Haeger A.,Radboud Institute for Molecular Life science |
Krause M.,Radboud Institute for Molecular Life science |
Wolf K.,Radboud Institute for Molecular Life science |
Friedl P.,Radboud Institute for Molecular Life science |
And 2 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2014
Background Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop either single-cell or collective invasion modes, however, the mechanical and molecular programs underlying such plasticity of mesenchymal invasion programs remain unclear. Methods To test how tissue anatomy determines invasion mode, spheroids of MV3 melanoma and HT1080 fibrosarcoma cells were embedded into 3D collagen matrices of varying density and stiffness and analyzed for migration type and efficacy with matrix metalloproteinase (MMP)-dependent collagen degradation enabled or pharmacologically inhibited. Results With increasing collagen density and dependent on proteolytic collagen breakdown and track clearance, but independent of matrix stiffness, cells switched from single-cell to collective invasion modes. Conversion to collective invasion included gain of cell-to-cell junctions, supracellular polarization and joint guidance along migration tracks. Conclusions The density of the extracellulair matrix (ECM) determines the invasion mode of mesenchymal tumor cells. Whereas fibrillar, high porosity ECM enables single-cell dissemination, dense matrix induces cell-cell interaction, leader-follower cell behavior and collective migration as an obligate protease-dependent process. General significance These findings establish plasticity of cancer invasion programs in response to ECM porosity and confinement, thereby recapitulating invasion patterns of mesenchymal tumors in vivo. The conversion to collective invasion with increasing ECM confinement supports the concept of cell jamming as a guiding principle for melanoma and fibrosarcoma cells into dense tissue. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. © 2014 Elsevier B.V.
Ghamari A.,Erasmus Medical Center |
van de Corput M.P.C.,Erasmus Medical Center |
Thongjuea S.,University of Bergen |
Van Cappellen W.A.,Erasmus Medical Center |
And 8 more authors.
Genes and Development | Year: 2013
Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments. © 2013 by Cold Spring Harbor Laboratory Press.
Jelluma N.,Cancer Genomics Center |
Dansen T.B.,Cancer Genomics Center |
Sliedrecht T.,Cancer Genomics Center |
Sliedrecht T.,Netherlands Proteomics Center |
And 4 more authors.
Journal of Cell Biology | Year: 2010
Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule-chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition. © 2010 Jelluma et al.
Medema R.H.,Cancer Genomics Center |
Lindqvist A.,Karolinska Institutet
Trends in Biochemical Sciences | Year: 2011
Reversible protein phosphorylation is an essential aspect of mitosis and forms the basis of nuclear envelope breakdown, chromosome condensation and spindle assembly. Through global phosphoproteomic analysis, it has become clear that overall protein phosphorylation and phosphosite occupancy is most abundant during mitosis. At mitotic exit, this abundant phosphorylation must be reversed, and this process requires massive and rapid protein dephosphorylation. In addition to this global shift in protein phosphorylation, careful spatial control of protein (de)phosphorylation is equally important for spindle assembly, chromosome disjunction and chromosome alignment. In this review, we discuss the underlying mechanisms that enforce the dramatic global shift in protein phosphorylation as well as the mechanisms that allow for highly localized substrate phosphorylation in mitosis. © 2011 Elsevier Ltd.
Gargiulo G.,Netherlands Cancer Institute |
Serresi M.,Netherlands Cancer Institute |
Cesaroni M.,Temple University |
Hulsman D.,Netherlands Cancer Institute |
And 2 more authors.
Nature Protocols | Year: 2014
Loss-of-function (LOF) experiments targeting multiple genes during tumorigenesis can be implemented using pooled shRNA libraries. RNAi screens in animal models rely on the use of multiple shRNAs to simultaneously disrupt gene function, as well as to serve as barcodes for cell fate outcomes during tumorigenesis. Here we provide a protocol for performing RNAi screens in orthotopic mouse tumor models, referring to glioma and lung adenocarcinoma as specific examples. The protocol aims to provide guidelines for applying RNAi to a diverse spectrum of solid tumors and to highlight crucial considerations when designing and performing these studies. It covers shRNA library assembly and packaging into lentiviral particles, and transduction into tumor-initiating cells (TICs), followed by in vivo transplantation, tumor DNA recovery, sequencing and analysis. Depending on the target genes and tumor model, tumor suppressors and oncogenes can be identified or biological pathways can be dissected in 6-9 weeks. © 2014 Nature America, Inc. All rights reserved.
