Publications by authors named "Wiljan Hendriks"

64 Publications

Biological and Technical Challenges in Unraveling the Role of N-Glycans in Immune Receptor Regulation.

Front Chem 2020 5;8:55. Epub 2020 Feb 5.

Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands.

N-glycosylation of membrane receptors is important for a wide variety of cellular processes. In the immune system, loss or alteration of receptor glycosylation can affect pathogen recognition, cell-cell interaction, and activation as well as migration. This is not only due to aberrant folding of the receptor, but also to altered lateral mobility or aggregation capacity. Despite increasing evidence of their biological relevance, glycosylation-dependent mechanisms of receptor regulation are hard to dissect at the molecular level. This is due to the intrinsic complexity of the glycosylation process and high diversity of glycan structures combined with the technical limitations of the current experimental tools. It is still challenging to precisely determine the localization and site-occupancy of glycosylation sites, glycan micro- and macro-heterogeneity at the individual receptor level as well as the biological function and specific interactome of receptor glycoforms. In addition, the tools available to manipulate N-glycans of a specific receptor are limited. Significant progress has however been made thanks to innovative approaches such as glycoproteomics, metabolic engineering, or chemoenzymatic labeling. By discussing examples of immune receptors involved in pathogen recognition, migration, antigen presentation, and cell signaling, this Mini Review will focus on the biological importance of N-glycosylation for receptor functions and highlight the technical challenges for examination and manipulation of receptor N-glycans.
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http://dx.doi.org/10.3389/fchem.2020.00055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013033PMC
February 2020

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness.

Theranostics 2020 1;10(3):1016-1032. Epub 2020 Jan 1.

IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier, Montpellier, F-34298, France.

Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. , the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.
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http://dx.doi.org/10.7150/thno.38537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956795PMC
April 2021

Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus.

Philos Trans R Soc Lond B Biol Sci 2019 08 1;374(1779):20180225. Epub 2019 Jul 1.

Department of Cell Biology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.

Directional cell migration in dense three-dimensional (3D) environments critically depends upon shape adaptation and is impeded depending on the size and rigidity of the nucleus. Accordingly, the nucleus is primarily understood as a physical obstacle; however, its pro-migratory functions by stepwise deformation and reshaping remain unclear. Using atomic force spectroscopy, time-lapse fluorescence microscopy and shape change analysis tools, we determined the nuclear size, deformability, morphology and shape change of HT1080 fibrosarcoma cells expressing the Fucci cell cycle indicator or being pre-treated with chromatin-decondensating agent TSA. We show oscillating peak accelerations during migration through 3D collagen matrices and microdevices that occur during shape reversion of deformed nuclei (recoil), and increase with confinement. During G1 cell-cycle phase, nucleus stiffness was increased and yielded further increased speed fluctuations together with sustained cell migration rates in confinement when compared to interphase populations or to periods of intrinsic nuclear softening in the S/G2 cell-cycle phase. Likewise, nuclear softening by pharmacological chromatin decondensation or after lamin A/C depletion reduced peak oscillations in confinement. In conclusion, deformation and recoil of the stiff nucleus contributes to saltatory locomotion in dense tissues. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
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http://dx.doi.org/10.1098/rstb.2018.0225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627020PMC
August 2019

Certainty-based marking in a formative assessment improves student course appreciation but not summative examination scores.

BMC Med Educ 2019 May 31;19(1):178. Epub 2019 May 31.

Department of Animal Ecology and Physiology, Institute for Water and Wetland Research, Radboud University, Nijmegen, The Netherlands.

Background: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation.

Methods: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs.

Results: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs.

Conclusions: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.
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http://dx.doi.org/10.1186/s12909-019-1610-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544949PMC
May 2019

Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

FASEB J 2019 01 12;33(1):557-571. Epub 2018 Jul 12.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1), resulting in metabolic stress. Although IDH gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDH gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1 mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1- and IDH-glioma xenografts. Gene set enrichment analyses of clinical IDH1 gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDH gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDH glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDH gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.
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http://dx.doi.org/10.1096/fj.201800907RRDOI Listing
January 2019

Proteinaceous Regulators and Inhibitors of Protein Tyrosine Phosphatases.

Molecules 2018 Feb 12;23(2). Epub 2018 Feb 12.

Biomarkers in Cancer Unit, Biocruces Health Research Institute, 48903 Barakaldo, Spain.

