Thomas Seufferlein

Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network.

Protein kinase D (PKD) binds to diacylglycerol (DAG) in the trans-Golgi network (TGN) and is activated by trimeric G-protein subunits βγ. This complex then regulates the formation of transport carriers in the TGN that traffic to the plasma membrane in non-polarized cells. Here we report specificity of different PKD isoforms in regulating protein trafficking from the TGN. Kinase-inactive forms of PKD1, PKD2 and PKD3 localize to the TGN in polarized and non-polarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.

Protein kinase D: an intracellular traffic regulator on the move

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

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.

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCμ, PKD2 and PKD3/PKCν. They all share a similar architecture with regulatory sub‐domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

Cancer of the pancreas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.

M. Ducreux1,2, A. Sa. Cuhna2,3, C. Caramella4, A. Hollebecque1,5, P. Burtin1, D. Goéré6, T. Seufferlein7, K. Haustermans8, J. L. Van Laethem9, T. Conroy10 & D. Arnold11, on behalf of the ESMO Guidelines Committee* Département de médecine, Gustave Roussy, Villejuif; Faculté de Médecine, Université Paris Sud, le Kremlin Bicêtre; Département de Chirugie Hépato-biliaire, Hopital Paul Brousse, Villejuif; Département d’imagerie; Département d’Innovation Thérapeutique; Département de Chirurgie Générale, Gustave Roussy, Villejuif, France; Department of Internal Medicine I, Ulm University Hospital Medical Center, Ulm, Germany; Department of Radiation Oncology, Leuven Kankerinstitute, Leuven; Departement of Gastroenterology, Hôpital Erasme, Cliniques Universitaires de Bruxelles, Brussels, Belgium; Département de médecine, Institut de Cancérologie de Lorraine, Vandoeuvre lés Nancy, France; Department of Medical Oncology, Tumor Biology Center, Freiburg, Germany

Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells.

Our results demonstrate that the novel vasoactive regulatory peptide adrenomedullin is a potent mitogen for Swiss 3T3 cells. Acting via a specific adrenomedullin receptor, it stimulates a dose‐dependent increase in DNA synthesis in synergy with insulin. Additionally, adrenomedullin stimulates further progression through the cell cycle resulting in cell proliferation, an effect that was further enhanced by the presence of insulin. Adrenomedullin rapidly induces accumulation of intracellular CAMP but does not stimulate an increase in intracellular Ca2+, activation of protein kinase C, or tyrosine phosphorylation of intracellular substrates. Adrenomedullin‐stimulated mitogenesis is markedly enhanced in Swiss 3T3 cells stably transfected with a constitutively activated Gsα, which are highly sensitive to agents that elevate cAMP, and is inhibited by the PKA inhibitor H‐89. Adrenomedullin is, thus, identified as a novel mitogenic regulatory peptide acting via cAMP.

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH2 terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

Gemcitabine and oxaliplatin with or without cetuximab in advanced biliary-tract cancer (BINGO): a randomised, open-label, non-comparative phase 2 trial

BACKGROUND Gemcitabine plus a platinum-based agent (eg, cisplatin or oxaliplatin) is the standard of care for advanced biliary cancers. We investigated the addition of cetuximab to chemotherapy in patients with advanced biliary cancers. METHODS In this non-comparative, open-label, randomised phase 2 trial, we recruited patients with locally advanced (non-resectable) or metastatic cholangiocarcinoma, gallbladder carcinoma, or ampullary carcinoma and a WHO performance status of 0 or 1 from 18 hospitals across France and Germany. Eligible patients were randomly assigned (1:1) centrally with a minimisation procedure to first-line treatment with gemcitabine (1000 mg/m(2)) and oxaliplatin (100 mg/m(2)) with or without cetuximab (500 mg/m(2)), repeated every 2 weeks until disease progression or unacceptable toxicity. Randomisation was stratified by centre, primary site of disease, disease stage, and previous treatment with curative intent or adjuvant therapy. Investigators who assessed treatment response were not masked to group assignment. The primary endpoint was the proportion of patients who were progression-free at 4 months, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00552149. FINDINGS Between Oct 10, 2007, and Dec 18, 2009, 76 patients were assigned to chemotherapy plus cetuximab and 74 to chemotherapy alone. 48 (63%; 95% CI 52-74) patients assigned to chemotherapy plus cetuximab and 40 (54%; 43-65) assigned to chemotherapy alone were progression-free at 4 months. Median progression-free survival was 6·1 months (95% CI 5·1-7·6) in the chemotherapy plus cetuximab group and 5·5 months (3·7-6·6) in the chemotherapy alone group. Median overall survival was 11·0 months (9·1-13·7) in the chemotherapy plus cetuximab group and 12·4 months (8·6-16·0) in the chemotherapy alone group. The most common grade 3-4 adverse events were peripheral neuropathy (in 18 [24%] of 76 patients who received chemotherapy plus cetuximab vs ten [15%] of 68 who received chemotherapy alone), neutropenia (17 [22%] vs 11 [16%]), and increased aminotransferase concentrations (17 [22%] vs ten [15%]). 70 serious adverse events were reported in 39 (51%) of 76 patients who received chemotherapy plus cetuximab (34 events in 19 [25%] patients were treatment-related), whereas 41 serious adverse events were reported in 25 (35%) of 71 patients who received chemotherapy alone (20 events in 12 [17%] patients were treatment-related). One patient died of atypical pneumonia related to treatment in the chemotherapy alone group. INTERPRETATION The addition of cetuximab to gemcitabine and oxaliplatin did not seem to enhance the activity of chemotherapy in patients with advanced biliary cancer, although it was well tolerated. Gemcitabine and platinum-based combination should remain the standard treatment option. FUNDING Institut National du Cancer, Merck Serono.

