Publications by authors named "Jörg Schrader"

34 Publications

Finding the Appropriate Therapeutic Strategy in Patients with Neuroendocrine Tumors of the Pancreas: Guideline Recommendations Meet the Clinical Reality.

J Clin Med 2021 Jul 7;10(14). Epub 2021 Jul 7.

Department of Gastroenterology and Endocrinology, University Hospital Marburg, Baldinger Strasse, D 35043 Marburg, Germany.

The systemic treatment of patients with pancreatic neuroendocrine tumors is based on placebo-controlled trials and long-established chemotherapy approaches. In addition, peptide receptor radionuclide therapy (PRRT) was approved as a parallel approach for pancreatic neuroendocrine tumors (NET), in addition to small bowel NET, after the NETTER-1 trial. The current ESMO and NCCN guidelines attempted to describe treatment algorithms for pancreatic NET based on the current data. In our survey, we recorded therapy decisions for the first- until the third-line of therapy in German-speaking countries (Germany, Austria, and Switzerland) using fictional case reports and discussed these in the context of the current ESMO guidelines. Compared with the recommendations of the guidelines, PRRT was used more frequently and earlier. In patients with NET G1/G2 Ki-67 < 10%, the therapy algorithm consisting of somatostatin analogs (SSA)-PRRT-targeted therapy is a relevant approach. In clinical situations where chemotherapy is primarily used (remission pressure, Ki-67 > 10%), second-line PRRT was found acceptance and was often considered prior to targeted therapies. Despite the lack of prospective controlled trials, our study demonstrated the pivotal impact of PRRT. Therefore, further studies should compare PRRT with chemotherapy in pancreatic NETs in different clinical settings in first- and second-line approaches.
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http://dx.doi.org/10.3390/jcm10143023DOI Listing
July 2021

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Cells 2021 Jun 6;10(6). Epub 2021 Jun 6.

Department of Internal Medicine I, Martin Luther University, D-06120 Halle (Saale), Germany.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.
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http://dx.doi.org/10.3390/cells10061408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228033PMC
June 2021

Prevalence and risk factors of undiagnosed diabetes mellitus among gastroenterological patients: a HbA1c-based single center experience.

Z Gastroenterol 2021 Jun 22. Epub 2021 Jun 22.

I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Diabetes mellitus is a major risk factor for microvascular disease, leading to chronic kidney injury or cardiovascular disease, but there is a tremendous proportion of patients worldwide who suffer from undiagnosed diabetes. Until now, little is known about the prevalence of undiagnosed diabetes in gastroenterology inpatients.

Objective: To improve detection of undiagnosed diabetes, a routine screening procedure for gastroenterology inpatients, based on hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) measurement, was established.

Methods: We conducted a retrospective analysis of the implemented diabetes screening. Diabetes mellitus was diagnosed according to the guideline of the German Diabetes Association in patients with an HbA1c of ≥6.5% anld/or fasting plasma glucose (FPG) ≥126 mg/dL. Univariate and multivariate analyses were performed to identify independent risk factors for undiagnosed diabetes.

Results: Within a 3-month period, 606 patients were eligible for a diabetes screening. Pre-existing diabetes was documented in 120 patients (19.8 %), undiagnosed diabetes was found in 24 (3.9%), and 162 patients (26.7%) met the definition for prediabetes. Steroid medication use, age, and liver cirrhosis due to primary sclerosing cholangitis (PSC) were identified as risk factors for undiagnosed diabetes.

Conclusion: The prevalence of undiagnosed diabetes in gastroenterology inpatients is markedly elevated in comparison to the general population, and a substantial number of inpatients are in a prediabetic status, underlining the need for diabetes screening. In addition to previously described risk factors of patient age and steroid medication use, we identified PSC-related liver cirrhosis (but not liver cirrhosis due to another etiology) as an independent risk factor for undiagnosed diabetes.
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http://dx.doi.org/10.1055/a-1482-8840DOI Listing
June 2021

PI3K/mTOR Dual Inhibitor PF-04691502 Is a Schedule-Dependent Radiosensitizer for Gastroenteropancreatic Neuroendocrine Tumors.

