[en] The infiltrative nature of Glioblastoma (GBM), the most aggressive primary brain tumor, critically prevents complete surgical resection and masks tumor cells behind the blood brain barrier reducing the efficacy of systemic treatment. Here, we use a genome-wide interference screen to determine invasion-essential genes and identify the AN1/A20 zinc finger domain containing protein 3 (ZFAND3) as a crucial driver of GBM invasion. Using patient-derived cellular models, we show that loss of ZFAND3 hampers the invasive capacity of GBM, whereas ZFAND3 overexpression increases motility in cells that were initially not invasive. At the mechanistic level, we find that ZFAND3 activity requires nuclear localization and integral zinc-finger domains. Our findings indicate that ZFAND3 acts within a nuclear protein complex to activate gene transcription and regulates the promoter of invasion-related genes such as COL6A2, FN1, and NRCAM. Further investigation in ZFAND3 function in GBM and other invasive cancers is warranted.
Research center :
Luxembourg Institute of Health
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Schuster, Anne
Klein, Eliane
Neirinckx, Virginie ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Département des sciences biomédicales et précliniques
Claes, A., Idema, A. J. & Wesseling, P. Diffuse glioma growth: a guerilla war. Acta Neuropathol. 114, 443–458 (2007). DOI: 10.1007/s00401-007-0293-7
Osswald, M. et al. Brain tumour cells interconnect to a functional and resistant network. Nature 528, 93–98 (2015). DOI: 10.1038/nature16071
Weil, S. et al. Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas. Neuro Oncol. 19, 1316–1326 (2017). DOI: 10.1093/neuonc/nox070
Cuddapah, V. A., Robel, S., Watkins, S. & Sontheimer, H. A neurocentric perspective on glioma invasion. Nat. Rev. Neurosci. 15, 455–465 (2014). DOI: 10.1038/nrn3765
de Gooijer, M. C. et al. Experimenter’s guide to glioblastoma invasion pathways. Trends Mol. Med. 24, 763–780 (2018). DOI: 10.1016/j.molmed.2018.07.003
Lefranc, F. et al. Glioblastoma quo vadis: will migration and invasiveness reemerge as therapeutic targets? Cancer Treat. Rev. 68, 145–154 (2018). DOI: 10.1016/j.ctrv.2018.06.017
Picariello, H. S. et al. Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner. Proc. Natl Acad. Sci. USA 116, 15550–15559 (2019). DOI: 10.1073/pnas.1902847116
Tome-Garcia, J. et al. Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma. Nat. Commun. 9, 4020 (2018). DOI: 10.1038/s41467-018-06258-2
Schuster, A. et al. RNAi/CRISPR screens: from a pool to a valid hit. Trends Biotechnol. 37, 38–55 (2019). DOI: 10.1016/j.tibtech.2018.08.002
Seo, M. et al. RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways. Nat. Commun. 5, 5217 (2014). DOI: 10.1038/ncomms6217
Agaesse, G. et al. A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion. Oncogene 36, 446–457 (2017). DOI: 10.1038/onc.2016.219
Prolo, L. M. et al. Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion. Sci. Rep. 9, 14020 (2019). DOI: 10.1038/s41598-019-50160-w
Lopez-Fernandez, L. A. & del Mazo, J. Characterization of genes expressed early in mouse spermatogenesis, isolated from a subtractive cDNA library. Mamm. Genome 7, 698–700 (1996). DOI: 10.1007/s003359900210
de Luis, O., Lopez-Fernandez, L. A. & del Mazo, J. Tex27, a gene containing a zinc-finger domain, is up-regulated during the haploid stages of spermatogenesis. Exp. Cell Res. 249, 320–326 (1999). DOI: 10.1006/excr.1999.4482
Ndiaye, F. K. et al. Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion. Mol. Metab. 6, 459–470 (2017). DOI: 10.1016/j.molmet.2017.03.011
