Subgroup: Tumor cell motility and secretion

Projects

The actin cytoskeleton in cancer and neutrophil motility

A major focus of our work is the molecular control of cancer cell motility. We are exploring how PDAC cells migrate in confined spatial environments and 3D-matrices. PDAC tumors are encapsulate by a highly dense fibrotic matrix. When tumor cells move in such an environment they adapt their mode of migration. Using micro channel systems we are studying factors that change the cellular mode of motility to amoeboid-like migration, which is independent of matrix degradation in confined 3D-matrices [1]. Also, the nucleus and its spatial restrictions during migration in confined environments are under investigation. Our experiments are conducted in cooperation with Professor Ralf Kemkemer (Hochschule Reutlingen/MPI for Intelligent Systems, Stuttgart).

Relevant publications:
  • Paňková, D. Rösel, M. Novotný, Jan Brábek. The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells Cell Mol Life Sci. 2010 Jan; 67(1): 63–71. Published online 2009 Aug 26. doi: 10.1007/s00018-009-0132-1

As part of the CRC 1149 project A6 we are investigating, how PKD1 regulates the extravasation of neutrophils during trauma by modulating the assembly of the subcortical actin network. Neutrophils are amongst the first immune cells to infiltrate a site of injury. During extravasation from blood vessels, neutrophils have to squeeze through very narrow passages. The deformability of neutrophils strongly depends of the coordinated disassembly and polymerization of the subcortical actin network [2]. In cooperation with Dr. Stephan Paschke (Department of Visceral Surgery) we are investigating, how these features are regulated by PKD1 [3] and its substrates in neutrophils and whether these features affect neutrophil swarming during trauma in vivo (https://www.uni-ulm.de/en/einrichtungen/crc-1149/research/projects/a6/).

  • Paschke S., Weidner A.F., Paust T., Marti O., Beil M., Ben-Chetrit E. Technical advance: Inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments. J Leukoc Biol. 2013 Nov;94(5):1091-6. doi: 10.1189/jlb.1012510. Epub 2013 Jul 30.
  • Eiseler T., Döppler H., Yan I.K., Kitatani K., Mizuno K., Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. Nat Cell Biol. 2009 May;11(5):545-56. doi: 10.1038/ncb1861. Epub 2009 Mar 29.
  • PKDs in epithelial and endothelial barrier function: The actin cytoskeleton and actin-binding proteins, such as Cortactin and Vinculin are major contributors to the molecular control of cell-cell adhesion [4, 5]. We have recently shown that Cortactin and its phosphorylation by PKD1 [6] guide the disassembly or formation (non-phosphorylated Cortactin) of adherens junctions by modulating the composition and F-actin linkage of the E-cadherin adhesion complex [7]. Similar mechanisms apply during the regulation of endothelial barrier integrity by VE-cadherin-mediated adhesion complexes. Efficient cell-cell adhesion is a pre-requisite for epithelial and endothelial barrier integrity. We are currently investigating, how these factors affect barrier function and trans-endothelial passage of neutrophils in several model systems and settings, including trauma models.
  • Helwani F.M., Kovacs E.M., Paterson A.D., Verma S., Ali R.G., Fanning A.S., Weed S.A., Yap A.S. Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J Cell Biol. 2004 Mar 15;164(6):899-910.
  • Peng X., Maiers J.L., Choudhury D., Craig S.W., DeMali K.A. α-Catenin uses a novel mechanism to activate vinculin. J Biol Chem. 2012 Mar 2;287(10):7728-37. doi: 10.1074/jbc.M111.297481. Epub 2012 Jan 10.
  • Eiseler T., Hausser A., De Kimpe L., Van Lint J., Pfizenmaier K. Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin. J Biol Chem. 2010 Jun 11;285(24):18672-83. doi: 10.1074/jbc.M109.093880. Epub 2010 Apr 2.
  • Sroka R., Van Lint J., Katz S.F., Schneider M.R., Kleger A., Paschke S., Seufferlein T., Eiseler T. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation. J Cell Sci. 2016 Jun 15;129(12):2416-29. doi: 10.1242/jcs.184721. Epub 2016 May 13.
Molecular control of secretion processes involved in cancer cell invasion

A second major focus of our group is the molecular control of secretion processes involved in the transport of cancer relevant cargo, e.g. secretion of matrix-metalloproteinases (MMPs) which facilitate ECM degradation and angiogenesis [8]. We also explore the role of exosome secretion during cancer cell invasion and metastasis as well as induced secretion of the secretogranin Chromogranin A (CgA) from BON neuroendocrine pancreatic cancer cells [9-11].