Proper control of the phosphotyrosine content in signal transduction proteins is essential for normal cell behavior and is lost in many pathologies. Attempts to normalize aberrant tyrosine phosphorylation levels in disease states currently involve either the application of small compounds that inhibit tyrosine kinases (TKs) or the addition of growth factors or their mimetics to boost receptor-type TK activity. Therapies that target the TK enzymatic counterparts, the multi-enzyme family of protein tyrosine phosphatases (PTPs), are still lacking despite their undisputed involvement in human diseases. Efforts to pharmacologically modulate PTP activity have been frustrated by the conserved structure of the PTP catalytic core, providing a daunting problem with respect to target specificity. Over the years, however, many different protein interaction-based regulatory mechanisms that control PTP activity have been uncovered, providing alternative possibilities to control PTPs individually. Here, we review these regulatory principles, discuss existing biologics and proteinaceous compounds that affect PTP activity, and mention future opportunities to drug PTPs via these regulatory concepts.
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http://dx.doi.org/10.3390/molecules23020395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016963PMC
February 2018

Selective MET Kinase Inhibition in MET-Dependent Glioma Models Alters Gene Expression and Induces Tumor Plasticity.

Mol Cancer Res 2017 11 27;15(11):1587-1597. Epub 2017 Jul 27.

Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands.

The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that transcriptomes differed significantly from those in control xenografts. Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling and urge for prudency with drug-testing strategies to validate monotherapies. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0177DOI Listing
November 2017

Psoriasis-Associated Late Cornified Envelope (LCE) Proteins Have Antibacterial Activity.

J Invest Dermatol 2017 11 17;137(11):2380-2388. Epub 2017 Jun 17.

Department of Dermatology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands. Electronic address:

Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.
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http://dx.doi.org/10.1016/j.jid.2017.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651197PMC
November 2017

Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

FASEB J 2017 10 8;31(10):4286-4294. Epub 2017 Jun 8.

Department of Dermatology, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands;

Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of (Cst6-deficient; ) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-) mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-) mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.
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http://dx.doi.org/10.1096/fj.201700267RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602906PMC
October 2017

Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma.

Acta Neuropathol Commun 2016 09 1;4(1):96. Epub 2016 Sep 1.

Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 26, 6525, GA, Nijmegen, The Netherlands.

The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy.
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http://dx.doi.org/10.1186/s40478-016-0372-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009684PMC
September 2016

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

PLoS One 2016 22;11(8):e0160972. Epub 2016 Aug 22.

Biomarkers in Cancer Unit, Biocruces Health Research Institute, Barakaldo, Spain.

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160972PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993582PMC
July 2017

Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

Methods Mol Biol 2016 ;1447:79-93

Biocruces Health Research Institute, Pza Cruces s/n, 48903, Barakaldo, Spain.

In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest.
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http://dx.doi.org/10.1007/978-1-4939-3746-2_5DOI Listing
January 2018

Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma.

Sci Rep 2016 07 27;6:30486. Epub 2016 Jul 27.

Department of Pathology, Radboudumc, Nijmegen, The Netherlands.

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1, and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP(+) to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1(R314C), which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1(R314C) lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP(+), and differs from the IDH1(R132) mutants in that it does not produce D-2-HG. Because IDH1(R314C) is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.
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http://dx.doi.org/10.1038/srep30486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962051PMC
July 2016

Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

Immunity 2015 Aug 28;43(2):277-88. Epub 2015 Jul 28.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pathology and Department of Medicine, New York University Langone Medical Center, New York, NY 10016, USA. Electronic address:

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation.
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http://dx.doi.org/10.1016/j.immuni.2015.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547994PMC
August 2015

Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein.

Acta Neuropathol 2015 Jul 11;130(1):131-44. Epub 2015 Apr 11.

Department of Pathology, Radboud University Medical Center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.
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http://dx.doi.org/10.1007/s00401-015-1420-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469304PMC
July 2015

Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression.

Mol Brain 2015 Jan 9;8. Epub 2015 Jan 9.

Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, Nijmegen, 6500, HB, The Netherlands.

Background: Regulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop.

Results: LTD in the cerebellum of Ptprr (-/-) mice is strongly impeded, in vitro and in vivo. Comparison of basal phospho-MAPK levels between wild-type and PTPRR deficient cerebellar slices revealed increased levels in mutants. This high basal phospho-MAPK level attenuated further increases in phospho-MAPK during chemical induction of LTD, essentially disrupting the positive feedback loop and preventing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation and endocytosis.

Conclusions: Our findings indicate an important role for PTPRR in maintaining low basal MAPK activity in Purkinje cells. This creates an optimal 'window' to boost MAPK activity following signals that induce LTD, which can then propagate through feed-forward signals to cause AMPAR internalization and LTD.
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http://dx.doi.org/10.1186/s13041-014-0092-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304614PMC
January 2015

PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

Methods 2015 May 22;77-78:147-56. Epub 2014 Oct 22.