Cells 2021 May 20;10(5). Epub 2021 May 20.

Markey Cancer Center, University of Kentucky, Lexington, KY 40536, USA.

Patients with advanced-stage gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have a poor overall prognosis despite chemotherapy and radiotherapy (e.g., peptide receptor radionuclide therapy (PRRT)). Better treatment options are needed to improve disease regression and patient survival. The purpose of this study was to examine a new treatment strategy by combining PI3K/mTOR dual inhibition and radiotherapy. First, we assessed the efficacy of two PI3K/mTOR dual inhibitors, PF-04691502 and PKI-402, to inhibit pAkt and increase apoptosis in NET cell lines (BON and QGP-1) and patient-derived tumor spheroids as single agents or combined with radiotherapy (XRT). Treatment with PF-04691502 decreased pAkt (Ser473) expression for up to 72 h compared with the control; in contrast, decreased pAkt expression was noted for less than 24 h with PKI-402. Simultaneous treatment with PF-04691502 and XRT did not induce apoptosis in NET cells; however, the addition of PF-04691502 48 h after XRT significantly increased apoptosis compared to PF-04691502 or XRT treatment alone. Our results demonstrate that schedule-dependent administration of a PI3K/mTOR inhibitor, combined with XRT, can enhance cytotoxicity by promoting the radiosensitivity of NET cells. Moreover, our findings suggest that radiotherapy, in combination with timed PI3K/mTOR inhibition, may be a promising therapeutic regimen for patients with GEP-NET.
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http://dx.doi.org/10.3390/cells10051261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160730PMC
May 2021

Leptomeningeal Carcinomatosis: A Clinical Dilemma in Neuroendocrine Neoplasms.

Biology (Basel) 2021 Mar 28;10(4). Epub 2021 Mar 28.

Clinic for Internal Medicine I, Martin-Luther University Halle/Wittenberg, Ernst-Grube-Straße 40, 06120 Halle, Germany.

Central nervous system (CNS) involvement by paraneoplastic syndromes, brain metastases, or leptomeningeal carcinomatosis (LC) in patients with neuroendocrine neoplasms (NEN) has only been described in individual case reports. We evaluated patients with LC in four neuroendocrine tumor (NET) centers (Halle/Saale, Hamburg, Heidelberg, and Marburg) and characterized them clinically. In the study, 17 patients with a LC were defined with respect to diagnosis, clinic, and therapy. The prognosis of a LC is very poor, with 10 months in median overall survival (mOS). This is reflected by an even worse course in neuroendocrine carcinoma (NEC) G3 Ki-67 >55%, with a mOS of 2 months. Motor and sensory deficits together with vigilance abnormalities were common symptoms. In most cases, targeted radiation or temozolomide therapy was used against the LC. LC appears to be similarly devastating to brain metastases in NEN patients. Therefore, the indication for CNS imaging should be discussed in certain cases.
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http://dx.doi.org/10.3390/biology10040277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066280PMC
March 2021

[Unusual cause of hypercalcemia in pregnancy].

Z Gastroenterol 2021 Feb 8;59(2):143-148. Epub 2021 Feb 8.

Klinik für Innere Medizin, Albertinen-Krankenhaus, Hamburg, Germany.

Background:  Neuroendocrine tumors (NET) diagnosed during pregnancy are extremely rare. This case report describes diagnosis and treatment of a metastasized pancreas NET that became symptomatic in the second trimester.

Case Description:  A 33-year-old patient presented to the emergency department in the 19 week of pregnancy (WOP) with persistent diarrhea. Laboratory tests showed a pronounced hypercalcemia (3.53 mmol/l). Imaging revealed a mass in the pancreatic corpus/tail with extensive liver metastasis. Histologically, a NET (G2, SSTR-positive) with paraneoplastic parathormone-related-peptide secretion was found to be the cause of hypercalcemia. Under a treatment with octreotide, calcium values normalized and diarrhea stopped. After delivery of a healthy child (32.WOP via cesarean section) tumor progress was found. The pancreatic mass was resected completely, the liver metastases as far as possible. Postoperatively, in a CT scan, residual suspicious liver lesions could be found, and a palliative therapy with lanreotide was initiated. With this treatment, the patient has been asymptomatic for one year, and serum calcium remained normal. The child developed normally.