Results: Highly invasive and metastatic pancreatic cancer poses a major challenge for the development of novel therapeutic strategies. The Protein Kinase D (PKD) isoforms PKD1 (PKCµ)/PKD2/PKD3 are involved in controlling tumor cell metastasis, e.g. by modulating cell motility, tumor angiogenesis and matrix degradation. Our research indicates, that in particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is comparatively lower. PKD1 and 2 control different invasive aspects of pancreatic cancer cells in an isoform-specific manner. PKD2 enhances invasion of pancreatic cancer cells (Fig. 1) by facilitating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). Combined knockdown of MMP7/9 blocked PKD2-mediated invasion in 3D-ECM assays and by tumors grown on the chorioallantois membrane of fertilized chicken eggs (CAM). Additionally, we were able to show that PKD2-mediated tumor angiogenesis is fostered by MMP-9, which liberates extracellular matrix-bound VEGF-A (Fig. 2). Specific knockdown of PKD1, on the other hand, enhanced pancreatic cancer cell invasion by driving cancer cell motility. Mimicking highly invasive PDAC with high PKD2 expression and loss of PKD1, we show that knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced their invasive phenotype in 3D-ECM systems (Fig. 3). Our data suggest that this particular feature is controlled by generating highly motile cells and we describe an isoform-specific interaction of PKD1 and not PKD2 with the actin regulatory Cofilin-phosphatase Slingshot1L (SSH1L) at peripheral dynamic actin structures. PKD1 inhibits SSH1L by direct phosphorylation at Serine 978 [12]. Active Cofilin controls generation of ‘barbed ends’ that drive actin polymerization and membrane protrusion during the initial response of cells towards a chemotactic stimulus. Loss of PKD1 thus may be beneficial for tumor cells to further enhance their matrix-invading abilities [8, 12, 13].

PKD kinases

PKD kinases are also known to drive vesicle fission from the trans-Golgi network [Review: 14]. We are investigating how specific PKD isoforms, in particular PKD2, regulate and execute different steps of vesicle budding and fission by a network of protein interactions involving the small GTPase Arf1 [15, 16] (Fig. 4). Also, regulation of Cortactin and actin polymerization during vesicle separation from Golgi membranes is investigated [17] (Fig. 5). In order to address these questions we are studying compartment-specific protein dydynamics, interactions and activation processes utilizing high-end confocal microscopy techniques, such as Fluorescence-Recovery-after-Photobleaching (FRAP) and Foerster Energy Transfer (FRET) complemented by biochemical assays.

p120-catenin family protein p0071

The p120-catenin family protein p0071 (also known as plakophilin 4 /PKP4) has been implicated in various cellular contexts such as intercellular adhesion, neurite outgrowth, cytoskeletal organization and cell division via controlling Rho-GTPase signalling [18-20]. During a yeast-two-hybrid screen by our collaborating partner Prof. Mechthild Hatzfeld (MLU, Halle) around 25% of the identified proteins were assigned to cellular transport processes and/or bind to transport vesicles [18]. Recent data from our lab revealed significantly impaired transport of various cargos, e.g. of the secretogranin Chromogranin A (CgA) from BON cells (Fig. 6) and matrix metalloproteinase-9 (MMP-9) from PDAC cells after p0071 depletion. Deregulated secretion of these factors is a hallmark of various cancer subtypes. Our work therefore aims to identify and characterize functions of p0071 within the respective cellular transport machinery.
During investigation of the above described projects we are also exploring how molecular alterations  impact on PDAC tumor cell invasion in a variety of different setups, e.g. by testing tumor cells for their ability to migrate towards chemotactic stimuli. We are evaluating invasion, tumor angiogenesis and immune cell infiltration in 2- and 3D culture systems. In vitro studies are further complemented by in vivo models, e.g. tumors grown on chorioallantois membranes (CAM) [9] as well as mouse model systems to understand the different contributions of tumor cell motility, MMP regulation and angiogenesis to tumor metastasis in vivo.