Centro de Investigación Príncipe Felipe, Valencia, Spain; BioCruces Health Research Institute, Barakaldo, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. Electronic address:

Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from scaffolding and regulatory proteins. Here, we review the current knowledge on PTEN-PDZ domain interactions and tumor suppressor networks, describe methodology suitable to analyze these interactions, and report the binding of PTEN and the PDZ domain-containing protein tyrosine phosphatase PTPN13. Yeast two-hybrid and GST pull-down analyses showed that PTEN binds to PDZ2/PTPN13 domain in a manner that depends on the specific PTPN13 PDZ domain arrangement involving the interdomain region between PDZ1 and PDZ2. Furthermore, a specific binding profile of PTEN to PDZ2/PTPN13 domain was observed by mutational analysis of the PTEN PDZ-BM. Our results disclose a PDZ-mediated physical interaction of PTEN and PTPN13 with potential relevance in tumor suppression and cell homeostasis.
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http://dx.doi.org/10.1016/j.ymeth.2014.10.017DOI Listing
May 2015

Intracellular and extracellular domains of protein tyrosine phosphatase PTPRZ-B differentially regulate glioma cell growth and motility.

Oncotarget 2014 Sep;5(18):8690-702

Department of Cell Biology , Radboud University Medical Center, Nijmegen, The Netherlands.

Gliomas are primary brain tumors for which surgical resection and radiotherapy is difficult because of the diffuse infiltrative growth of the tumor into the brain parenchyma. For development of alternative, drug-based, therapies more insight in the molecular processes that steer this typical growth and morphodynamic behavior of glioma cells is needed. Protein tyrosine phosphatase PTPRZ-B is a transmembrane signaling molecule that is found to be strongly up-regulated in glioma specimens. We assessed the contribution of PTPRZ-B protein domains to tumor cell growth and migration, via lentiviral knock-down and over-expression using clinically relevant glioma xenografts and their derived cell models. PTPRZ-B knock-down resulted in reduced migration and proliferation of glioma cells in vitro and also inhibited tumor growth in vivo. Interestingly, expression of only the PTPRZ-B extracellular segment was sufficient to rescue the in vitro migratory phenotype that resulted from PTPRZ-B knock-down. In contrast, PTPRZ-B knock-down effects on proliferation could be reverted only after re-expression of PTPRZ-B variants that contained its C-terminal PDZ binding domain. Thus, distinct domains of PTPRZ-B are differentially required for migration and proliferation of glioma cells, respectively. PTPRZ-B signaling pathways therefore represent attractive therapeutic entry points to combat these tumors.
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http://dx.doi.org/10.18632/oncotarget.2366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226714PMC
September 2014

Analysis of protein-protein interaction between late cornified envelope proteins and corneodesmosin.

Exp Dermatol 2014 Oct;23(10):769-71

Department of Dermatology, Radboud University Medical Center, Nijmegen, The Netherlands.

Deletion of two members of the late cornified envelope (LCE) family, LCE3B and LCE3C (LCE3C_LCE3B-del), has been identified as risk factor for psoriasis with a possible role in skin barrier function. Moreover, genetic interaction between LCE3C_LCE3B-del and HLA-C*06, located in the psoriasis susceptibility regions 4 and 1 (PSORS4 and 1), has been reported in several populations. Because of high linkage disequilibrium between the PSORS1 genes HLA-C*06 and corneodesmosin (CDSN), both genes are potentially involved in psoriasis. As corneodesmosin and LCE proteins are both constituents of the stratum corneum, we investigated potential direct protein-protein interactions between six LCE proteins and two corneodesmosin sequence variants. Partial colocalization of LCE2 and CDSN was observed in normal and psoriasis skin using immunofluorescence microscopy. Co-expression of eCFP-LCE and mRFP-CDSN proteins in COS-1 cells and human adult keratinocytes, and GST pull-down results did not provide evidence for direct interactions between LCE proteins and CDSN variants.
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http://dx.doi.org/10.1111/exd.12524DOI Listing
October 2014

Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory.

Behav Brain Res 2014 May 18;265:111-20. Epub 2014 Feb 18.

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Electronic address:

Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal cortex but their precise role in these regions remains to be determined. Here, we evaluated phenotypic consequences of loss of PTPRR activity and found that basal smell was normal for Ptprr(-/-) mice. Also, spatial learning and fear-associated contextual learning were unaffected. PTPRR deficiency, however, resulted in impaired novel object recognition and a striking increase in exploratory activity in a new environment. The data corroborate the importance of proper control of MAPK signaling in cerebral functions and put forward PTPRR as a novel target to modulate synaptic processes.
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http://dx.doi.org/10.1016/j.bbr.2014.02.014DOI Listing
May 2014

PTPs emerge as PIPs: protein tyrosine phosphatases with lipid-phosphatase activities in human disease.