Discussion:  This unusual case shows that even in extensively metastasized symptomatic NETs during pregnancy, there may be sufficient diagnostic and therapeutic options that allow for a continuation of pregnancy in close interdisciplinary cooperation under careful risk-benefit assessment for mother and child.
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http://dx.doi.org/10.1055/a-1340-0452DOI Listing
February 2021

Gastric NET Subtypes: Do We Need An Additional One?

Z Gastroenterol 2021 Mar 27;59(3):255-258. Epub 2021 Jan 27.

I. Medizinische Klinik und Poliklinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Depending on etiology, prognosis and malignant potential, recent S2k guideline differentiates gastric neuroendocrine tumors (gNET) in 4 types with different treatment implications.We report on a 55-year-old patient with the accidental finding of a 15 mm gNET. Apart from a prolonged use of proton pump inhibitors (PPI) for 20 years as a treatment for gastroesophageal reflux disease, there were no other associations or risk factors for gNETs. Formally, this patient would have been classified as a type III gNET, implicating gastric surgery. From a pathophysiological point of view, however, the assumed prolonged gastrin hypersecretion would have justified an assignment as a type I gNET. The gNET was resected by ESD, but histology showed an R1 situation. After cessation of PPIs, there is no recurrence so far. Besides, the initially documented numerous and large gland polyps showed an impressive regression only a few weeks after cessation of PPI.This case points to a probably underestimated gap in the present gNET classification. On the basis of present literature, the therapeutic dilemma of PPI-associated gNETs is discussed. A new assignment of PPI associated gNETs as type Ib could help to overcome this dilemma.
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http://dx.doi.org/10.1055/a-1348-2727DOI Listing
March 2021

Possible tumour cell reimplantation during curative endoscopic therapy of superficial Barrett's carcinoma.

Gut 2021 Jan 13. Epub 2021 Jan 13.

Department of Interdisciplinary Endoscopy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background And Aims: Endoscopic resection has been established as curative therapy for superficial cancer arising from Barrett's oesophagus (BE); recurrences are very rare. Based on a case series with unusual and massive early recurrences, we analyse the issue of tumour cell reimplantation.

Methods: This hypothesis was developed on the basis of two out of seven patients treated by circumferential (n=6) or nearly circumferential (n=1) en bloc and R0 endoscopic resection of T1 neoplastic BE. Subsequently, a prospective histocytological analysis of endoscope channels and accessories was performed in 2 phases (cytohistological analysis; test for cell viability) in 22 different oesophageal carcinoma patients undergoing endoscopy. Finally, cultures from two oesophageal adenocarcinoma cell lines were incubated with different triamcinolone concentrations (0.625-10 mg/mL); cell growth was determined on a Multiwell plate reader.

Results: Cancer regrowth in the two suspicious cases (male, 78/71 years) occurred 7 and 1 months, respectively, after curative tumour resection. Subsequent surgery showed advanced tumours (T2) with lymph node metastases; one patient died. On cytohistological examinations of channels and accessories, suspicious/neoplastic cells were found in 4/10 superficial and in all 5 advanced cancers. Further analyses in seven further advanced adenocarcinoma cases showed viable cells in two channel washing specimens. Finally, cell culture experiments demonstrated enhanced tumour cell growth by triamcinolone after 24 hours compared with controls.

Conclusions: Tumour cell reimplanation from contaminated endoscopes and accessories is a possible cause of local recurrence after curative endoscopic therapy for superficial Barrett carcinoma; also, corticosteroid injection could have promoted tumour regrowth in these cases.
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http://dx.doi.org/10.1136/gutjnl-2020-322723DOI Listing
January 2021

Professional Assessment of the Impact of COVID-19 on Handling NET Patients.

J Clin Med 2020 Nov 11;9(11). Epub 2020 Nov 11.