Relevant publications:
  • Wille C., Köhler C., Armacki M., Jamali A., Gössele U., Pfizenmaier K., Seufferlein T., Eiseler T. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases. Mol Biol Cell. 2014 Feb;25(3):324-36. doi: 10.1091/mbc.E13-06-0334. Epub 2013 Dec 11.
  • von Wichert G., Edenfeld T., von Blume J., Krisp H., Krndija D., Schmid H., Oswald F., Lother U., Walther P., Adler G., and Seufferlein T. (2008). Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells. Cell Signal 20, 925-934.
  • von Wichert G., Haeussler U., Greten F.R., Kliche S., Dralle H., Bohm B.O., Adler G., and Seufferlein T. (2005). Regulation of cyclin D1 expression by autocrine IGF-I in human BON neuroendocrine tumour cells. Oncogene 24, 1284-1289.
  • von Wichert G., Jehle P.M., Hoeflich A., Koschnick S., Dralle H., Wolf E., Wiedenmann B., Boehm B.O., Adler G., and Seufferlein T. (2000). Insulin-like growth factor-I is an autocrine regulator of chromogranin A secretion and growth in human neuroendocrine tumor cells. Cancer Res 60, 4573-4581.
  • Eiseler T., Döppler H., Yan I.K., Kitatani K., Mizuno K., Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. Nat Cell Biol. 2009 May;11(5):545-56. doi: 10.1038/ncb1861.
  • Wille C., Seufferlein T., Eiseler T. Protein Kinase D family kinases: roads start to segregate. Bioarchitecture. 2014;4(3):111-5. doi: 10.4161/bioa.29273. Epub 2014 May 21.
  • Malhotra V., Campelo F. PKD regulates membrane fission to generate TGN to cell surface transport carriers. Cold Spring Harb Perspect Biol. 2011 Feb 1;3(2). pii: a005280. doi: 10.1101/cshperspect.a005280. Review.
  • Pusapati G.V., Krndija D., Armacki M., von Wichert G., von Blume J., Malhotra V., Adler G., Seufferlein T. Role of the second cysteine-rich domain and Pro275 in protein kinase D2 interaction with ADP-ribosylation factor 1, trans-Golgi network recruitment, and protein transport. Mol Biol Cell. 2010 Mar 15;21(6):1011-22. doi: 10.1091/mbc.E09-09-0814. Epub 2010 Jan 20.
  • Eiseler T., Wille C., Koehler C., Illing A., Seufferlein T. Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion. J Biol Chem. 2016 Jan 1;291(1):462-77. doi: 10.1074/jbc.M115.673582. Epub 2015 Oct 27.
  • Cao H., Weller S., Orth J.D., Chen J., Huang B., Chen J.L., Stamnes M., McNiven M.A. Actin and Arf1-dependent recruitment of a cortactin-dynamin complexto the Golgi regulates post-Golgi transport. Nat Cell Biol. 2005 May;7(5):483-92. Epub 2005 Apr 10.
  • Keil R., Schulz J., Hatzfeld M. p0071/PKP4, a multifunctional protein coordinating cell adhesion with cytoskeletal organization. J. Biol Chem. 2013 Aug;394(8):1005-17. doi: 10.1515/hsz-2013-0114. Review.
  • Wolf A., Keil R., Götzl O., Mun A., Schwarze K., Lederer M., Hüttelmaier S., Hatzfeld M. The armadillo protein p0071 regulates Rho signalling during cytokinesis. Nat Cell Biol. 2006 Dec;8(12):1432-40.
  • Keil R., Kiessling C., Hatzfeld M. Targeting of p0071 to the midbody depends on KIF3. J Cell Sci. 2009 Apr 15;122(Pt 8):1174-83. doi: 10.1242/jcs.045377.