Cho, Y. S. et al. Meta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians. Nat. Genet. 44, 67–72 (2011). DOI: 10.1038/ng.1019
Bougnaud, S. et al. Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma. Oncotarget 7, 31955–31971 (2016). DOI: 10.18632/oncotarget.7454
Wang, Q. et al. Tumor evolution of glioma-intrinsic gene expression subtypes associates with immunological changes in the microenvironment. Cancer Cell 32, 42–56 (2017). e46. DOI: 10.1016/j.ccell.2017.06.003
Konig, R. et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat. Methods 4, 847–849 (2007). DOI: 10.1038/nmeth1089
Luo, B. et al. Highly parallel identification of essential genes in cancer cells. Proc. Natl Acad. Sci. USA 105, 20380–20385 (2008). DOI: 10.1073/pnas.0810485105
Li, W. et al. Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol. 16, 281 (2015). DOI: 10.1186/s13059-015-0843-6
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014). DOI: 10.1186/s13059-014-0554-4
Diaz, A. A., Qin, H., Ramalho-Santos, M. & Song, J. S. HiTSelect: a comprehensive tool for high-complexity-pooled screen analysis. Nucleic Acids Res. 43, e16 (2015). DOI: 10.1093/nar/gku1197
Bowman, R. L., Wang, Q., Carro, A., Verhaak, R. G. & Squatrito, M. GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol. 19, 139–141 (2017). DOI: 10.1093/neuonc/now247
Tang, Z. et al. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 45, W98–W102 (2017). DOI: 10.1093/nar/gkx247
Consortium, G. T. The genotype-tissue expression (GTEx) project. Nat. Genet 45, 580–585 (2013). DOI: 10.1038/ng.2653
Roux, K. J., Kim, D. I. & Burke, B. BioID: a screen for protein-protein interactions. Curr. Protoc. Protein Sci. 74, 23 (2013). Unit 19. DOI: 10.1002/0471140864.ps1923s74
Schaefer, U., Schmeier, S. & Bajic, V. B. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins. Nucleic Acids Res. 39, D106–D110 (2011). DOI: 10.1093/nar/gkq945
Cassandri, M. et al. Zinc-finger proteins in health and disease. Cell Death Disco. 3, 17071 (2017). DOI: 10.1038/cddiscovery.2017.71
Osorio, F. G. et al. Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling. Nat. Med. 22, 91–96 (2016). DOI: 10.1038/nm.4013
Kurihara-Shimomura, M. et al. Zinc finger AN1-type containing 4 is a novel marker for predicting metastasis and poor prognosis in oral squamous cell carcinoma. J. Clin. Pathol. 71, 436–441 (2018). DOI: 10.1136/jclinpath-2017-204770
Suarez-Canto, J. et al. Distinct expression and clinical significance of zinc finger AN-1-type containing 4 in oral squamous cell carcinomas. J. Clin. Med. 7 (2018).
Pizzato Scomazzon, S. et al. The zinc-finger AN1-type domain 2a gene acts as a regulator of cell survival in human melanoma: role of E3-ligase cIAP2. Mol. Cancer Res. 17, 2444–2456 (2019). DOI: 10.1158/1541-7786.MCR-19-0243
Turakhiya, A. et al. ZFAND1 recruits p97 and the 26S proteasome to promote the clearance of arsenite-induced stress granules. Mol. Cell 70, 906–919 (2018).e907. DOI: 10.1016/j.molcel.2018.04.021
Rossi, A. et al. The proteasome inhibitor bortezomib is a potent inducer of zinc finger AN1-type domain 2a gene expression: role of heat shock factor 1 (HSF1)-heat shock factor 2 (HSF2) heterocomplexes. J. Biol. Chem. 289, 12705–12715 (2014). DOI: 10.1074/jbc.M113.513242
Lee, D., Takayama, S. & Goldberg, A. L. ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation. Proc. Natl. Acad. Sci. USA 115, E9550–E9559 (2018). DOI: 10.1073/pnas.1809934115