I. Medical Department-Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.

The treatment and monitoring of patients with neuroendocrine tumors (NET) has been a major challenge during the COVID-19 pandemic. In a survey, we investigated the influence of COVID-19 on the care of NET patients in the German speaking countries Germany, Austria and Switzerland. The multidisciplinarity of all treating physicians in the outpatient and inpatient sector was reflected in our survey. Furthermore, we were able to present findings pertaining to the university and non-university medical care. Overall, only a minority of appointments were cancelled, mostly as a result of medical prioritization and less for fear of infection by patients. In the university sector, longer delays for diagnostic measures were observed in comparison to non-university care. During the COVID-19 crisis, NET patients rarely changed their current therapy, but the pandemic impacted the assessment of the different treatment modalities at risk of developing severe COVID-19 disease. This survey provides the first real-world data on the treatment of NET patients from the physicians' perspective during the COVID-19 crisis. Despite delays in diagnostic procedures and outpatient appointments, only a minority of physicians foresee a major impact of COVID-19 on NET patient care.
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http://dx.doi.org/10.3390/jcm9113633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696769PMC
November 2020

A Comprehensive Molecular Characterization of the Pancreatic Neuroendocrine Tumor Cell Lines BON-1 and QGP-1.

Cancers (Basel) 2020 Mar 14;12(3). Epub 2020 Mar 14.

First Department of Medicine, University Hospital Schleswig-Holstein, Campus Lübeck, D-23538 Lübeck, Germany.

Experimental models of neuroendocrine tumor disease are scarce, with only a few existing neuroendocrine tumor cell lines of pancreatic origin (panNET). Their molecular characterization has so far focused on the neuroendocrine phenotype and cancer-related mutations, while a transcription-based assessment of their developmental origin and malignant potential is lacking. In this study, we performed immunoblotting and qPCR analysis of neuroendocrine, epithelial, developmental endocrine-related genes as well as next-generation sequencing (NGS) analysis of microRNAs (miRs) on three panNET cell lines, BON-1, QGP-1, and NT-3. All three lines displayed a neuroendocrine and epithelial phenotype; however, while insulinoma-derived NT-3 cells preferentially expressed markers of mature functional pancreatic β-cells (i.e., , ), both BON-1 and QGP-1 displayed high expression of genes associated with immature or non-functional β/δ-cells genes (i.e., ), or pancreatic endocrine progenitors (i.e., ). NGS-based identification of miRs in BON-1 and QGP-1 cells revealed the presence of all six members of the miR-17-92 cluster, which have been implicated in b-cell function and differentiation, but also have roles in cancer being both oncogenic or tumor suppressive. Notably, both BON-1 and QGP-1 cells expressed several miRs known to be negatively associated with epithelial-mesenchymal transition, invasion or metastasis. Moreover, both cell lines failed to exhibit migratory activity in vitro. Taken together, NT-3 cells resemble mature functional β-cells, while both BON-1 and QGP-1 are more similar to immature/non-functional pancreatic β/δ-cells or pancreatic endocrine progenitors. Based on the recent identification of three transcriptional subtypes in panNETs, NT-3 cells resemble the "islet/insulinoma tumors" (IT) subtype, while BON-1 and QGP-1 cells were tentatively classified as "metastasis-like/primary" (MLP). Our results provide a comprehensive characterization of three panNET cell lines and demonstrate their relevance as neuroendocrine tumor models.
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http://dx.doi.org/10.3390/cancers12030691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140066PMC
March 2020

Homogeneous MMR Deficiency Throughout the Entire Tumor Mass Occurs in a Subset of Colorectal Neuroendocrine Carcinomas.

Endocr Pathol 2020 Jun;31(2):182-189

General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)-based analysis using the "Bethesda Panel" confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass.
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http://dx.doi.org/10.1007/s12022-020-09612-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250944PMC
June 2020

MMR Deficiency is Homogeneous in Pancreatic Carcinoma and Associated with High Density of Cd8-Positive Lymphocytes.

Ann Surg Oncol 2020 Oct 27;27(10):3997-4006. Epub 2020 Feb 27.

Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Microsatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking.

Methods: To study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6.

Results: In six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (p < 0.0001), suggesting a role of MSI for immune response.

