GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence

Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAPα is downregulated in higher-grade tumours, leading to increased dominance of the GFAPδ isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAPδ and GFAPα isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAPδ-depleted cells migrate randomly through the brain tissue, whereas GFAPα-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAPnetwork lead to specific migratory dynamics and behaviours of gliomas.

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation

Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6–1.0) between the gene expression levels for platelet-derived growth factor β(PDGFβ), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 μM), DMPQ (200 nM), and burixafor (1 μM) identified EGF, PDGFβ, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.

Microglial-stimulation of glioma invasion involves the EGFR ligand amphiregulin

High grade glioma is one of the deadliest human cancers with a median survival rate of only one year following diagnosis. The highly motile and invasive nature of high grade glioma makes it difficult to completely remove surgically. Therefore, increasing our knowledge of the mechanisms glioma cells use to invade normal brain is of critical importance in designing novel therapies. It was previously shown by our laboratory that tumor-associated microglia (TAMs) stimulate glioma cell invasion and this process is dependent on CSF-1R signaling. In this study, we seek to identify pro-invasive factors that are upregulated in microglia in a CSF-1R-dependent manner. We assayed cDNA and protein from microglia treated with conditioned media from the murine glioma cell line GL261, and discovered that several EGFR ligands including amphiregulin (AREG) are strongly upregulated. This upregulation is blocked by addition of a pharmacological CSF-1R inhibitor. Using RNA interference, we show that AREG-depleted microglia are less effective at promoting invasion of GL261 cells into Matrigel-coated invasion chambers. In addition, an AREG blocking antibody strongly attenuates the ability of THP-1 macrophages to activate human glioma cell line U87 invasion. Furthermore, we have identified a signaling pathway which involves CSF-1 signaling through ERK to upregulate AREG expression in microglia. Interfering with ERK using pharmacological inhibitors prevents AREG upregulation in microglia and microglia-stimulated GL261 invasion. These data highlight AREG as a key factor in produced by tumor associated microglia in promoting glioma invasion.

A Ferroptosis-Related Prognostic Risk Score Model to Predict Clinical Significance and Immunogenic Characteristics in Glioblastoma Multiforme

Background. Ferroptosis is a recently identified cell death pathway, and the susceptibility to ferroptosis inducers varies among cancer cell types. There have been recent attempts to clarify the mechanisms implicated in ferroptosis, glioma invasion, and the immune microenvironment but little is known about ferroptosis regulation in GBM. Methods. Screening ferroptosis-related genes from published reports and existing databases, we constructed an integrated model based on the RNA-sequencing data in GBM. The association of FRGPRS and overall survival is identified and validated across several different datasets. Genomic and clinical characteristics, immune infiltration, enriched pathways, pan-cancer, drug resistance, and immune checkpoint inhibitor therapy are compared among various FRGPRS subgroups. Results. We identified and confirmed the influences of five ferroptosis key hub genes in the FRGPRS model. The FRGPRS model could serve to predict overall survival and progression-free survival in GBM patients, and high FRGPRS was associated with comparatively stronger immunity, higher proportions of tumour tissue, and good cytolytic immune and chemotherapeutics response in GBM patients. Conclusions. The five ferroptosis key hub genes constituting the FRGPRS model could serve to predict overall survival and progression-free survival in patients with GBM and help guide timely and efficacious therapeutic strategies customised and optimised for each individual patient. This discovery may lay the foundation for the development and optimisation of other iterations of this model for the improved forecasting, detection, and treatment of other malignancies notorious for their drug resistance and immune escape.

TAMI-45. MICROGLIAL CYTOKINES INDUCE INVASIVENESS AND PROLIFERATION THROUGH PYK2 AND FAK ACTIVATION IN HUMAN GLIOBLASTOMA CELL