Fenner, B. J., Scannell, M. & Prehn, J. H. Identification of polyubiquitin binding proteins involved in NF-kappaB signaling using protein arrays. Biochim Biophys. Acta 1794, 1010–1016 (2009). DOI: 10.1016/j.bbapap.2009.02.013
He, G., Sun, D., Ou, Z. & Ding, A. The protein Zfand5 binds and stabilizes mRNAs with AU-rich elements in their 3’-untranslated regions. J. Biol. Chem. 287, 24967–24977 (2012). DOI: 10.1074/jbc.M112.362020
Sa-Moura, B. et al. A conserved protein with AN1 zinc finger and ubiquitin-like domains modulates Cdc48 (p97) function in the ubiquitin-proteasome pathway. J. Biol. Chem. 288, 33682–33696 (2013). DOI: 10.1074/jbc.M113.521088
Hishiya, A. et al. A novel ubiquitin-binding protein ZNF216 functioning in muscle atrophy. EMBO J. 25, 554–564 (2006). DOI: 10.1038/sj.emboj.7600945
Sun, D., Lei, W., Hou, X., Li, H. & Ni, W. PUF60 accelerates the progression of breast cancer through downregulation of PTEN expression. Cancer Manag. Res. 11, 821–830 (2019). DOI: 10.2147/CMAR.S180242
Mao, Y. Q. & Houry, W. A. The role of pontin and reptin in cellular physiology and cancer etiology. Front Mol. Biosci. 4, 58 (2017). DOI: 10.3389/fmolb.2017.00058
Serrano, F. et al. A novel human pluripotent stem cell-derived neural crest model of treacher collins syndrome shows defects in cell death and migration. Stem Cells Dev. 28, 81–100 (2019). DOI: 10.1089/scd.2017.0234
Gollapalli, K. et al. Subventricular zone involvement in Glioblastoma - A proteomic evaluation and clinicoradiological correlation. Sci. Rep. 7, 1449 (2017). DOI: 10.1038/s41598-017-01202-8
Machado, R. A. C. et al. CHD7 promotes glioblastoma cell motility and invasiveness through transcriptional modulation of an invasion signature. Sci. Rep. 9, 3952 (2019). DOI: 10.1038/s41598-019-39564-w
Geng, F., Wenzel, S. & Tansey, W. P. Ubiquitin and proteasomes in transcription. Annu Rev. Biochem. 81, 177–201 (2012). DOI: 10.1146/annurev-biochem-052110-120012
Dirkse, A. et al. Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment. Nat. Commun. 10, 1787 (2019). DOI: 10.1038/s41467-019-09853-z
Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996). DOI: 10.1126/science.272.5259.263
Abdul Rahim, S. A. et al. Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A. Br. J. Cancer 117, 813–825 (2017). DOI: 10.1038/bjc.2017.263
Langmead, B., Wilks, C., Antonescu, V. & Charles, R. Scaling read aligners to hundreds of threads on general-purpose processors. Bioinformatics 35, 421–432 (2019). DOI: 10.1093/bioinformatics/bty648
Anders, S., Pyl, P. T. & Huber, W. HTSeq-a python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). DOI: 10.1093/bioinformatics/btu638
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). DOI: 10.1186/s13059-014-0550-8
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008). DOI: 10.1038/nbt.1511
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011). DOI: 10.1021/pr101065j
Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteom. 13, 2513–2526 (2014). DOI: 10.1074/mcp.M113.031591
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015). DOI: 10.1093/nar/gkv007
Agoston, Z. & Schulte, D. Meis2 competes with the Groucho co-repressor Tle4 for binding to Otx2 and specifies tectal fate without induction of a secondary midbrain-hindbrain boundary organizer. Development 136, 3311–3322 (2009). DOI: 10.1242/dev.037770
Hau, A. C. et al. MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1. J. Cell Biol. 216, 2715–2729 (2017). DOI: 10.1083/jcb.201701154
Varnaite, R. & MacNeill, S. A. Meet the neighbors: mapping local protein interactomes by proximity-dependent labeling with BioID. Proteomics 16, 2503–2518 (2016). DOI: 10.1002/pmic.201600123