Staff

Postdoc / Principal Investigator

Profilbild von Dr. Tim Eiseler

Dr. Tim Eiseler

Current group members

Dr. Christoph Wille

Robert Sroka

Alexander Becher

Claudia Ruhland

Funding

Deutsche Forschungsgemeinschaft (DFG)

Cooperation partners

  • Prof. Dr. Johan Van Lint, Katholieke Universiteit Leuven, Belgium
  • Prof. Dr. med. Alexander Kleger, Department for Internal Medicine I, University of Ulm
  • Prof. Dr. Franz Oswald, Department for Internal Medicine I, University of Ulm
  • Prof. Dr. Mechthild Haztfeld, Division of Pathobiochemistry, Martin-Luther University, Halle
  • Dr. Martin Müller, Center for Internal Medicine I, University of Ulm
  • Dr. Angelika Rück, Core Facility for Confocal and Multiphoton Microscopy
  • Dr. Stephan Paschke, Department of Visceral Surgery, University of Ulm
  • Prof. Dr. Ralf Kemkemer, Max Planck Institute for Intelligent Systems, Stuttgart/University Reutlingen
  • Dr. Julia von Blume, Max Planck Institute for Biochemistry, Munich

Selected publications

  • Sroka R., Seufferlein T., Eiseler T. Detection and quantitative analysis of protein interactions in large multi-protein complexes by acceptor-photobleach-FRET. JoVE in press.
  • Hohwieler M., Illing A., Hermann P.C., Mayer T., Stockmann M., Perkhofer L., Eiseler T., Antony J.S., Müller M., Renz S., Kuo C.C., Lin Q., Sendler M., Breunig M., Kleiderman S.M., Lechel A., Zenker M., Leichsenring M., Rosendahl J., Zenke M., Sainz B. Jr., Mayerle J., Costa I.G., Seufferlein T., Kormann M., Wagner M., Liebau S., Kleger A. Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut. 2016 Sep 15. pii: gutjnl-2016-312423. doi: 10.1136/gutjnl-2016-312423.
  • Perkhofer L., Walter K., Costa I.G., Carrasco M.C., Eiseler T., Hafner S., Genze F., Zenke M., Bergmann W., Illing A., Hohwieler M., Köhntop R., Lin Q., Holzmann K.H., Seufferlein T., Wagner M., Liebau S., Hermann P.C., Kleger A., Müller M. Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness. Stem Cell Res. 2016 Aug 10;17(2):367-378. doi: 10.1016/j.scr.2016.08.007.
  • Sroka R., Van Lint J., Katz S.F., Schneider M.R., Kleger A., Paschke S., Seufferlein T., Eiseler T. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation. J Cell Sci. 2016 Jun 15;129(12):2416-29. doi: 10.1242/jcs.184721.
  • Russell R., Ilg M., Lin Q., Wu G., Lechel A., Bergmann W., Eiseler T., Linta L., Kumar P. P., Klingenstein M., Adachi K., Hohwieler M., Sakk O., Raab S., Moon A., Zenke M., Seufferlein T., Schöler H.R., Illing A., Liebau S., Kleger A. A Dynamic Role of TBX3 in the Pluripotency Circuitry. Stem Cell Reports. 2015 Dec 8;5(6):1155-70. doi: 10.1016/j.stemcr.2015.11.003.
  • Eiseler T*., Wille C.*, Koehler C., Illing A., Seufferlein T. Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion. J Biol Chem. 2016 Jan 1;291(1):462-77. doi: 10.1074/jbc.M115.673582 (shared first author)
  • Müller M., Schröer J., Azoitei N., Eiseler T., Bergmann W., Köhntop R., Lin Q., Costa I.G., Zenke M., Genze F., Weidgang C., Seufferlein T., Liebau S., Kleger A. A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells. Sci Rep. 2015 Jul 7;5:11742. doi: 10.1038/srep11742.