Conclusions: Our data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.
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http://dx.doi.org/10.1245/s10434-020-08209-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471097PMC
October 2020

Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice.

Exp Mol Med 2018 10 10;50(10):1-13. Epub 2018 Oct 10.

Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg- Eppendorf (UKE), Hamburg, Germany.

Cosmc is ubiquitously expressed and acts as a specific molecular chaperone assisting the folding and stability of core 1 synthase. Thus, it plays a crucial role in the biosynthesis of O-linked glycosylation of proteins. Here, we show that ablation of Cosmc in the exocrine pancreas of mice causes expression of truncated O-glycans (Tn antigen), resulting in exocrine pancreatic insufficiency with decreased activities of digestive enzymes and diabetes. To understand the molecular causes of the pleiotropic phenotype, we used Vicia villosa agglutinin to enrich Tn antigen-modified proteins from Cosmc-KO pancreatic lysates and performed a proteomic analysis. Interestingly, a variety of proteins were identified, of which bile salt-activated lipase (also denoted carboxyl-ester lipase, Cel) was the most abundant. In humans, frameshift mutations in CEL cause maturity-onset diabetes of the young type 8 (MODY8), a monogenic syndrome of diabetes and pancreatic exocrine dysfunction. Here, we provide data suggesting that differentially O-glycosylated Cel could negatively affect beta cell function. Taken together, our findings demonstrate the importance of correct O-glycan formation for normal exocrine and endocrine pancreatic function, implying that aberrant O-glycans might be relevant for pathogenic mechanisms of the pancreas.
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http://dx.doi.org/10.1038/s12276-018-0157-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180059PMC
October 2018

The role of GSK3 and its reversal with GSK3 antagonism in everolimus resistance.

Endocr Relat Cancer 2018 10 12;25(10):893-908. Epub 2018 Jun 12.

Medizinische Klinik und Poliklinik IVInterdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System (GEPNET-KUM), Klinikum der Universität München (KUM), Ludwig-Maximilians-University, Munich, Germany

Pancreatic neuroendocrine tumors (panNETs) are often inoperable at diagnosis. The mTORC1 inhibitor everolimus has been approved for the treatment of advanced NETs. However, the regular development of resistance to everolimus limits its clinical efficacy. We established two independent everolimus-resistant panNET (BON1) cell lines (BON1 RR1, BON1 RR2) to find potential mechanisms of resistance. After 24 weeks of permanent exposure to 10 nM everolimus, BON1 RR1 and BON1 RR2 showed stable resistance with cellular survival rates of 96.70% (IC = 5200 nM) and 92.30% (IC = 2500 nM), respectively. The control cell line showed sensitivity to 10 nM everolimus with cellular survival declining to 54.70% (IC = 34 nM). Both resistant cell lines did not regain sensitivity over time and showed persistent stable resistance after a drug holiday of 13 weeks. The mechanisms of resistance in our cell line model included morphological adaptations, G1 cell cycle arrest associated with reduced CDK1(cdc2) expression and decreased autophagy. Cellular migration potential was increased and indirectly linked to c-Met activation. GSK3 was over-activated in association with reduced baseline IRS-1 protein levels. Specific GSK3 inhibition strongly decreased BON1 RR1/RR2 cell survival. The combination of everolimus with the PI3Kα inhibitor BYL719 re-established everolimus sensitivity through GSK3 inhibition and restoration of autophagy. We suggest that GSK3 over-activation combined with decreased baseline IRS-1 protein levels and decreased autophagy may be a crucial feature of everolimus resistance, and hence, a possible therapeutic target.
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http://dx.doi.org/10.1530/ERC-18-0159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439002PMC
October 2018

Establishment of the First Well-differentiated Human Pancreatic Neuroendocrine Tumor Model.

Mol Cancer Res 2018 03 12;16(3):496-507. Epub 2018 Jan 12.