Impact of tumor cell cytoskeleton organization on invasiveness and migration: a microchannel-based approach

Cell migration is a fundamental feature of the interaction of cells with their surrounding. The cells stiffness and ability to deform itself are two major characteristics that rule migration behavior especially in three-dimensional tissue. We simulate this situation making use of a micro-fabricated migration chip to test the active invasive behavior of pancreatic cancer cells (Panc-1) into narrow channels. At a channel width of 7 µm cell migration through the channels was significantly impeded due to size exclusion. A striking increase in cell invasiveness was observed once the cells were treated with the bioactive lipid sphingosylphosphorylcholine (SPC) that leads to a reorganization of the cells keratin network, an enhancement of the cells deformability, and also an increase in the cells migration speed on flat surfaces. The migration speed of the highly deformed cells inside the channels was three times higher than of cells on flat substrates but was not affected upon SPC treatment. Cells inside the channels migrated predominantly by smooth sliding while maintaining constant cell length. In contrast, cells on adhesion mediating narrow lines moved in a stepwise way, characterized by fluctuations in cell length. Taken together, with our migration chip we demonstrate that the dimensionality of the environment strongly affects the migration phenotype and we suggest that the spatial cytoskeletal keratin organization correlates with the tumor cells invasive potential.

Sphingosylphosphorylcholine Rapidly Induces Tyrosine Phosphorylation of p125FAK and Paxillin, Rearrangement of the Actin Cytoskeleton and Focal Contact Assembly REQUIREMENT OF p21rho IN THE SIGNALING PATHWAY

Sphingosylphosphorylcholine (SPC), a potent mitogen for Swiss 3T3 cells, rapidly induced tyrosine phosphorylation of multiple substrates including bands of Mr 110,000-130,000 and Mr 70,000-80,000 in Swiss 3T3 cells. Focal adhesion kinase (p125FAK) and paxillin were identified as prominent substrates for SPC-stimulated tyrosine phosphorylation. An increase in tyrosine phosphorylation of p125FAK was detected as soon as 30 s after SPC stimulation, reaching a maximum after 2.5 min. SPC induced tyrosine phosphorylation of p125FAK in a concentration-dependent fashion; a half-maximum effect occurred at 250 nM. Tyrosine phosphorylation of p125FAK induced by SPC could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. SPC induced a unique pattern of reorganization of the actin cytoskeleton with a rapid appearance of actin microspikes at the plasma membrane that was followed by the formation of actin stress fibers. This pattern of cytoskeletal changes was clearly distinguishable from that induced by bombesin and 1oleoyl-lysophosphatidic acid. Formation of microspikes and actin stress fibers were accompanied by striking assembly of focal adhesion plaques. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented SPC-induced tyrosine phosphorylation of p125FAK. In addition, tyrosine phosphorylation of p125FAK was markedly inhibited in the presence of platelet-derived growth factor at a concentration (30 ng/ml) that disrupts actin stress fibers. Finally, microinjection of Clostridium botulinum C3 exoenzyme, which inactivates p21rho, prevented SPC-induced formation of actin stress fibers, focal adhesion assembly, and tyrosine phosphorylation. Thus, p21rho is upstream of both cytoskeletal reorganization and tyrosine phosphorylation in SPC-treated cells.