Conditioned Medium From Reactive Astrocytes Inhibits Proliferation, Resistance, and Migration of p53-Mutant Glioblastoma Spheroid Through GLI-1 Downregulation.

Glioblastoma (GBM) aggressiveness is partly driven by the reactivation of signaling pathways such as Sonic hedgehog (SHH) and the interaction with its microenvironment. SHH pathway activation is one of the phenomena behind the glial transformation in response to tumor growth. The reactivation of the SHH signaling cascade during GBM-astrocyte interaction is highly relevant to understanding the mechanisms used by the tumor to modulate the adjacent stroma. The role of reactive astrocytes considering SHH signaling during GBM progression is investigated using a 3D in vitro model. T98G GBM spheroids displayed significant downregulation of SHH (61.4 ± 9.3%), GLI-1 (6.5 ± 3.7%), Ki-67 (33.7 ± 8.1%), and mutant MTp53 (21.3 ± 10.6%) compared to the CONTROL group when incubated with conditioned medium of reactive astrocytes (CM-AST). The SHH pathway inhibitor, GANT-61, significantly reduced previous markers (SHH = 43.0 ± 12.1%; GLI-1 = 9.5 ± 3.4%; Ki-67 = 31.9 ± 4.6%; MTp53 = 6.5 ± 7.5%) compared to the CONTROL, and a synergistic effect could be observed between GANT-61 and CM-AST. The volume (2.0 ± 0.2 × 107 µm³), cell viability (80.4 ± 3.2%), and migration (41 ± 10%) of GBM spheroids were significantly reduced in the presence of GANT-61 and CM-AST when compared to CM-AST after 72 h (volume = 2.3 ± 0.4 × 107 µm³; viability = 92.2 ± 6.5%; migration = 102.5 ± 14.6%). Results demonstrated that factors released by reactive astrocytes promoted a neuroprotective effect preventing GBM progression using a 3D in vitro model potentiated by SHH pathway inhibition.

Endothelial cell ferroptosis influences IDH wild-type glioblastoma growth in recurrent glioblastoma multiforme patients.

Glioblastomas are known for their poor clinical prognosis, with recurrent tumors often exhibiting greater invasiveness and faster growth rates compared to primary tumors. To understand the intratumoral changes driving this phenomenon, we employed single-cell sequencing to analyze the differences between two pairs of primary and recurrent glioblastomas. Our findings revealed an upregulation of ferroptosis in endothelial cells within recurrent tumors, identified by the significant overexpression of the NOX4 gene. Further analysis indicated that knocking down NOX4 in endothelial cells reduced the activity of the ferroptosis pathway. Utilizing conditioned media from endothelial cells with lower ferroptosis activity, we observed a decrease in the growth rate of glioblastoma cells. These results highlighted the complex role of ferroptosis within tumors and suggested that targeting ferroptosis in the treatment of glioblastomas requires careful consideration of its effects on endothelial cells, as it may otherwise produce counterproductive outcomes.

Enhancing Therapeutic Approaches in Glioblastoma with Pro-Oxidant Treatments and Synergistic Combinations: In Vitro Experience of Doxorubicin and Photodynamic Therapy.

Glioblastoma (GBM) is an aggressive brain cancer characterized by significant molecular and cellular heterogeneity, which complicates treatment efforts. Current standard therapies, including surgical resection, radiation, and temozolomide (TMZ) chemotherapy, often fail to achieve long-term remission due to tumor recurrence and resistance. A pro-oxidant environment is involved in glioma progression, with oxidative stress contributing to the genetic instability that leads to gliomagenesis. Evaluating pro-oxidant therapies in brain tumors is crucial due to their potential to selectively target and eradicate cancer cells by exploiting the elevated oxidative stress levels inherent in these malignant cells, thereby offering a novel and effective strategy for overcoming resistance to conventional therapies. This study investigates the therapeutic potential of doxorubicin (DOX) and photodynamic therapy (PDT) with Me-ALA, focusing on their effects on redox homeostasis. Basal ROS levels and antioxidant gene expression (NFE2L2, CAT, GSR) were quantitatively assessed across GBM cell lines, revealing significant variability probably linked to genetic differences. DOX and PDT treatments, both individually and in combination, were analyzed for their efficacy in inducing oxidative stress and cytotoxicity. An in silico analysis further explored the relationship between gene mutations and oxidative stress in GBM patients, providing insights into the molecular mechanisms underlying treatment responses. Our findings suggest that pro-oxidant therapies, such as DOX and PDT in combination, could selectively target GBM cells, highlighting a promising avenue for improving therapeutic outcomes in GBM.

