2+-activated Cl− channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca2+ and voltage-dependent Cl− currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1598-8) contains supplementary material, which is available to authorized users.

EROD activity and cytochrome P4501A induction in liver and gills of Senegal sole Solea senegalensis from a polluted Huelva Estuary (SW Spain)

EROD activity and induction cytochrome P4501A in liver and gills of Senegal sole, Solea senegalensis, from a heavy metal and PAH polluted estuary, was studied. Liver and gill CYP1A catalytic activity was assessed at the enzyme activity level-measured as 7-ethoxyresorufin-O-deethylase and cellular localization of CYP1A in the liver was studied using immunohistochemistry. Liver EROD was correlated with phenanthrene-type metabolites in liver and copper concentrations in water. Strong CYP1A occurrence was observed in acinar pancreatic cells, pancreatic duct epithelium and vascular system endothelium and negative/rare induction were observed in hepatocytes and sinusoidal endothelium. In gills, EROD activity showed a significant correlation with different fractions of heavy metals in sediment but no correlation was observed between EROD activity and PAHs. Strongly positive CYP1A associated staining of the vascular system endothelia and primary filament cells and a moderate staining of pillar cells in gills were observed. The results substantiated the utility of EROD activity and CYP1A induction measurement as biomarkers for use by aquatic toxicologists and indicate that catalytic assays and immunohistochemical assays appear to be sensitive to different kinds of pollutants being the use of both methods recommended for monitoring programs.

Особенности гистоструктуры поджелудочной железы при аутоиммунном панкреатите I и II типов: связь с уровнем IgG4-положительных плазматических клеток

Aims/hypothesis. The study was undertaken to examine the special aspects of pancreatic histopathology in patients with autoimmune pancreatitis (AIP) type I and II, in connection with the level of IgG4-positive plasma cells (PPC) in the tissue.Material and methods. The study was conducted on 54 patients with chronic pancreatitis, of whom15 cases with autoimmune pancreatitis were selected. Detailed histological evaluation in three topographical zones of the pancreas (head, body and tail) was carried out for each case.Results and interpretation. AIP type I was found in five cases. In all cases in pancreas (all three zones) stages III-IV of AIP were observed with characteristic dense lymphocytic and plasma cells infiltration, multilevel fibrosis and obliterating phlebitis. In the liver hepatitis was observed with a slight fibrosis and plasma cells infiltration (up to 5-PPC per high power field), IgG4-PPC were located in dilated portal tracts. The high level of IgG-PPC and IgG4-PPC in the pancreatic tissue (≥ 30 IgG4-PPC per high power field) also confirmed the diagnosis of AIP type I, as IgG4-associated sclerosing disease. These patients had a diffuse damage of the pancreas at III (3 cases) and IV (2 cases) stages. All cases were marked by complicated disease course of AIP: in 60% (3 people) – a chronic fibrocystic pancreatitis, and 40% (2 people) – a chronic calculus pancreatopathy. In all the patients with AIP type I the dense plasma cells infiltrate around main and interlobular ducts and pancreatic lymph follicles was found with compact clusters of IgG4-PPC (≥ 30 IgG4-PPC per high power field).AIP type II was found in other 10 cases. Two of them were characterized by diffuse, and eight – by the focal form of AIP. Stage IV of AIP was detected in one patient with the total damage of pancreas, and the AIP on II stage of development in another. AIP activity in these patients was minimal. Focal form of AIP type II which was located in the body of the pancreas (II-III stage AIP) was observed in three cases, in the tail (I and II Stage AIP) – in two cases, and in the head – in one case (I stage AIP). Another two cases showed conjoined destruction of the body and tail (II stage).In the focal form of AIP type II in epithelium of main pancreatic duct, collecting ducts and acini single and multiple neutrophilic leukocytes were observed, including the formation of microabscesses. Areas of desquamation, epithelial mitotic figures were observed in ductal epithelium. Suchlike high activity of AIP type II was observed in half of the cases. Lymphocytic and plasma cells infiltration was located not only in the ducts area but also in the stroma and lobules. For AIP type II at stages III-IV obliterating phlebitis located in the fibrous tissue was typical. In periductal fibrous tissue clusters of IgG PPC were identified. All cases show focal compact clusters of IgG4-PPC in periductal, perilobular and fibrotic tissue. IgG4-PPC were counted to the limit of 5-10 per high power field.Conclusions. Autoimmune pancreatitis type I is marked by dense lymphocytic and plasma cells infiltrate in periductal zone, multilevel fibrosis, obliterating venulitis and a high level of IgG4-PPC in the pancreas (≥ 30 IgG4-PPC per high power field). Autoimmune pancreatitis type II, in addition to the known histopathologic features of AIP, was defined by neutrophilic leukocyte epithelial damage of pancreatic ducts and low level of IgG4-PPC in the pancreas. For AIP type II in 80% of cases focal damage of the pancreas at stage I-III was typical. In perspective those findings will lead to developing of simple and affordable diagnostic for AIP type I and II.

Mucus secretion of pancreatic ductal epithelium increases in chronic pancreatitis

Anita Bal azs , Duerr Julia , Zhe Zhou-Suckow , Jolanthe Schatterny , Istv an Bal azs N emeth , Tiszlavicz L aszl o , Zolt an Rakonczay , Marcus A. Mall , P eter Hegyi a a First Department of Medicine, University of Szeged, Szeged, Hungary Department of Translational Pulmonology, Translational Lung Research Center, University of Heidelberg, Heidelberg, Germany Department of Pathology University of Szeged, Szeged, Hungary

L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression

Regulatory T cell (T-reg) enrichment in the tumor microenvironment is regarded as an important mechanism of tumor immune escape. Hence, the presence of T-regs in highly malignant pancreatic ductal adenocarcinoma (PDAC) is correlated with short survival. Likewise, the adhesion molecule L1CAM is upregulated during PDAC progression in the pancreatic ductal epithelium also being associated with poor prognosis. To investigate whether L1CAM contributes to enrichment of T-regs in PDAC, human CD4+CD25+CD127−CD49d− T-regs and CD4+CD25− T-effector cells (T-effs) were isolated by magnetic bead separation from blood of healthy donors. Their phenotype and functional behavior were analyzed in dependence on human premalignant (H6c7) or malignant (Panc1) pancreatic ductal epithelial cells, either exhibiting or lacking L1CAM expression. T cells derived from blood and tumors of PDAC patients were analyzed by flow cytometry and findings were correlated with clinical parameters. Predominantly T-regs but not T-effs showed an increased migration on L1CAM expressing H6c7 and Panc1 cells. Whereas proliferation of T-regs did not change in the presence of L1CAM, T-effs proliferated less, exhibited a decreased CD25 expression and an increased expression of CD69. Moreover, these T-effs exhibited a regulatory phenotype as they inhibited proliferation of autologous T cells. Accordingly, CD4+CD25−CD69+ T cells were highly abundant in PDAC tissues compared to blood being associated with nodal invasion and higher grading in PDAC patients. Overall, these data point to an important role of L1CAM in the enrichment of immunosuppressive T cells in particular of a CD4+CD25−CD69+-phenotype in PDAC providing a novel mechanism of tumor immune escape which contributes to tumor progression.