Volker Ellenrieder

Overexpression of c‐myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway

The nuclear factor of activated T cell (NFAT) proteins are a family of Ca2+/calcineurin‐responsive transcription factors primarily recognized for their central roles in T lymphocyte activation and cardiac valve development. We demonstrate that NFATc1 is commonly overexpressed in pancreatic carcinomas and enhances the malignant potential of tumor cells through transcriptional activation of the c‐myc oncogene. Activated NFATc1 directly binds to a specific element within the proximal c‐myc promoter and upregulates c‐myc transcription, ultimately resulting in increased cell proliferation and enhanced anchorage‐independent growth. Conversely, c‐myc transcription and anchorage‐dependent and ‐independent cell growth is significantly attenuated by inhibition of Ca2+/calcineurin signaling or siRNA‐mediated knock down of NFATc1 expression. Together, these results demonstrate that ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway is an important mechanism of oncogenic c‐myc activation in pancreatic cancer.

Role of MT‐MMPs and MMP‐2 in pancreatic cancer progression

Activation of matrix metalloproteinase‐2 (MMP‐2) by the membrane‐type matrix metalloproteinases (MT‐MMPs) has been associated with tumor progression. In the present study, we examined the role of MMP‐2 and its activators MT1‐MMP, MT2‐MMP and MT3‐MMP in pancreatic tumor cell invasion and the development of the desmoplastic reaction characteristic of pancreatic cancer tissues. Northern blot analyses revealed that transcript levels of MT1‐MMP and MT2‐MMP, but not MT3‐MMP, were enhanced in pancreatic cancer tissues (n = 18) compared with both chronic pancreatitis (n = 9) and healthy pancreas (n = 9). A good correlation was found between MT1‐MMP and both MMP‐2 expression (p < 0.01) and activity in pancreatic cancer tissues. In addition, expression and activation of MMP‐2 were strongly associated with the extent of the desmoplastic reaction in pancreatic cancer tissues. Invasion assays showed a good correlation between MMP‐2 expression and activity and the invasive potential of pancreatic cancer cell lines. In cell lines with high levels of MMP‐2 expression and activity, the MMP inhibitor Batimastat led to significant reduction of the number of invading cells. Our results suggest that MT1‐MMP is involved in the progression of pancreatic cancer via activation of MMP‐2. MMP‐2 itself plays an important role in tumor cell invasion and appears to be associated with the development of the characteristic desmoplastic reaction in pancreatic cancer. Int. J. Cancer 85:14–20, 2000.

TGF‐β–induced invasiveness of pancreatic cancer cells is mediated by matrix metalloproteinase‐2 and the urokinase plasminogen activator system

TGF‐β strongly promotes local tumor progression in advanced epithelial tumors, though the underlying mechanisms are poorly understood. In the present study, we demonstrate the potential of TGF‐β to increase the invasiveness of the pancreatic cancer cell lines PANC‐1 and IMIM‐PC1. TGF‐β–induced tumor cell invasion occurred in a time‐dependent manner, started after 12 hr and continued to increase even 48 hr after a single application of the growth factor. Blocking of secreted TGF‐β1 by application of neutralizing antibodies 24 hr after TGF‐β treatment completely prevented the sustained effects of TGF‐β on tumor cell invasion. Together with our previous observation that TGF‐β1 up‐regulates its own expression in both cell lines, our data suggest that TGF‐β1 acts in an autocrine manner to maintain tumor cell invasion. As measured by Northern blot hybridization and zymography, TGF‐β treatment of PANC‐1 and IMIM‐PC1 cells resulted in strong up‐regulation of expression and activity of both matrix metalloproteinase‐2 (MMP‐2) and the urokinase plasminogen activator (uPA) system. Treatment with MMP inhibitors or inhibitors of the uPA system caused significant reduction of TGF‐β–induced invasiveness in both cell lines. In contrast, expression and activity of MMP‐2 and uPA as well as tumor cell invasiveness remained unaffected in cell lines with defects of the TGF‐β type II receptor (MiaPaca2) or the Smad4 gene (IMIM‐PC2 and CAPAN‐1). In these cell lines, TGF‐β also failed to auto‐induce its own expression. In conclusion, our results suggest that TGF‐β1 is a strong promotor of pancreatic cancer progression. TGF‐β thereby acts in an autocrine manner to induce tumor cell invasion, which is mediated by MMP‐2 and the uPA system.