Mutations in glioblastoma proteins do not disrupt epitope presentation and recognition, maintaining a specific CD8 T cell immune response potential.

Antigen-specific cytotoxic CD8 T cells are extremely effective in controlling tumor growth and have been the focus of immunotherapy approaches. We leverage in silico tools to investigate whether the occurrence of mutations in proteins previously described as immunogenic and highly expressed by glioblastoma multiforme (GBM), such as Epidermal Growth Factor Receptor (EGFR), Isocitrate Dehydrogenase 1 (IDH1), Phosphatase and Tensin homolog (PTEN) and Tumor Protein 53 (TP53), may be contributing to the differential presentation of immunogenic epitopes. We recovered Class I MHC binding information from wild-type and mutated proteins using the Immune Epitope Database (IEDB). After that, we built peptide-MHC (pMHC-I) models in HLA-arena, followed by hierarchical clustering analysis based on electrostatic surface features from each complex. We identified point mutations that are determinants for the presentation of a set of peptides from TP53 protein. We point to structural features in the pMHC-I complexes of wild-type and mutated peptides, which may play a role in the recognition of CD8 T cells. To further explore these features, we performed 100 ns molecular dynamics simulations for the peptide pairs (wt/mut) selected. In pursuit of novel therapeutic targets for GBM treatment, we selected peptides where our predictive results indicated that mutations would not disrupt epitope presentation, thereby maintaining a specific CD8 T cell immune response. These peptides hold potential for future GBM interventions, including peptide-based or mRNA vaccine development applications.

Network-Based Transcriptome Analysis Reveals FAM3C as a Novel Potential Biomarker for Glioblastoma.

Glioblastoma (GBM) is the most common form of malignant primary brain tumor with a high mortality rate. The aim of the present study was to investigate the clinical significance of Family with Sequence Similarity 3, Member C, FAM3C, in GBM using bioinformatic-integrated analysis. First, we performed the transcriptomic integration analysis to assess the expression profile of FAM3C in GBM using several data sets (RNA-sequencing and scRNA-sequencing), which were obtained from TCGA and GEO databases. By using the STRING platform, we investigated FAM3C-coregulated genes to construct the protein-protein interaction network. Next, Metascape, Enrichr, and CIBERSORT databases were used. We found FAM3C high expression in GBM with poor survival rates. Further, we observed, via FAM3C coexpression network analysis, that FAM3C plays key roles in several hallmarks of cancer. Surprisingly, we also highlighted five FAM3C­coregulated genes overexpressed in GBM. Specifically, we demonstrated the association between the high expression of FAM3C and the abundance of the different immune cells, which may markedly worsen GBM prognosis. For the first time, our findings suggest that FAM3C not only can be a new emerging biomarker with promising therapeutic values to GBM patients but also gave a new insight into a potential resource for future GBM studies.

Temozolomide resistance mechanisms: unveiling the role of translesion DNA polymerase kappa in glioblastoma spheroids in vitro.

Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 µM); (iii) exerts antiproliferative effects (≤25 µM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared with WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Unveiling the Dynamics behind Glioblastoma Multiforme Single-Cell Data Heterogeneity.