I. Medical Department - Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Clinical options for systemic therapy of neuroendocrine tumors (NET) are limited. Development of new drugs requires suitable representative and model systems. So far, the unavailability of a human model with a well-differentiated phenotype and typical growth characteristics has impaired preclinical research in NET. Herein, we establish and characterize a lymph node-derived cell line (NT-3) from a male patient with well-differentiated pancreatic NET. Neuroendocrine differentiation and tumor biology was compared with existing NET cell lines BON and QGP-1. growth was assessed in a xenograft mouse model. The neuroendocrine identity of NT-3 was verified by expression of multiple NET-specific markers, which were highly expressed in NT-3 compared with BON and QGP-1. In addition, NT-3 expressed and secreted insulin. Until now, this well-differentiated phenotype is stable since 58 passages. The proliferative labeling index, measured by Ki-67, of 14.6% ± 1.0% in NT-3 is akin to the original tumor (15%-20%), and was lower than in BON (80.6% ± 3.3%) and QGP-1 (82.6% ± 1.0%). NT-3 highly expressed somatostatin receptors (SSTRs: 1, 2, 3, and 5). Upon subcutaneous transplantation of NT-3 cells, recipient mice developed tumors with an efficient tumor take rate (94%) and growth rate (139% ± 13%) by 4 weeks. Importantly, morphology and neuroendocrine marker expression of xenograft tumors resembled the original human tumor. High expression of somatostatin receptors and a well-differentiated phenotype as well as a slow growth rate qualify the new cell line as a relevant model to study neuroendocrine tumor biology and to develop new tumor treatments. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0163DOI Listing
March 2018

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models.

PLoS One 2017 11;12(8):e0182852. Epub 2017 Aug 11.

Dept. of Gastroenterology, CC13 (CBF and CVK), Charité - Universitätsmedizin Berlin, Berlin, Germany.

Background/aims: The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines.

Methods: Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA.

Results: BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately.

Conclusion: Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182852PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553670PMC
October 2017

Activation of the Akt-CREB signalling axis by a proline-rich heptapeptide confers resistance to stress-induced cell death and inflammation.

Immunology 2017 08 16;151(4):474-480. Epub 2017 May 16.

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

Cell stress of various kinds can lead to the induction of cell death and a damaging inflammatory response. Hence, a goal of therapeutic cell-stress management is to develop agents that might effectively regulate undesirable cell death and inflammation. To that end, we developed a synthetic peptide of seven amino acids based on structural mimicry to a functional domain of p53, a key factor in the responses of cells to stressful stimuli. This heptapeptide, which we term Stressin-1, was found to inhibit both cell death and the secretion of inflammatory mediators by various cell types in response to different stressful agents in vitro. The combined anti-inflammatory and anti-apoptotic activities of Stressin-1 were associated with a cellular signalling cascade that induced activation of Akt kinase and activation of the cAMP response element-binding protein (CREB) transcription factor. These immediate signalling events led to the inhibition of the signal transducer and activator of transcription and nuclear factor-κB pathways 24 hr later. Unexpectedly, we found no evidence for a direct involvement of p53 in the effects produced by Stressin-1. Intraperitoneal administration of 100 μg of Stressin-1 to lethally irradiated mice significantly protected them from death. These findings show that activating the Akt-CREB axis with Stressin-1 can counteract some of the undesirable effects of various cell stresses. Stressin-1 may have clinical usefulness.
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http://dx.doi.org/10.1111/imm.12745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506396PMC
August 2017

Matrix conditions and KLF2-dependent induction of heme oxygenase-1 modulate inhibition of HCV replication by fluvastatin.

PLoS One 2014 6;9(5):e96533. Epub 2014 May 6.

Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background & Aims: HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.

Methods: HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.

Results: All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.

Conclusions: Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011762PMC
January 2015

The role of MDSCs in hepatocellular carcinoma--in vivo veritas?

Authors:
Jörg Schrader

J Hepatol 2013 Nov 16;59(5):921-3. Epub 2013 Aug 16.

I. Medical Department, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2013.08.003DOI Listing
November 2013

Targeting aurora kinases with danusertib (PHA-739358) inhibits growth of liver metastases from gastroenteropancreatic neuroendocrine tumors in an orthotopic xenograft model.

Clin Cancer Res 2012 Sep 2;18(17):4621-32. Epub 2012 Jul 2.

