Postinfectious T-lymphocytic enteral leiomyositis as a rare cause of chronic intestinal pseudoobstruction.

T-lymphocytic enteral leiomyositis (T-lel) is a rare disorder causing chronic intestinal pseudo-obstruction (CIPO), with cases predominantly being reported in the field of veterinary and pediatric medicine. Here, we present a case of T-lel-associated CIPO in an adult female, who initially presented with a paralytic ileus 2 weeks after a common gastroenteritis. The histological diagnosis was established through full-thickness bowel biopsy, exhibiting a dense lymphocytic infiltrate in the lamina muscularis of the intestinal wall. This case shows that T-lel can be a cause of chronic intestinal pseudo-obstruction not only in children but also in adults. A subsequent induction of an immunosuppressive therapy with steroids, azathioprine, and ultimately TNF-alpha-inhibiting antibodies led to a slow recovery and stable disease.

[Motility disorder and weight loss in a 71-year-old male patient]. / Motilitätsstörung und Gewichtsverlust bei einem 71-jährigen Patienten.

A 71-year-old man presented to this clinic for evaluation of an unclear abdominal tumor. He complained of abdominal pain, weight loss and motility disorders, which began some weeks previously. Ultrasound and computed tomography (CT) scans showed a large mesenterial space-occupying lesion with accompanying lymphadenopathy, slight accumulation of ascites and venous congestion. For confirmation of the suspected diagnosis of a sclerosing mesenteritis and exclusion of a lymphoma a laparoscopy was carried out with excision of tissue. The material was not adequately representative so that a laparotomy was carried out for removal of a new tissue specimen. The tissue specimen confirmed the rare diagnosis of sclerosing mesenteritis and due to the complaints a pharmaceutical treatment with prednisone and tamoxifen was initiated.

IGF-1 drives chromogranin A secretion via activation of Arf1 in human neuroendocrine tumour cells.

Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells.

Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.

The phosphatase of regenerating liver 3 (PRL-3) promotes cell migration through Arf-activity-dependent stimulation of integrin α5 recycling.

The formation of metastasis is one of the most critical problems in oncology. The phosphatase of regenerating liver 3 (PRL-3) is a new target in colorectal cancer, mediating metastatic behavior through a promigratory function. However, detailed explanations for this effect have remained elusive. Here we show that PRL-3 interacts with the ADP-ribosylation factor 1 (Arf1). PRL-3 colocalizes with Arf1 in an endosomal compartment and associates with transmembrane proteins such as the transferrin receptor and α5 integrins. PRL-3 interacts with Arf1 through a distinct motif and regulates activation of Arf1. PRL-3-mediated migration depends on expression and activation of Arf1 and is sensitive to treatment with Brefeldin A. We also demonstrate that PRL-3 modulates recycling of α5 integrins and that its phosphatase activity as well as Arf activation and compartmentalization with Arf1 are required for this effect. In summary our data identify a new function for PRL-3 and show that Arf1 is a new PRL-3-dependent mediator of enhanced migration of cancer cells through enhanced recycling of matrix receptors.

Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells.

Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8-serine(431) and keratin 18-serine(52) and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild-type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18-serine(52) was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin-myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18-serine(52) might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18-serine (52) in the cell periphery points at a particular role of the peripheral keratin network in this regard.

Protein kinase D regulates RhoA activity via rhotekin phosphorylation.

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells.

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

LICC: L-BLP25 in patients with colorectal carcinoma after curative resection of hepatic metastases: a randomized, placebo-controlled, multicenter, multinational, double-blinded phase II trial.

BACKGROUND: 15-20% of all patients initially diagnosed with colorectal cancer develop metastatic disease and surgical resection remains the only potentially curative treatment available. Current 5-year survival following R0-resection of liver metastases is 28-39%, but recurrence eventually occurs in up to 70%. To date, adjuvant chemotherapy has not improved clinical outcomes significantly. The primary objective of the ongoing LICC trial (L-BLP25 In Colorectal Cancer) is to determine whether L-BLP25, an active cancer immunotherapy, extends recurrence-free survival (RFS) time over placebo in colorectal cancer patients following R0/R1 resection of hepatic metastases. L-BLP25 targets MUC1 glycoprotein, which is highly expressed in hepatic metastases from colorectal cancer. In a phase IIB trial, L-BLP25 has shown acceptable tolerability and a trend towards longer survival in patients with stage IIIB locoregional NSCLC. METHODS/DESIGN: This is a multinational, phase II, multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 159 patients from 20 centers in 3 countries. Patients with stage IV colorectal adenocarcinoma limited to liver metastases are included. Following curative-intent complete resection of the primary tumor and of all synchronous/metachronous metastases, eligible patients are randomized 2:1 to receive either L-BLP25 or placebo. Those allocated to L-BLP25 receive a single dose of 300 mg/m2 cyclophosphamide (CP) 3 days before first L-BLP25 dose, then primary treatment with s.c. L-BLP25 930 µg once weekly for 8 weeks, followed by s.c. L-BLP25 930 µg maintenance doses at 6-week (years 1&2) and 12-week (year 3) intervals unless recurrence occurs. In the control arm, CP is replaced by saline solution and L-BLP25 by placebo. Primary endpoint is the comparison of recurrence-free survival (RFS) time between groups. Secondary endpoints are overall survival (OS) time, safety, tolerability, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. The primary endpoint will be assessed in Q3 2016. Follow-up will end Q3 2017. Interim analyses are not planned. DISCUSSION: The design and implementation of such a vaccination study in colorectal cancer is feasible. The study will provide recurrence-free and overall survival rates of groups in an unbiased fashion. TRIAL REGISTRATION: EudraCT Number 2011-000218-20.

Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells.

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

[EBV-positive MTX-associated lymphoproliferative disorder and Ig M myeloma in rheumatoid arthritis]. / EBV-positive MTX-assoziierte lymphoproliferative Erkrankung und IgM-Myelom bei rheumatoider Arthritis.

HISTORY: An 80-year-old female patient arrived with a pronounced lymphadenopathy and weight loss. 6 years ago she had been diagnosed with rheumatoid arthritis. At the time of arrival, she was administered Methotrexate (MTX) 10 mg/week. FINDINGS AND DIAGNOSIS: By lymph node biopsy, a clonal population of both EBV-positive B and T cells was seen. Newly occurring anemia (Hb 10 g/dl), monoclonal gammopathy of the Ig M isotype and detection of 40 % EBV-positive plasma cells in the bone marrow were consistent with the diagnosis of Ig M myeloma. We interpret these findings as a biclonal Epstein Barr Virus-positive Methotrexate-associated lymphoproliferative disorder (MTX-LPD). TREATMENT AND COURSE: The clinical condition improved immediately after MTX discontinuation. In the follow-up after 4 months, the gamma globulin concentration in serum was significantly reduced (from 51.1 to 34.7 %) and a renewed immune electrophoresis of the serum was without evidence of monoclonal gammopathy. CONCLUSION: Based on this case, the association of RA with lymphoproliferative disorders can be confirmed - here as an association of RA with biclonal MTX-LPD or multiple myeloma. Therapy with MTX and reactivation of EBV infection are important influencing factors.

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system.

BACKGROUND: Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions. RESULTS: We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells. CONCLUSION: We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.

Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo.

Activation of the inhibitor of NF-kappaB kinase/NF-kappaB (IKK/NF-kappaB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-kappaB target genes, including mediators of the inflammatory response such as TNF-alpha and ICAM-1. Indeed, inhibition of TNF-alpha activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-kappaB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages.

Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin. RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin.

Tumor biology and cancer therapy - an evolving relationship.

The aim of palliative chemotherapy is to increase survival whilst maintaining maximum quality of life for the individual concerned. Although we are still continuing to explore the optimum use of traditional chemotherapy agents, the introduction of targeted therapies has significantly broadened the therapeutic options. Interestingly, the results from current trials put the underlying biological concept often into a new, less favorable perspective. Recent data suggested that altered pathways underlie cancer, and not just altered genes. Thus, an effective therapeutic agent will sometimes have to target downstream parts of a signaling pathway or physiological effects rather than individual genes. In addition, over the past few years increasing evidence has suggested that solid tumors represent a very heterogeneous group of cells with different susceptibility to cancer therapy. Thus, since therapeutic concepts and pathophysiological understanding are continuously evolving a combination of current concepts in tumor therapy and tumor biology is needed. This review aims to present current problems of cancer therapy by highlighting exemplary results from recent clinical trials with colorectal and pancreatic cancer patients and to discuss the current understanding of the underlying reasons.

Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells.

Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the Golgi apparatus is a subject of intense research. The protein kinase D (PKD) family of serine threonine kinases has so far been implicated in the regulation of constitutive secretion in epithelial cells. Here we examined whether PKD2 expression and activity could also play a role in the release of secretory granules from the trans Golgi network (TGN) in neuroendocrine tumour cells and hence be a target to block autonomous secretion by these tumours. Our data show that expression and catalytic activity of PKD2 are required for the release of chromogranin A containing secretory vesicles. Inhibition of PKD2 activity or siRNA knockdown of PKD2 resulted in a marked perinuclear retention of chromogranin A immunofluorescence in the trans Golgi network and led to a marked reduction in basal as well as phorbol ester stimulated secretion of chromogranin A into the supernatant of cells. Thus, PKD2 controls the release of secretory granules in neuroendocrine tumour cells at the level of the Golgi apparatus and could hence serve as a novel target to block hormone secretion in functional neuroendocrine tumours.

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.

Focal adhesion kinase mediates defects in the force-dependent reinforcement of initial integrin-cytoskeleton linkages in metastatic colon cancer cell lines.

Micro-environmental clues, including the biophysical interpretation of the extracellular matrix, are critical to proliferation, apoptosis and migration. Here, we show that metastatic human colon cancer cell lines display altered matrix interaction. Interaction of colon cancer cells with collagen I depends on integrins (mainly alpha(1)/beta(1)) but metastatic cells display delayed spreading and reduced extension of lamellipodia. In addition, cells show defective strengthening of integrin-cytoskeleton linkages upon mechanical stimulation, as determined by laser trapping experiments and binding of large beads to the cell surface. However, adhesion to pliable surfaces is ameliorated in metastatic variants. These changes are caused by constitutive activation of focal adhesion kinase (FAK) and can be modulated by changing expression and/or activity of FAK via RNA-interference or expression of inhibitory constructs, respectively. In addition, consistent with defective strengthening of integrin-cytoskeleton linkages, metastatic cell lines show reduced random motility. Taken together these data suggest that constitutive activation of FAK causes defects in spreading, reinforcement of integrin-cytoskeleton linkages and migration and at the same time could ameliorate the adhesion of metastatic cells to suboptimal surfaces.

Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling.

Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.