Glioblastoma Multiforme is a brain tumor distinguished by its aggressiveness. We suggested that this aggressiveness leads single-cell RNA-sequence data (scRNA-seq) to span a representative portion of the cancer attractors domain. This conjecture allowed us to interpret the scRNA-seq heterogeneity as reflecting a representative trajectory within the attractor's domain. We considered factors such as genomic instability to characterize the cancer dynamics through stochastic fixed points. The fixed points were derived from centroids obtained through various clustering methods to verify our method sensitivity. This methodological foundation is based upon sample and time average equivalence, assigning an interpretative value to the data cluster centroids and supporting parameters estimation. We used stochastic simulations to reproduce the dynamics, and our results showed an alignment between experimental and simulated dataset centroids. We also computed the Waddington landscape, which provided a visual framework for validating the centroids and standard deviations as characterizations of cancer attractors. Additionally, we examined the stability and transitions between attractors and revealed a potential interplay between subtypes. These transitions might be related to cancer recurrence and progression, connecting the molecular mechanisms of cancer heterogeneity with statistical properties of gene expression dynamics. Our work advances the modeling of gene expression dynamics and paves the way for personalized therapeutic interventions.

Ethnicity-Based Variations in Focal Adhesion Kinase Signaling in Glioblastoma Gene Expression: A Study of the Puerto Rican Hispanic Population.

Glioblastoma (GBM), an aggressive form of brain cancer, has a higher incidence in non-Hispanics when compared to the US Hispanic population. Using data from RT-PCR analysis of 21 GBM tissue from Hispanic patients in Puerto Rico, we identified significant correlations in the gene expression of focal adhesion kinase and proline-rich tyrosine kinase (PTK2 and PTK2B) with NGFR (nerve growth factor receptor), PDGFRB (platelet-derived growth factor receptor B), EGFR (epithelial growth factor receptor), and CXCR1 (C-X-C motif chemokine receptor 1). This study further explores these correlations found in gene expression while accounting for sex and ethnicity. Statistically significant (p < 0.05) correlations with an r value > ±0.7 were subsequently contrasted with mRNA expression data acquired from cBioPortal for 323 GBM specimens. Significant correlations in Puerto Rican male patients were found between PTK2 and PTK2B, NGFR, PDGFRB, EGFR, and CXCR1, which did not arise in non-Hispanic male patient data. The data for Puerto Rican female patients showed correlations in PTK2 with PTK2B, NGFR, PDGFRB, and EGFR, all of which did not appear in the data for non-Hispanic female patients. The data acquired from cBioPortal for non-Puerto Rican Hispanic patients supported the correlations found in the Puerto Rican population for both sexes. Our findings reveal distinct correlations in gene expression patterns, particularly involving PTK2, PTK2B, NGFR, PDGFRB, and EGFR among Puerto Rican Hispanic patients when compared to non-Hispanic counterparts.

Integrated transcriptomics uncovers an enhanced association between the prion protein gene expression and vesicle dynamics signatures in glioblastomas.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor that exhibits resistance to current treatment, making the identification of novel therapeutic targets essential. In this context, cellular prion protein (PrPC) stands out as a potential candidate for new therapies. Encoded by the PRNP gene, PrPC can present increased expression levels in GBM, impacting cell proliferation, growth, migration, invasion and stemness. Nevertheless, the exact molecular mechanisms through which PRNP/PrPC modulates key aspects of GBM biology remain elusive. METHODS: To elucidate the implications of PRNP/PrPC in the biology of this cancer, we analyzed publicly available RNA sequencing (RNA-seq) data of patient-derived GBMs from four independent studies. First, we ranked samples profiled by bulk RNA-seq as PRNPhigh and PRNPlow and compared their transcriptomic landscape. Then, we analyzed PRNP+ and PRNP- GBM cells profiled by single-cell RNA-seq to further understand the molecular context within which PRNP/PrPC might function in this tumor. We explored an additional proteomics dataset, applying similar comparative approaches, to corroborate our findings. RESULTS: Functional profiling revealed that vesicular dynamics signatures are strongly correlated with PRNP/PrPC levels in GBM. We found a panel of 73 genes, enriched in vesicle-related pathways, whose expression levels are increased in PRNPhigh/PRNP+ cells across all RNA-seq datasets. Vesicle-associated genes, ANXA1, RAB31, DSTN and SYPL1, were found to be upregulated in vitro in an in-house collection of patient-derived GBM. Moreover, proteome analysis of patient-derived samples reinforces the findings of enhanced vesicle biogenesis, processing and trafficking in PRNPhigh/PRNP+ GBM cells. CONCLUSIONS: Together, our findings shed light on a novel role for PrPC as a potential modulator of vesicle biology in GBM, which is pivotal for intercellular communication and cancer maintenance. We also introduce GBMdiscovery, a novel user-friendly tool that allows the investigation of specific genes in GBM biology.

Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma.

Glioblastomas derived from malignant astrocytes are the most common primary tumors of the central nervous system in humans, exhibiting very bad prognosis. Treatment with surgery, radiotherapy, and chemotherapy (mainly using temozolomide), generates as much one-year survival. The circadian clock controls different aspects of tumor development, and its role in GBM is beginning to be explored. Here, the role of the canonic circadian clock gene bmal1 was studied in vivo in a nude mice model bearing human GBMs from LN229 cells xenografted orthotopically in the dorsal striatum. For that aim, a bmal1 knock-down was generated in LN229 cells by CRISPR/Cas9 gene editing tool, and tumor progression was followed in male mice by measuring survival, tumor growth, cell proliferation and prognosis with CD44 marker, as well as astrocyte activation in the tumor microenvironment with GFAP and nestin markers. Disruption of bmal1 in the tumor decreased survival, increased tumor growth and CD44 expression, worsened motor performance, as well as increased GFAP expression in astrocytes at tumor microenvironment. In addition, survival and tumor progression was not affected in mice bearing LN229 wild type GBM that underwent circadian disruption by constant light, as compared to mice synchronized to 12:12 light-dark cycles. These results consistently demonstrate in an in vivo orthotopic model of human GBM, that bmal1 has a key role as a tumor suppressor gene regulating GBM progression.

Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

Upregulation of the canonical signaling pathway of interferon-gamma is associated with glioblastoma progression.

BACKGROUND: Glioblastoma is a brain malignant tumor grade IV, highly invasive. Alterations in several signaling pathways are involved in glioblastoma development. In this work, we evaluated the IFN-γ canonical signaling pathway in glioblastoma cells and its effect on cell viability and migration. METHODS: The levels of STAT1/pSTAT1, IRF1, and PD-L1 in LN-18 glioblastoma cells were analyzed using western blotting. Cell viability was evaluated by calcein-AM/propidium iodide assays, and a wound healing assay was used to study the migration of glioblastoma cells treated with IFN-γ. Our aim was to determine the expression of IFN-γ signaling elements in cell lines and tissue from glioblastoma samples and examine the relationship between these elements and the survival of glioblastoma patients. The following platforms were utilized for analysis: the CCLE (Cancer Cell Line Encyclopedia), UALCAN (University of Alabama at Birmingham Cancer data analysis Portal), GEPIA (Gene Expression Profiling Interactive Analysis), and GENT2 (Gene Expression patterns across Normal and Tumor tissues). RESULTS: Our results evidenced that IFN-γ signaling increases non-phosphorylated and phosphorylated STAT1 levels and promotes the upregulation of IRF1 and PD-L1 in glioblastoma cells. The activation of IFN-γ signaling increased cell migration without affecting the viability of glioblastoma cells. Furthermore, in silico analysis showed that the elements of IFN-γ signaling pathways (IFNGR1/IFNGR2/STAT1/IRF1) are upregulated in human glioblastoma samples. The upregulation of IFN-γ signaling was associated with shorter survival in glioblastoma patients. CONCLUSION: IFN-γ signaling pathway is upregulated in glioblastoma, displaying pro-tumor activity. Thus, IFN-γ signaling elements may be potential biomarkers and targets for treating glioblastoma.

PI3K Signaling Pathways as a Molecular Target for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common type of cancer that affects the central nervous system (CNS). It currently accounts for about 2% of diagnosed malignant tumors worldwide, with 296,000 new cases reported per year. The first-choice treatment consists of surgical resection, radiotherapy, and adjuvant chemotherapy, which increases patients' survival by 15 months. New clinical and pre-clinical research aims to improve this prognosis by proposing the search for new drugs that effectively eliminate cancer cells, circumventing problems such as resistance to treatment. One of the promising therapeutic strategies in the treatment of GBM is the inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway, which is closely related to the process of tumor carcinogenesis. This review sought to address the main scientific studies of synthetic or natural drug prototypes that target specific therapy co-directed via the PI3K pathway, against human glioblastoma.