I. Medizinische Klinik, Diagnostische und Interventionelle Radiologie, II. Medizinische Klinik, Onkologisches Zentrum, Universitätsklinikum Hamburg-Eppendorf; Labor Lademannbogen, Hamburg, Germany.

Purpose: Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies.

Experimental Design: Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo.

Results: The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases.

Conclusions: These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-2968DOI Listing
September 2012

Chronic liver inflammation dominated by interferon-γ can prevent hepatocarcinogenesis.

Oncoimmunology 2012 Mar;1(2):222-223

Department of Medicine I; University Medical Centre Hamburg-Eppendorf; Hamburg, Germany.

Inflammation is a major stimulus for carcinogenesis; however inflammation can also inhibit tumor growth and deplete malignant cells. The differences between cancer-promoting and cancer-inhibitory inflammation are not clear. We identified Interferon-γ as a major mediator of cancer-inhibitory inflammation that promotes anti-cancer immunity in the liver and sensitizes malignant hepatocytes for apoptosis.
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http://dx.doi.org/10.4161/onci.1.2.18114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376979PMC
March 2012

Chronic inflammatory IFN-γ signaling suppresses hepatocarcinogenesis in mice by sensitizing hepatocytes for apoptosis.

Cancer Res 2011 Jun 21;71(11):3763-71. Epub 2011 Apr 21.

Department of Medicine I, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

Chronic liver inflammation is a critical component of hepatocarcinogenesis. Indeed, inflammatory mediators are believed to promote liver cancer by upholding compensatory proliferation of hepatocytes in response to tissue damage. However, inflammation can also mediate the depletion of malignant cells, but the difference between tumor-suppressive and tumor-promoting inflammation is not defined at the molecular level. Here, we analyzed the role of the major inflammatory mediator IFN-γ in chemical hepatocarcinogenesis of transgenic mice that overexpress IFN-γ in the liver; these mice manifest severe chronic inflammatory liver damage and lasting compensatory regeneration. We found that chronic exposure to IFN-γ suppressed chemical hepatocarcinogenesis, despite overt liver injury. Indeed, IFN-γ-transgenic mice had significantly fewer and significantly less advanced malignant lesions than nontransgenic mice. This tumor-suppressive effect of IFN-γ seemed to be mediated in part by its known immune activating function, indicated by infiltration of IFN-γ-transgenic livers with CD8 T cells, natural killer T cells, and natural killer cells. However, IFN-γ seemed to prevent carcinogenesis also by activating the cell-intrinsic p53 tumor suppressor pathway. Indeed, exposure to IFN-γ in vivo or in vitro was associated with accumulation of p53 in hepatocytes and the sensitization of hepatocytes to apoptosis induced by genotoxic stress. The IFN-γ-induced increase in apoptosis of hepatocytes seemed to be p53 dependent. Thus, chronic inflammation dominated by IFN-γ may prevent hepatocarcinogenesis, despite continued inflammatory liver injury and regeneration. Therefore, the carcinogenic potential of inflammation seems to be determined by type and composition of its mediators and manipulating the type of chronic inflammation may serve the prevention of cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3232DOI Listing
June 2011

Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells.

Hepatology 2011 Apr;53(4):1192-205

Medical Research Council (MRC) Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; Department of Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Unlabelled: There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of "mechanically tunable" matrix-coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold and 12.2-fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness-dependent regulation of basal and hepatocyte growth factor-stimulated mitogenic signaling through extracellular signal-regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. β1-Integrin and focal adhesion kinase were found to modulate stiffness-dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c-kit, cysteine-X-cysteine receptor 4, octamer-4 (CXCR4), and NANOG.

Conclusion: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;).
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http://dx.doi.org/10.1002/hep.24108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076070PMC
April 2011

The inflammatory microenvironment of HCC - the plot becomes complex.

J Hepatol 2011 May 23;54(5):853-5. Epub 2010 Dec 23.

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http://dx.doi.org/10.1016/j.jhep.2010.12.014DOI Listing
May 2011

Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown.

BMC Cancer 2009 Dec 28;9:464. Epub 2009 Dec 28.

Department of Neurology, University of Marburg, Marburg, Germany.

