Research paper
CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

https://doi.org/10.1016/j.ejcb.2019.151046Get rights and content

Highlights

  • CRN2 knock-out mice display neurological and behavioural alterations.

  • glioblastoma cells overexpressing CRN2 more effectively encase capillaries.

  • CRN2 interacts with TIMP4 as well as MMP14.

  • TIMP4 secretion and MMP14 activity are increased by CRN2.

  • CRN2 is present in exosome fractions.

Abstract

CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.

Introduction

The coronin protein CRN2 (synonyms: coronin 1C, coronin 3, CRNN4) is a ubiquitously expressed member of the coronin family of proteins (Clemen et al., 2008), which belongs to the super family of eukaryotic-specific WD40-repeat domain proteins (Smith, 2008). Since the first description of a coronin protein in Dictyostelium discoideum (de Hostos et al., 1991), the coronin family of conserved actin cytoskeleton regulator proteins meanwhile has been studied in various model organisms. Phylogenetic analyses determined seventeen coronin subfamilies including alternatively spliced forms of specific coronins, coronin gene duplications in certain phylogenetic branches, a subfamily of chimeric coronins, and the most well-known seven coronin paralogs in mammals (Eckert et al., 2011; Morgan and Fernandez, 2008; Xavier et al., 2008, 2009). The 474 amino acid coronin protein CRN2 with an apparent molecular mass of 57 kDa harbours a basic N-terminal signature motif (Rybakin and Clemen, 2005) followed by seven WD40-repeats which adopt the fold of a seven-bladed β-propeller (Appleton et al., 2006; McArdle and Hofmann, 2008), a linker domain, and a C-terminal coiled coil mediating trimerization (Kammerer et al., 2005; Spoerl et al., 2002).

CRN2 is present in the cytoplasm and enriched at actin filaments, lamellipodia and membrane ruffles (Spoerl et al., 2002). It plays a role in multiple, actin filament-dependent cellular functions like proliferation, migration, formation of cellular protrusions, endocytosis, and secretion (Rosentreter et al., 2007). CRN2 binds to actin filaments via different actin binding sites (Chan et al., 2012; Xavier et al., 2012). Its actin filament bundling, inhibition of actin polymerization, and Arp2/3 complex binding capacities are disabled by protein kinase CK2 dependent phosphorylation at serine residue 463 within the coiled coil region (Xavier et al., 2012). CRN2 function likely is also regulated by protein-tyrosine phosphatase 1B (PTP1B), as tyrosine-phosphorylated CRN2 has been identified as a substrate of PTP1B (Mondol et al., 2014). In addition to its direct effects on actin filaments, CRN2 modifies the actin cytoskeleton in conjunction with small G-proteins. Binding of CRN2 to GDP-Rac1 and RCC2 leads to an enrichment of GTP-Rac1 at membrane protrusions via vesicular trafficking (Williamson et al., 2014, 2015), and its interaction with GDP-Rab27a increased its F-actin bundling activity associated with endocytosis of the insulin secretory membrane for recycling in pancreatic beta-cells (Kimura et al., 2010).

Several studies demonstrated an involvement of CRN2 in the progression of different forms of human cancer. For example, it was identified as a potential marker for melanoma progression, possibly via the Erk mitogen-activated protein kinase cascade (Roadcap et al., 2008; Shields et al., 2007). Similarly, a marked increase of CRN2 expression was reported in hepatocellular carcinoma cells giving rise to pulmonary metastases (Wu et al., 2010). CRN2 has also been reported to be associated with a poor prognosis of gastric cancer (Cheng et al., 2019) and to promote the metastatic behaviour of gastric cancer cells including their migration and invasion (Ren et al., 2012). Furthermore, immunohistochemistry studies revealed a strong expression of CRN2 in the majority of primary effusion lymphoma cells (Luan et al., 2010). Notably, the expression of CRN2 was reported to correlate with the malignant phenotype of diffuse gliomas (Thal et al., 2008). In this respect, CRN2 knock-down in U373 and A172 human glioblastoma cells led to reduced levels of cell proliferation, cell motility and invasion into the extracellular matrix as compared to control cells (Thal et al., 2008). In contrast, CRN2 overexpression as well as expression of a S463A phosphorylation-resistant CRN2 variant in U373 glioblastoma cells increased proliferation, matrix degradation and invasion but decreased adhesion and formation of invadopodia-like extensions (Ziemann et al., 2013).

Section snippets

Generation of CRN2 knock-out mice and crossbreeding with a glioblastoma mouse model

Generation of CRN2 knock-out mice was performed according to (Behrens et al., 2016). Validation of the correct gene targeting event, the CRN2 knock-out at the mRNA level, and the lack of CRN2 protein isoforms (Xavier et al., 2009) as well as potential truncated protein species was done by Southern blotting and PCR genotyping, RT-PCR in conjunction with sequencing, and immunoblotting using several mono- and polyclonal CRN2-specific antibodies, respectively (Fig. 1 and data not shown).

Comprehensive phenotyping of CRN2 knock-out mice

A cohort of 15 female and 7 male homozygous CRN2 knock-out mice (reporter insertion allele, Fig. 1A) and 8 female and 7 male wild-type siblings were subjected to a comprehensive phenotyping (“primary screen”) at the German Mouse Clinic, Munich. Analyses started with mice aged 9 weeks and ended with age of 21 weeks. The conducted tests and results are summarised, and values that are increased or decreased as compared to the wild-type controls are highlighted in green or orange, respectively (

Discussion

The goal of the present study was to further explore the functional role of the actin filament-binding protein CRN2 in the context of glioblastoma multiforme. For this purpose, we crossbred our CRN2 knock-out mice with an inducible Tp53/Pten knock-out glioblastoma mouse model (Chow et al., 2011; Maire and Ligon, 2011). Our prior comprehensive analysis of the CRN2 knock-out mouse line revealed mild neurological and behavioural alterations. A reduced hearing sensitivity had been described for

Conclusions

In the present study, we further explored the functional role of the actin filament-binding protein CRN2 in the context of glioblastoma multiforme. We performed a multi-scale analysis of the tumour-promoting effects of CRN2 in vivo, ex vivo, and in vitro. Key findings of our study are, a) the detection of neurological and behavioural alterations in CRN2 knock-out mice, b) a higher tumour cell encasement of murine brain slice capillaries by glioblastoma cells overexpressing CRN2, c) the

Funding

Grant support by the German Research Foundation (DFG) (grants NO 113/22-2 to AAN and CSC, and CL 381/2-1 to CSC) and the German Federal Ministry of Education and Research (Infrafrontier grant 01KX1012 to MHdA) is gratefully acknowledged.

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The glioblastoma mouse model was kindly provided by Suzanne J. Baker, St. Jude Children's Research Hospital, Memphis, USA, and was generated using a GFAP-creER allele generated by the Baker lab, combined with a Tp53 allele kindly provided by Anton Berns, Netherlands Cancer Institute, Amsterdam, The Netherlands, and a Pten allele kindly provided by Tak W. Mak, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada. We thank Marija Marko for technical assistance and helpful

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