  • Qin R., Schmid H., Münzberg C., Maass U., Krndija D., Adler G., Seufferlein T., Liedert A., Ignatius A., Oswald F., Eiseler T., von Wichert G. Phosphorylation and turnover of paxillin in focal contacts is controlled by force and defines the dynamic state of the adhesion site. Cytoskeleton (Hoboken). 2015 Feb;72(2):101-12. doi: 10.1002/cm.21209.
  • Wille C., Seufferlein T., Eiseler T. Protein Kinase D family kinases: roads start to segregate. Bioarchitecture. 2014;4(3):111-5. doi: 10.4161/bioa.29273.
  • Wille C., Köhler C., Armacki M., Jamali A., Gössele U., Pfizenmaier K., Seufferlein T., Eiseler T. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases. Mol Biol Cell. 2014 Feb;25(3):324-36. doi: 10.1091/mbc.E13-06-0334.
  • Döppler H., Bastea L.I., Eiseler T., Storz P. Neuregulin mediates F-actin-driven cell migration through inhibition of protein kinase D1 via Rac1 protein. J Biol Chem. 2013 Jan 4;288(1):455-65. doi: 10.1074/jbc.M112.397448.
  • Eiseler T., Köhler C., Nimmagadda S.C., Jamali A., Funk N., Joodi G., Storz P., Seufferlein T. Protein kinase D1 mediates anchorage-dependent and -independent growth of tumor cells via the zinc finger transcription factor Snail1. J Biol Chem. 2012 Sep 21;287(39):32367-80.
  • Busch T., Armacki M., Eiseler T., Joodi G., Temme C., Jansen J., von Wichert G., Omary M.B., Spatz J., Seufferlein T. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells. J Cell Sci. 2012 May 1;125(Pt 9):2148-59. doi: 10.1242/jcs.080127.
  • Pusapati G.V.*, Eiseler T.*, Rykx A., Vandoninck S., Derua R., Waelkens E., Van Lint J., von Wichert G., Seufferlein T. Protein kinase D regulates RhoA activity via rhotekin phosphorylation. J Biol Chem. 2012 Mar 16;287(12):9473-83. doi: 10.1074/jbc.M112.339564 (shared first author).
  • Ziegler S., Eiseler T., Scholz R.P., Beck A., Link G., Hausser A. A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration. Mol Biol Cell. 2011 Mar 1;22(5):570-80. doi: 10.1091/mbc.E10-05-0427.
  • Eiseler T., Hausser A., De Kimpe L., Van Lint J., Pfizenmaier K. Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin. J Biol Chem. 2010 Jun 11;285(24):18672-83. doi: 10.1074/jbc.M109.093880.
  • Eiseler T., Döppler H., Yan I.K., Kitatani K., Mizuno K., Storz P. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot. Nat Cell Biol. 2009 May;11(5):545-56. doi: 10.1038/ncb1861.
  • Eiseler T., Döppler H., Yan I.K., Goodison S., Storz P. Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion. Breast Cancer Res. 2009;11(1):R13. doi: 10.1186/bcr2232.
  • Cowell C.F., Yan I.K., Eiseler T., Leightner A.C., Döppler H., Storz P. Loss of cell-cell contacts induces NF-kappaB via RhoA-mediated activation of protein kinase D1. J Cell Biochem. 2009 Mar 1;106(4):714-28. doi: 10.1002/jcb.22067.
  • Eiseler T., Schmid M.A., Topbas F., Pfizenmaier K., Hausser A. PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration. FEBS Lett. 2007 Sep 4;581(22):4279-87.
  • Brändlin I., Eiseler T., Salowsky R., Johannes F.J. Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon). J Biol Chem. 2002 Nov 22;277(47):45451-7.
  • Brändlin I., Hübner S., Eiseler T., Martinez-Moya M., Horschinek A., Hausser A., Link G., Rupp S., Storz P., Pfizenmaier K., Johannes F.J. Protein kinase C (PKC)eta-mediated PKC mu activation modulates ERK and JNK signal pathways. J Biol Chem. 2002 Feb 22;277(8):6490-6.