Background: Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis.

Results: Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1.

Conclusion: Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition.
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http://dx.doi.org/10.1186/1471-2407-9-464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810303PMC
December 2009

Aurora kinase inhibitor PHA-739358 suppresses growth of hepatocellular carcinoma in vitro and in a xenograft mouse model.

Neoplasia 2009 Sep;11(9):934-44

I. Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50 nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G(2)/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735802PMC
http://dx.doi.org/10.1593/neo.09664DOI Listing
September 2009

Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells.

J Immunol 2009 Apr;182(8):4633-40

Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

TGF-beta induces the conversion of CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg). Activin A is a pleiotropic TGF-beta family member and is expressed in response to inflammatory signals. In this study, we report on the effects of activin A on the conversion of CD4(+)CD25(-) T cells into Foxp3-expressing induced Treg (iTreg). Activin A was able to promote the conversion of CD4(+)CD25(-) T cells into iTreg in a dose-dependent manner in vitro. Activin A together with TGF-beta1 had synergistic effects on the rate of iTreg conversion in vitro. Intact TGF-beta1 signaling seemed to be essential for the effects of activin A on iTreg generation because cells overexpressing a dominant negative TGF-beta type II receptor could not be converted by activin A in vitro. In vivo, the frequency of peripheral, but not central, Treg was increased in transgenic mice with elevated activin A serum levels and the in vivo conversion rate of CD4(+)CD25(-) T cells into Foxp3-expressing iTreg was increased as compared with wild type mice. These data suggest a role for activin A as a promoter of the TGF-beta dependent conversion of CD4(+)CD25(-) T cells into iTreg in vitro and in vivo. Therefore, besides promoting inflammation, activin A may contribute to the regulation of inflammation via the expansion of peripheral Treg.
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http://dx.doi.org/10.4049/jimmunol.0803143DOI Listing
April 2009

Senescence of activated stellate cells: not just early retirement.

Hepatology 2009 Mar;49(3):1045-7

MRC Centre for Inflammation Research University of Edinburgh, Edinburgh, UK.

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http://dx.doi.org/10.1002/hep.22832DOI Listing
March 2009

Loss of nuclear p27 expression and its prognostic role in relation to cyclin E and p53 mutation in gastroenteropancreatic neuroendocrine tumors.

Clin Cancer Res 2008 Nov;14(22):7378-84

Medizinische Klinik I, Gastroenterologie/Infektiologie/Rheumatologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.

Purpose: Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are classified by the WHO, yet its prognostic value needs to be confirmed. Therefore, we aimed to determine the prognostic role of cell cycle key regulatory genes p53, p27kip1 (p27), and cyclin E in this tumor entity.

Experimental Design: Tumor specimen from 89 patients with a complete follow-up were studied immunohistochemically for p27 and cyclin E expression and for p53 mutations. The functional relevance of p27 was evaluated in the neuroendocrine cell lines BON1 (human) and INS1 (rat) by the use of small interfering RNA.

Results: Twenty-six of 29 benign, well-differentiated endocrine tumors (WHO class 1) showed a high expression (> 50%) of p27, whereas all 10 poorly differentiated endocrine carcinomas (WHO class 3) displayed a low expression of p27. Metastatic well-differentiated endocrine carcinomas (WHO class 2) showed a low p27 expression in 20 of 50 (40%) patients, which conferred a poor prognosis (median survival, 57 versus 140 months; P = 0.037). This prognostic dichotomy was improved by the use of a combination of p27 and cyclin E (high cyclin E/low p27 versus low cyclin E/high p27: median survival 53 months versus not reached; P = 0.0044). p53 mutations were rare (1 of 10 poorly differentiated endocrine carcinomas).

Conclusions: Loss of p27 and overexpression of cyclin E play a critical role in the aggressiveness of gastroenteropancreatic neuroendocrine tumors. This coincides with increased cell cycle progression. We propose a discussion whether to incorporate the immunohistochemical expression of p27 into a revised classification to individualize therapeutic strategies in this tumor entity.
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http://dx.doi.org/10.1158/1078-0432.CCR-08-0698DOI Listing
November 2008
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