Research paperCRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells
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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A full list of authors and their affiliations is provided in the Appendix section.