Hum Mol Genet 2013 Oct 29;22(R1):R66-76. Epub 2013 Jul 29.

Protein tyrosine phosphatases (PTPs) constitute a family of key homeostatic regulators, with wide implications on physiology and disease. Recent findings have unveiled that the biological activity of PTPs goes beyond the dephosphorylation of phospho-proteins to shut down protein tyrosine kinase-driven signaling cascades. Substrates dephosphorylated by clinically relevant PTPs extend to phospholipids and phosphorylated carbohydrates as well. In addition, non-catalytic functions are also used by PTPs to regulate essential cellular functions. Consequently, PTPs have emerged as novel potential therapeutic targets for human diseases, including cancer predispositions, myopathies and neuropathies. In this review, we highlight recent advances on the multifaceted role of lipid-phosphatase PTPs in human pathology, with an emphasis on hereditary diseases. The involved PTP regulatory networks and PTP modulatory strategies with potential therapeutic application are discussed.
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http://dx.doi.org/10.1093/hmg/ddt347DOI Listing
October 2013

Inactivation of LAR family phosphatase genes Ptprs and Ptprf causes craniofacial malformations resembling Pierre-Robin sequence.

Development 2013 Aug 17;140(16):3413-22. Epub 2013 Jul 17.

Goodman Cancer Research Centre, Department of Biochemistry, McGill University, Montreal, 1160 Pine Avenue W. Montreal, QC H3A 1A3, Canada.

Leukocyte antigen related (LAR) family receptor protein tyrosine phosphatases (RPTPs) regulate the fine balance between tyrosine phosphorylation and dephosphorylation that is crucial for cell signaling during development and tissue homeostasis. Here we show that LAR RPTPs are required for normal development of the mandibular and maxillary regions. Approximately half of the mouse embryos lacking both Ptprs (RPTPσ) and Ptprf (LAR) exhibit micrognathia (small lower jaw), cleft palate and microglossia/glossoptosis (small and deep tongue), a phenotype closely resembling Pierre-Robin sequence in humans. We show that jaw bone and cartilage patterning occurs aberrantly in LAR family phosphatase-deficient embryos and that the mandibular arch harbors a marked decrease in cell proliferation. Analysis of signal transduction in embryonic tissues and mouse embryonic fibroblast cultures identifies an increase in Bmp-Smad signaling and an abrogation of canonical Wnt signaling associated with loss of the LAR family phosphatases. A reactivation of β-catenin signaling by chemical inhibition of GSK3β successfully resensitizes LAR family phosphatase-deficient cells to Wnt induction, indicating that RPTPs are necessary for normal Wnt/β-catenin pathway activation. Together these results identify LAR RPTPs as important regulators of craniofacial morphogenesis and provide insight into the etiology of Pierre-Robin sequence.
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http://dx.doi.org/10.1242/dev.094532DOI Listing
August 2013

Protein tyrosine phosphatase variants in human hereditary disorders and disease susceptibilities.

Biochim Biophys Acta 2013 Oct 23;1832(10):1673-96. Epub 2013 May 23.

Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism to steer normal development and physiological functioning of multicellular organisms. Phosphotyrosine dephosphorylation is exerted by members of the super-family of protein tyrosine phosphatase (PTP) enzymes and many play such essential roles that a wide variety of hereditary disorders and disease susceptibilities in man are caused by PTP alleles. More than two decades of PTP research has resulted in a collection of PTP genetic variants with corresponding consequences at the molecular, cellular and physiological level. Here we present a comprehensive overview of these PTP gene variants that have been linked to disease states in man. Although the findings have direct bearing for disease diagnostics and for research on disease etiology, more work is necessary to translate this into therapies that alleviate the burden of these hereditary disorders and disease susceptibilities in man.
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http://dx.doi.org/10.1016/j.bbadis.2013.05.022DOI Listing
October 2013

Effects of dual targeting of tumor cells and stroma in human glioblastoma xenografts with a tyrosine kinase inhibitor against c-MET and VEGFR2.

PLoS One 2013 4;8(3):e58262. Epub 2013 Mar 4.

Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058262PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587584PMC
September 2013

Valosin containing protein (VCP/p97) is a novel substrate for the protein tyrosine phosphatase PTPL1.

Exp Cell Res 2013 Jan 24;319(1):1-11. Epub 2012 Sep 24.