Glioblastoma (GBM) is the most aggressive and highly invasive primary brain tumor in adults. Evidence suggests that microglia create a microenvironment favoring glioma invasion and proliferation. Indeed, previous reports indicate the involvement of focal adhesion kinase (FAK) signaling cascades in glioma cell proliferation. Besides, studies from our laboratory support a critical role of Pyk2, a relative of FAK, in glioma invasion by tumor-infiltrating microglia. However, the microglial-released factors modulating Pyk2 and FAK signaling pathways are unknown. In this study, 20 human GBM specimens were evaluated to identify the cytokine expression patterns in purified microglia and FAK and Pyk2 phosphorylation in glioma cell fraction by RT-PCR and western blot. A Pierson correlation test demonstrated a high correlation (0.8-1.0) of gene expression for PDGFα, PDGFβ, SDF-1α, IL-6, IL-8, and EGF in percoll-purified microglia, and pPyk2(Y579/580) and pFAK(Y925) levels in glioma cell fraction. The role of cytokines in cell invasion and proliferation by Pyk2/FAK activation was further investigated in primary cell lines from three patients. Thirty percent up-regulation of pPyk2 and pFAK was detected in glioma cells treated (2 hrs.) with microglia conditioned media (MCM) compared to control cells. siPyk2 or siFAK knockdown identified IL-6 (100 μM) and EGF (1 μM) as key factors of Pyk2- and FAK-dependent activation in all glioma cell lines. Similar results with siPyk2 or siFAK were observed for matrix degradation, invadopodia formation, cell viability, and mitosis. Indeed, Tocilizumab (IL-6R blocker, 100 ng/mL) and Gefitinib (EGFR blocker, 1 μM) reversed the effect of MCM on glioma cell proliferation and invasion in all cell lines evaluated. These findings support a pivotal role of Pyk2 and FAK in enhancing proliferation and invasion of glioma tumors through IL-6 and EGF-dependent pathways. The latter could be of clinical relevance for new therapeutic developments in GBM patients.

CircGLIS3 Promotes High-Grade Glioma Invasion via Modulating Ezrin Phosphorylation

High-grade glioma is highly invasive and malignant, resistant to combined therapies, and easy to relapse. A better understanding of circular RNA (circRNA) biological function in high-grade glioma might contribute to the therapeutic efficacy. Here, a circRNA merely upregulated in high-grade glioma, circGLIS3 (hsa_circ_0002874, originating from exon 2 of GLIS3), was validated by microarray and Real-time quantitative reverse transcription PCR (qRT-PCR). The role of circGLIS3 in glioma was assessed by functional experiments both in vitro and in vivo. Fluorescence in situ hybridization (FISH), RNA pull-down, RNA immunoprecipitation (RIP), and immunohistochemical staining were performed for mechanistic study. Cocultured brain endothelial cells with glioma explored the role of exosome-derived circGLIS3 in the glioma microenvironment. We found that upregulation of circGLIS3 promoted glioma cell migration and invasion and showed aggressive characteristics in tumor-bearing mice. Mechanistically, we found that circGLIS3 could promote the Ezrin T567 phosphorylation level. Moreover, circGLIS3 could be excreted by glioma through exosomes and induced endothelial cell angiogenesis. Our findings indicate that circGLIS3 is upregulated in high-grade glioma and contributes to the invasion and angiogenesis of glioma via modulating Ezrin T567 phosphorylation.

PPFIBP1 induces glioma cell migration and invasion through FAK/Src/JNK signaling pathway

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, with a 5-year survival ratio <5%. Invasive growth is a major determinant of the poor prognosis in GBM. In this study, we demonstrate that high expression of PPFIA binding protein 1 (PPFIBP1) correlates with remarkable invasion and poor prognosis of GBM patients. Using scratch and transwell assay, we find that the invasion and migration of GBM cells are promoted by overexpression of PPFIBP1, while inhibited by knockdown of PPFIBP1. Then, we illustrate that overexpression of PPFIBP1 facilitates glioma cell infiltration and reduces survival in xenograft models. Next, RNA-Seq and GO enrichment analysis reveal that PPFIBP1 regulates differentially expressed gene clusters involved in the Wnt and adhesion-related signaling pathways. Furthermore, we demonstrate that PPFIBP1 activates focal adhesion kinase (FAK), Src, c-Jun N-terminal kinase (JNK), and c-Jun, thereby enhancing Matrix metalloproteinase (MMP)-2 expression probably through interacting with SRCIN1 (p140Cap). Finally, inhibition of phosphorylation of Src and FAK significantly reversed the augmentation of invasion and migration caused by PPFIBP1 overexpression in GBM cells. In conclusion, these findings uncover a novel mechanism of glioma invasion and identify PPFIBP1 as a potential therapeutic target of glioma.

GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence.

Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAPα is downregulated in higher-grade tumours, leading to increased dominance of the GFAPδ isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAPδ and GFAPα isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAPδ-depleted cells migrate randomly through the brain tissue, whereas GFAPα-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAP-network lead to specific migratory dynamics and behaviours of gliomas.