Chronophin Is a Glial Tumor Modifier Involved in the Regulation Of
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Oncogene (2016) 35, 3163–3177 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Chronophin is a glial tumor modifier involved in the regulation of glioblastoma growth and invasiveness M Schulze1,2,9, O Fedorchenko3,10, TG Zink1, CB Knobbe-Thomsen4, S Kraus5, S Schwinn5, A Beilhack5, G Reifenberger4, CM Monoranu6,7, A-L Sirén8, E Jeanclos1,2 and A Gohla1,2 Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion. Oncogene (2016) 35, 3163–3177; doi:10.1038/onc.2015.376; published online 9 November 2015 INTRODUCTION their upstream activators p21-activated kinases and Rho- Glioblastoma is the most common and aggressive primary brain associated coiled-coil kinases (ROCK), and slingshot (SSH), 18,19 tumor in adults, with a median survival rate of 12–15 months after chronophin and PP1/PP2A-type cofilin phosphatases. diagnosis.1,2 This dismal outcome is attributed to rapid tumor Actin dynamics drive most forms of cell migration and cell 20–22 growth and diffuse intracerebral spread, coupled with poor invasion, and both pro- and anti-invasive functions of responses to chemo- and radiotherapy.3,4 elevated phosphocofilin levels have been found in different tumor 23–27 Based on recent large-scale molecular profiling approaches, entities. Intrigued by the observation that the cofilin- distinct molecular subtypes of glioblastomas have been defined, activating phosphatase chronophin/PDXP maps to a candidate and a characteristic landscape of chromosomal, (epi)genetic and glial tumor gene locus on 22q, we have investigated malignant transcriptional aberrations has been reported.5–7 Despite this glioma tissues for molecular alterations affecting the PDXP gene, impressive progress, the relevant genes associated with some of and studied functional implications of chronophin modulation on the recurrent chromosomal alterations are still unknown. For glioma cells in vitro and in vivo. example, although losses on the long arm of chromosome 22 occur with an overall frequency of 33% in the proneural, mesenchymal and classical glioblastoma subgroups,8 glial tumor RESULTS genes such as LZTR1 on 22q119 have only recently been identified. We investigated the RNA expression levels of cofilin-1 (CFL1), Older studies suggested additional tumor suppressor regions at LIMK1 and -2 (LIMK1, -2), slingshot 1-3 (SSH1-3) and chronophin 22q12.3–13.2 and 22q13.31.10–15 (PDXP) in glioblastoma tumor samples deposited in the The gene encoding the haloacid dehalogenase-type phospha- REMBRANDT database. Table 1 shows that PDXP transcript levels tase chronophin16 (gene name: pyridoxal phosphatase, or PDXP) were markedly reduced, whereas SSH or CFL1 genes were not maps to the candidate glial tumor region at 22q12.3–13.2, but a significantly changed. The RNA levels of LIMK1 were reduced, role of chronophin in brain tumors has not yet been explored. whereas LIMK2 RNA levels were increased. Analysis of our own Chronophin targets the actin-remodeling protein cofilin.17 Cofilin collection of glial tumors by quantitative real-time PCR (qPCR) phosphorylation on serine-3 (Ser-3) is an important mechanism to confirmed and extended these results (Figure 1a). In particular, a regulate the cofilin/actin association, and is accomplished by the reduced PDXP expression was found in diffuse astrocytomas (AII), opposing activities of cofilin kinases, such as LIM kinases (LIMK), anaplastic astrocytomas (AAIII) and primary glioblastomas (GBIV) 1Institute for Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; 2Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; 3Institute for Biochemistry and Molecular Biology II, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 4Department of Neuropathology, Heinrich Heine University, Düsseldorf, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany; 5Department of Internal Medicine II and Center for Interdisciplinary Clinical Research, University of Würzburg, Würzburg, Germany; 6Department of Neuropathology, Institute of Pathology, University of Würzburg, Würzburg, Germany; 7Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany and 8Department of Neurosurgery, University of Würzburg, Würzburg, Germany. Correspondence: Professor A Gohla, Department of Pharmacology, University of Würzburg, Versbacher Strasse 9, Würzburg 97078, Bavaria, Germany. E-mail: [email protected] 9Current address: Department of Neuropathology, University of Regensburg, Regensburg, Germany. 