Departments of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.

Identification of Protein Tyrosine Phosphatase (PTP) substrates is critical in understanding cellular role in normal cells as well as cancer cells. We have previously shown that reduction of PTPL1 protein levels in Ewings sarcoma (ES) inhibit cell growth and tumorigenesis. Therefore, we sought to identify novel PTPL1 substrates that may be important for tumorigenesis. In this current work, we demonstrated that mouse embryonic fibroblasts without PTPL1 catalytic activity fail to form foci when transfected with oncogenes. We proved that catalytic activity of PTPL1 is important for ES cell growth. Using a substrate-trapping mutant of PTPL1 we identified putative PTPL1 substrates by mass-spectrometry. One of these putative substrates was characterized as Valosin Containing Protein (VCP/p97). Using multiple biochemical assays we validated VCP as a novel substrate of PTPL1. We also provide evidence that tyrosine phosphorylation of VCP might be important for its midbody localization during cytokinesis. In conclusion, our work identifies VCP as a new substrate for PTPL1, which may be important in cellular transformation. Our investigation link an oncogenic transcription factor EWS-FLI1, with a key transcriptional target protein tyrosine phosphatase PTPL1, and its substrate VCP. Given our observation that PTPL1 catalytic activity is important for cell transformation, our results may also suggest that VCP regulation by PTPL1 might be important for tumorigenesis.
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http://dx.doi.org/10.1016/j.yexcr.2012.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638858PMC
January 2013

Protein tyrosine phosphatases in health and disease.

FEBS J 2013 Jan 1;280(2):708-30. Epub 2012 Oct 1.

Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Protein tyrosine phosphatases (PTPs) represent a super-family of enzymes that play essential roles in normal development and physiology. In this review, we will discuss the PTPs that have a causative role in hereditary diseases in humans. In addition, recent progress in the development and analysis of animal models expressing mutant PTPs will be presented. The impact of PTP signaling on health and disease will be exemplified for the fields of bone development, synaptogenesis and central nervous system diseases. Collectively, research on PTPs since the late 1980's yielded the cogent view that development of PTP-directed therapeutic tools is essential to further combat human disease.
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http://dx.doi.org/10.1111/febs.12000DOI Listing
January 2013

Phosphorylation target site specificity for AGC kinases DMPK E and Lats2.

J Cell Biochem 2012 Jun;113(6):2126-35

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Serine/threonine kinases of the AGC group are important regulators of cell growth and motility. To examine the candidate substrate profile for two members of this group, DMPK E and Lats2, we performed in vitro kinase assays on peptide arrays. Substrate peptides for both kinases exhibited a predominance of basic residues surrounding the phosphorylation target site. 3D homology modeling of the kinase domains of DMPK E and Lats2 indicated that presence of two negative pockets in the peptide binding groove provides an explanation for the substrate preference. These findings will aid future research toward signaling functions of Lats2 and DMPK E within cells.
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http://dx.doi.org/10.1002/jcb.24086DOI Listing
June 2012

Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes.

Eur J Cell Biol 2012 Aug 15;91(8):629-39. Epub 2012 Mar 15.

Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Myosin II is a hexameric protein complex consisting of two myosin heavy chains, two myosin essential light chains and two myosin regulatory light chains. Multiple subunit isoforms exist, allowing great diversity in myosin II composition which likely impacts on its contractile properties. Little is known about the evolutionary origin, expression pattern and function of myosin regulatory light chain (MLC2) isoforms. We analysed the evolutionary relationship between smooth muscle (sm), nonmuscle (nm) and nonmuscle-like (nml) MLC2 genes, which encode three homologous proteins expressed in nonmuscle cells. The three genes arose by successive gene duplication events. The high sequence similarity between the tandemly arranged nm- and nml-MLC2 genes is best explained by gene conversion. Urea/glycerol-polyacrylamide gel electrophoresis and RNA analysis were employed to monitor expression of sm-, nm- and nml-MLC2 in human and mouse cell lines. Conspicuous differences between transformed and non-transformed cells were observed, with sm-MLC2 being suppressed in Ras-transformed cells. Our findings shed light on the evolutionary history of three homologous MLC2 proteins and point to isoform-specific cell growth-related roles in nonmuscle cell myosin II contractility.
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http://dx.doi.org/10.1016/j.ejcb.2012.02.001DOI Listing
August 2012

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers.

PLoS One 2012 18;7(1):e30447. Epub 2012 Jan 18.

Cancer Biology Research Center, Sanford Research/University of South Dakota, Sioux Falls, South Dakota, United States of America.

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030447PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261204PMC
July 2012
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