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Mutations in the WWOX gene cause a broad range of ultra-rare neurodevelopmental and brain degenerative disorders, associated with a high likelihood of premature death in animal models as well as in humans. The encoded Wwox protein is a WW domain-containing oxidoreductase that participates in crucial biological processes including tumor suppression, cell growth/differentiation and regulation of steroid metabolism, while its role in neural development is less understood. We analyzed the exomes of a family affected with multiple pre- and postnatal anomalies, including cerebellar vermis hypoplasia, severe neurodevelopmental impairment and refractory epilepsy, and identified a segregating homozygous WWOX mutation leading to a premature stop codon. Abnormal cerebral cortex development due to a defective architecture of granular and molecular cell layers was found in the developing brain of a WWOX-deficient human fetus from this family. A similar disorganization of cortical layers was identified in lde/lde rats (carrying a homozygous truncating mutation which disrupts the active Wwox C-terminal domain) investigated at perinatal stages. Transcriptomic analyses of Wwox-depleted human neural progenitor cells showed an impaired expression of a number of neuronal migration-related genes encoding for tubulins, kinesins and associated proteins. These findings indicate that loss of Wwox may affect different cytoskeleton components and alter prenatal cortical development, highlighting a regulatory role of the WWOX gene in migrating neurons across different species.

IntroductionGlioblastoma (GBM) is notorious for its clinical and molecular heterogeneity, contributing to therapeutic failure and a grim prognosis. WWOX is one of the tumor suppressor genes important in nervous tissue or related pathologies, which was scarcely investigated in GBM for reliable associations with prognosis or disease progression despite known alterations. Recently, we observed a phenotypic heterogeneity between GBM cell lines (U87MG, T98G, U251MG, DBTRG-05MG), among which the anti-GBM activity of WWOX was generally corresponding, but colony growth and formation were inconsistent in DBTRG-05MG. This prompted us to investigate the molecular landscapes of these cell lines, intending to translate them into the clinical context.MethodsU87MG/T98G/U251MG/DBTRG-05MG were subjected to high-throughput sequencing, and obtained data were explored via weighted gene co-expression network analysis, differential expression analysis, functional annotation, and network building. Following the identification of the most relevant DBTRG-distinguishing driver genes, data from GBM patients were employed for, e.g., differential expression analysis, survival analysis, and principal component analysis.ResultsAlthough most driver genes were unique for each cell line, some were inversely regulated in DBTRG-05MG. Alongside driver genes, the differentially-expressed genes were used to build a WWOX-related network depicting protein–protein interactions in U87MG/T98G/U251MG/DBTRG-05MG. This network revealed processes distinctly regulated in DBTRG-05MG, e.g., microglia proliferation or neurofibrillary tangle assembly. POLE4 and HSF2BP were selected as DBTRG-discriminating driver genes based on the gene significance, module membership, and fold-change. Alongside WWOX, POLE4 and HSF2BP expression was used to stratify patients into cell lines-resembling groups that differed in, e.g., prognosis and treatment response. Some differences from a WWOX-related network were certified in patients, revealing genes that clarify clinical outcomes. Presumably, WWOX overexpression in DBTRG-05MG resulted in expression profile change resembling that of patients with inferior prognosis and drug response. Among these patients, WWOX may be inaccessible for its partners and does not manifest its anti-cancer activity, which was proposed in the literature but not regarding glioblastoma or concerning POLE4 and HSF2BP.ConclusionCell lines data enabled the identification of patients among which, despite high expression of WWOX tumor suppressor, no advantageous outcomes were noted due to the cancer-promoting profile ensured by other genes.

The brain is the most functionally organized structure of all organs. It manages behavior, perception and higher cognitive functions. The WWOX gene is non-classical tumor suppressor gene, which has been shown to have an impact on proliferation, apoptosis and migration processes. Moreover, genetic aberrations in WWOX induce severe neuropathological phenotypes in humans and rodents. The aim of the present study was to investigate in detail the impact of WWOX on human neural progenitor cell (hNPC) maintenance and how depletion of WWOX disturbs signaling pathways playing a pivotal role in neuronal differentiation and central nervous system (CNS) organogenesis. hNPC with a silenced WWOX gene exhibited lowered mitochondrial redox potential, enhanced adhesion to fibronectin and extracellular matrix protein mixture, downregulation of MMP2/9 expression and impaired 3D growth. Global transcriptome analysis using cap analysis of gene expression (CAGE) found that WWOX downregulation significantly changes the expression of multiple genes engaged in cytoskeleton organization, adhesion, cell signaling and chromatin remodeling. The massive changes in gene expression caused by WWOX silencing may strongly affect the differentiation and migration of neurons in organogenesis, brain injury, cancerogenesis or neurodifferentiation. WWOX gene appears to be an important regulator of neural tissue architecture and function.