Suijkerbuijk S.J.E.,Cancer Genomics Center |
Van Osch M.H.J.,Cancer Genomics Center |
Bos F.L.,University Utrecht |
Hanks S.,Institute of Cancer Research |
And 2 more authors.
Cancer Research | Year: 2010
Genetic mutations in the mitotic regulatory kinase BUBR1 are associated with the cancer-susceptible disorder mosaic variegated aneuploidy (MVA). In patients with biallelic mutations, a missense mutation pairs with a truncating mutation. Here, we show that cell lines derived from MVA patients with biallelic mutations have an impaired mitotic checkpoint, chromosome alignment defects, and low overall BUBR1 abundance. Ectopic expression of BUBR1 restored mitotic checkpoint activity, proving that BUBR1 dysfunction causes chromosome segregation errors in the patients. Combined analysis of patient cells and functional protein replacement shows that all MVA mutations fall in two distinct classes: those that impose specific defects in checkpoint activity or microtubule attachment and those that lower BUBR1 protein abundance. Low protein abundance is the direct result of the absence of transcripts from truncating mutants combined with high protein turnover of missense mutants. In this group of missense mutants, the amino acid change consistently occurs in or near the BUBR1 kinase domain. Our findings provide a molecular explanation for chromosomal instability in patients with biallelic genetic mutations in BUBR1. ©2010 AACR.
Lens S.M.A.,Cancer Genomics Center |
Voest E.E.,Cancer Genomics Center |
Medema R.H.,Cancer Genomics Center
Nature Reviews Cancer | Year: 2010
Large numbers of inhibitors for polo-like kinases and aurora kinases are currently being evaluated as anticancer drugs. Interest in these drugs is fuelled by the idea that these kinases have unique functions in mitosis. Within the polo-like kinase family, the emphasis for targeted therapies has been on polo-like kinase 1 (PLK1), and in the aurora kinase family drugs have been developed to specifically target aurora kinase A (AURKA; also known as STK6) and/or aurora kinase B (AURKB; also known as STK12). Information on the selectivity of these compounds in vivo is limited, but it is likely that off-target effects within the same kinase families will affect efficacy and toxicity profiles. In addition, it is becoming clear that interplay between polo-like kinases and aurora kinases is much more extensive than initially anticipated, and that both kinase families are important factors in the response to classical chemotherapeutics that damage the genome or the mitotic spindle. In this Review we discuss the implications of these novel insights on the clinical applicability of polo-like kinase and aurora kinase inhibitors. © 2010 Macmillan Publishers Limited. All rights reserved.
Holthausen J.T.,Cancer Genomics Center |
Wyman C.,Cancer Genomics Center |
Wyman C.,Erasmus Medical Center |
Kanaar R.,Cancer Genomics Center |
Kanaar R.,Erasmus Medical Center
DNA Repair | Year: 2010
Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate. © 2010 Elsevier B.V.
Ristic D.,Cancer Genomics Center |
Kanaar R.,Cancer Genomics Center |
Kanaar R.,Erasmus University Rotterdam |
Wyman C.,Cancer Genomics Center |
Wyman C.,Erasmus University Rotterdam
Nucleic Acids Research | Year: 2011
The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51-DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3′end of ssDNA or from a region adjacent to the ss-ds junction. RAD51 filaments covering joint ss-dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange. © 2010 The Author(s).
De Jong M.,Erasmus University Rotterdam |
Essers J.,Cancer Genomics Center |
Van Weerden W.M.,Erasmus University Rotterdam
Nature Reviews Cancer | Year: 2014
Recent developments and improvements of multimodal imaging methods for use in animal research have substantially strengthened the options of in vivo visualization of cancer-related processes over time. Moreover, technological developments in probe synthesis and labelling have resulted in imaging probes with the potential for basic research, as well as for translational and clinical applications. In addition, more sophisticated cancer models are available to address cancer-related research questions. This Review gives an overview of developments in these three fields, with a focus on imaging approaches in animal cancer models and how these can help the translation of new therapies into the clinic. © 2014 Macmillan Publishers Limited. All rights reserved.