An mSin3A interaction domain links the transcriptional activity of KLF11 with its role in growth regulation.

KLF11 is a biochemical paradigm for a subset of proteins that repress transcription via a Mad1‐like mSin3A interacting domain (SID). The biological role of these proteins and the significance of their biochemical activity, however, remain to be established. We report that KLF11 is downregulated in human cancers, inhibits cell growth in vitro and in vivo, and suppresses neoplastic transformation. Transgenic mice for KLF11 display a downregulation of genes encoding the oxidative stress scavengers SOD2 and Catalase1. Chromatin immunoprecipitation assays confirm that, indeed, these genes are bonafide targets of KLF11. KLF11 expression renders cells more sensitive to oxidative drugs, an effect that is rescued by infection with recombinant adenoviruses expressing SOD2 and Catalase1. KLF11‐regulated functions require the Mad1‐like SID, indicating that these target genes involved in these phenomena are regulated via this corepressor system. These results demonstrate that SID‐containing KLF repressor proteins can inhibit cell growth and neoplastic transformation, and outline for the first time cellular and molecular mechanisms by which these functions may be achieved.

SPARC independent drug delivery and antitumour effects of nab-paclitaxel in genetically engineered mice

Design Pharmacokinetic and pharmacodynamic parameters of cremophor-paclitaxel, nab-paclitaxel (human-albumin-bound paclitaxel, Abraxane) and a novel mouse-albumin-bound paclitaxel (m-nab-paclitaxel) were evaluated in genetically engineered mouse models (GEMMs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), histological and biochemical analysis. Preclinical evaluation of m-nab-paclitaxel included assessment by three-dimensional high-resolution ultrasound and molecular analysis in a novel secreted protein acidic and rich in cysteine (SPARC)-deficient GEMM of pancreatic ductal adenocarcinoma (PDA). Results nab-Paclitaxel exerted its antitumoural effects in a dose-dependent manner and was associated with less toxicity compared with cremophor-paclitaxel. SPARC nullizygosity in a GEMM of PDA, KrasG12D;p53flox/−;p48Cre (KPfC), resulted in desmoplastic ductal pancreas tumours with impaired collagen maturation. Paclitaxel concentrations were significantly decreased in SPARC null plasma samples and tissues when administered as low-dose m-nab-paclitaxel. At the maximally tolerated dose, SPARC deficiency did not affect the intratumoural paclitaxel concentration, stromal deposition and the immediate therapeutic response. Conclusions nab-Paclitaxel accumulates and acts in a dose-dependent manner. The interaction of plasma SPARC and albumin-bound drugs is observed at low doses of nab-paclitaxel but is saturated at therapeutic doses in murine tumours. Thus, this study provides important information for future preclinical and clinical trials in PDA using nab-paclitaxel in combination with novel experimental and targeted agents.

Sp1 is required for transforming growth factor-beta-induced mesenchymal transition and migration in pancreatic cancer cells.

Transition from a sessile epithelial phenotype to a migrating mesenchymal phenotype is a crucial step in transforming growth factor-beta (TGF-beta)-induced pancreatic cancer cell migration and invasion. These profound morphologic and functional alterations are associated with characteristic changes in TGF-beta-regulated gene expression, defined by rapid repression of epithelial markers and a strong and sustained transcriptional induction of mesenchymal markers such as the intermediate filament vimentin. In this study, we have analyzed the role of the transcription factor Sp1 in TGF-beta-induced and Smad-mediated gene regulation during epithelial to mesenchymal transition (EMT) and migration of pancreatic cancer cells. Here, we show that Sp1 is required for TGF-beta-induced EMT, and that this function is especially mediated through transcriptional induction of vimentin. Our results emphasize the functional relevance of vimentin in TGF-beta-induced EMT because prevention of its induction strongly reduces cell migration. Altogether, this study helps to better understand the role of Sp1 in TGF-beta-induced progression of pancreatic cancer. It suggests that Sp1, via transcriptional induction of vimentin, cooperates with activated Smad complexes in mesenchymal transition and migration of pancreatic cancer cells upon TGF-beta stimulation.

Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype.

Pancreatic stellate cells (PSCs) are thought to be the primary source of the extensive fibrotic reaction characteristic of pancreatic cancer and chronic pancreatitis in humans. PSCs share many morphological and functional characteristics with hepatic stellate cells (HSCs), whose central role in liver fibrosis is well established. However, it has remained unclear if hepatic and pancreatic stellate cells are derived from a common cell lineage and if they are completely similar or if they possess organ-specific features. We have analysed the transcriptomes of HSCs, PSCs and skin fibroblasts to assess how the transcriptional phenotype of stellate cells differs from that of a typical fibroblast lineage cell and if there is evidence for a common stellate cell precursor. To this end, we have performed expression profiling of primary cultures of human HSCs, PSCs and skin fibroblasts using 23,000-feature ‘whole genome’ oligonucleotide micro-arrays. Expression data were verified using real-time PCR. The expression profiles of HSCs and PSCs displayed a great extent of similarity, clearly separating them from the fibroblasts. Predominantly extracellular and cell surface genes, but also signalling molecules, transcription factors and novel neural markers, were concordantly expressed in both stellate cell types. Despite this high degree of similarity, distinct differences in expression patterns were observed between HSCs and PSCs, reflecting organ-specific variations of the common stellate cell-specific phenotype.

NFAT-Induced Histone Acetylation Relay Switch Promotes c-Myc-Dependent Growth in Pancreatic Cancer Cells

BACKGROUND & AIMS Induction of immediate early transcription factors (ITF) represents the first transcriptional program controlling mitogen-stimulated cell cycle progression in cancer. Here, we examined the transcriptional mechanisms regulating the ITF protein c-Myc and its role in pancreatic cancer growth in vitro and in vivo. METHODS Expression of ITF proteins was examined by reverse-transcription polymerase chain reaction and immunoblotting, and its implications in cell cycle progression and growth was determined by flow cytometry and [(3)H]-thymidine incorporation. Intracellular Ca(2+) concentrations, calcineurin activity, and cellular nuclear factor of activated T cells (NFAT) distribution were analyzed. Transcription factor complex formations and promoter regulation were examined by immunoprecipitations, reporter gene assays, and chromatin immunoprecipitation. Using a combination of RNA interference knockdown technology and xenograft models, we analyzed the significance for pancreatic cancer tumor growth. RESULTS Serum promotes pancreatic cancer growth through induction of the proproliferative NFAT/c-Myc axis. Mechanistically, serum increases intracellular Ca(2+) concentrations and activates the calcineurin/NFAT pathway to induce c-Myc transcription. NFAT binds to a serum responsive element within the proximal promoter, initiates p300-dependent histone acetylation, and creates a local chromatin structure permissive for the inducible recruitment of Ets-like gene (ELK)-1, a protein required for maximal activation of the c-Myc promoter. The functional significance of this novel pathway was emphasized by impaired c-Myc expression, G1 arrest, and reduced tumor growth upon NFAT depletion in vitro and in vivo. CONCLUSIONS Our study uncovers a novel mechanism regulating cell growth and identifies the NFAT/ELK complex as modulators of early stages of mitogen-stimulated proliferation in pancreatic cancer cells.

HCV and HGV in B-cell non-Hodgkin's lymphoma

BACKGROUND/AIMS A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkins lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkins lymphoma. METHODS Serum samples from 69 patients with non-Hodgkins lymphoma were studied. Diagnosis of non-Hodgkins lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS Three of 69 (4.3%) patients with B-cell non-Hodgkins lymphoma were HCV-infected and nine non-Hodgkins lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkins lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS HCV seems to have no significance for the pathogenesis of non-Hodgkins lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkins lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Epigenetic regulation of autophagy by the methyltransferase G9a.

ABSTRACT Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.