10Current address: Department I of Internal Medicine, Center for Integrated Oncology Cologne-Bonn, University of Cologne, Cologne, Germany. Received 23 January 2015; revised 30 July 2015; accepted 4 September 2015; published online 9 November 2015 Chronophin is a glial tumor modifier M Schulze et al 3164 interference-induced chronophin depletion in GBM6840 cells Table 1. Gene expression analysis based on Rembrandt microarray increased Ser3-phosphocofilin levels by ~ 45%, whereas overall data sets Ser3-phosphocofilin levels in chronophin-depleted DBTRG cells 32 Gene expression in glioblastomas Gene Probe were only slightly increased. PhosTag -based separation of relative to non-neoplastic brain tissues symbol set phosphorylated and unphosphorylated cofilin (Supplementary Figure S3) also indicated that chronophin suppression in GBM6840 0.66 LIMK1 204356_at cells elevated phosphocofilin levels. This effect was not observed 2.27 LIMK2 202193_at in DBTRG cells, and chronophin reconstitution in U87MG cells only 1.11 CFL1 1555730_a_at led to a small reduction of phosphocofilin levels (Figure 4b). 1.06 CFL1 200021_at Quantitative real-time PCR analysis of LIMK1 and LIMK2 RNA 1.24 SSH1 1554274_a_at expression levels suggested that LIMKs contribute to the high 1.30 SSH1 1555624_a_at fi 0.90 SSH1 221752_at fraction of phosphorylated co lin seen in DBTRG and U87MG cells 0.93 SSH1 221753_at (LIMK1: GBM6840 1 ± 0.06, DBTRG 4.6 ± 0.23, U87MG 1.9 ± 0.21; 1.10 SSH2 1555423_at LIMK2: GBM6840 1 ± 0.12 DBTRG 3.0 ± 0.22, U87MG 0.7 ± 0.23; 0.89 SSH2 226080_at values are glyceraldehyde 3-phosphate dehydrogenase (GAPDH)- 1.08 SSH3 51192_at normalized mean values ± s.d., n = 3). 0.36 PDXP 223290_at Given the minor effects of chronophin depletion or over- fi Gene expression ratios (median expression intensities) in 221 glioblastoma expression on overall co lin phosphorylation in DBTRG or U87MG samples were compared with the geometric mean of 28 non-neoplastic cells, we examined the subcellular distribution of Ser3- brain samples to calculate the relative gene expression levels. phosphocofilin. Figure 4c shows that DBTRG control cells had Ser3-phosphocofilin and LIMK1-positive, F-actin-rich cell protru- sions. Chronophin knockdown increased leading edge formation, compared with non-neoplastic brain tissue samples. Finally, and Ser3-phosphocofilin was readily detectable in these struc- analysis of the TCGA database5 revealed that PDXP and LIMK1 tures. In U87MG control cells, Ser3-phosphocofilin was mostly expression was reduced in all glioblastoma subtypes, whereas diffusely distributed in the cytosol. Chronophin overexpression led CFL1 and LIMK2 expression levels were not altered (Supplementary to a breakdown of actin stress fibers and to the formation of Figure S1). Importantly, patients with glioblastomas characterized multipolar cells with F-actin-rich protrusions that stained positive by lower than median PDXP expression levels had a significantly for Ser3-phosphocofilin (Figure 4d). Chronophin depletion also shorter survival, whereas no association of LIMK1, LIMK2 or CFL1 increased F-actin levels in GBM6840 cells, whereas chronophin transcript levels with patient survival was found (Figure 1b). reconstitution in U87MG cells reduced steady-state actin poly- Immunohistochemistry revealed that the average fraction of merization (Figures 4e and f). Taken together, these findings show chronophin-positive cells per glial tumor progressively decreased that chronophin functions as a cofilin regulatory phosphatase that from grade I pilocytic astrocytomas (PAI) to AII, AIII and GBIV affects the subcellular distribution of Ser3-phosphocofilin and the tumors (Figure 2a). Supporting these results,