Role of Glycolytic and Glutamine Metabolism Reprogramming on the Proliferation, Invasion, and Apoptosis Resistance through Modulation of Signaling Pathways in Glioblastoma.

Glioma cells exhibit genetic and metabolic alterations that affect the deregulation of several cellular signal transduction pathways, including those related to glucose metabolism. Moreover, oncogenic signaling pathways induce the expression of metabolic genes, increasing the metabolic enzyme activities and thus the critical biosynthetic pathways to generate nucleotides, amino acids, and fatty acids, which provide energy and metabolic intermediates that are essential to accomplish the biosynthetic needs of glioma cells. In this review, we aim to explore how dysregulated metabolic enzymes and their metabolites from primary metabolism pathways in glioblastoma (GBM) such as glycolysis and glutaminolysis modulate anabolic and catabolic metabolic pathways as well as pro-oncogenic signaling and contribute to the formation, survival, growth, and malignancy of glioma cells. Also, we discuss promising therapeutic strategies by targeting the key players in metabolic regulation. Therefore, the knowledge of metabolic reprogramming is necessary to fully understand the biology of malignant gliomas to improve patient survival significantly.

A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway.

PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 in vitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.

ROCK inhibition reduces the sensitivity of mutant p53 glioblastoma to genotoxic stress through a Rac1-driven ROS production.

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown completely eliminated ROCKi's p53-dependent actions, since ROCK inhibition specifically elevated Rac-GTP levels only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor in improving the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.

PDGFRA, KIT, and KDR Gene Amplification in Glioblastoma: Heterogeneity and Clinical Significance.

Glioblastoma (GBM) is the most frequent tumor of the central nervous system, and its heterogeneity is a challenge in treatment. This study examined tumoral heterogeneity involving PDGFRA, KIT, and KDR gene amplification (GA) in 4q12 and its association with clinical parameters. Specimens from 22 GBM cases with GA for the 4q12 amplicon detected by FISH were investigated for homogeneous or heterogeneous coamplification patterns, diffuse or focal distribution of cells harboring GA throughout tumor sections, and pattern of clustering of fluorescence signals. Sixteen cases had homogenously amplification for all three genes (45.5%), for PDGFRA and KDR (22.7%), or only for PDGFRA (4.6%); six cases had heterogeneous GA patterns, with subpopulations including GA for all three genes and for two genes - PDGFRA and KDR (13.6%), or GA for all three and for only one gene - PDGFRA (9.1%) or KIT (4.6%). In 6 tumors (27.3%), GA was observed in focal tumor areas, while in the remaining 16 tumors (72.7%) it was diffusely distributed throughout the pathological specimen. Amplification was universally expressed as double minutes and homogenously stained regions. Coamplification of all three genes PDGFRA, KIT, and KDR, age ≥ 60 years, and total tumor resection were statistically associated with poor prognosis. FISH proved effective for detailed interpretation of molecular heterogeneity. The study uncovered an even more diverse range of amplification patterns involving the 4q12 oncogenes in GBM than previously described, thus highlighting a complex tumoral heterogeneity to be considered when devising more effective therapies.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG.

BACKGROUND: HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model. METHODS: Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry. RESULTS: The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG. CONCLUSIONS: We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Downregulation of the Rho GTPase pathway abrogates resistance to ionizing radiation in wild-type p53 glioblastoma by suppressing DNA repair mechanisms.

Glioblastoma (GBM), the most common aggressive brain tumor, is characterized by rapid cellular infiltration and is routinely treated with ionizing radiation (IR), but therapeutic resistance inevitably recurs. The actin cytoskeleton of glioblastoma cells provides their high invasiveness, but it remains unclear whether Rho GTPases modulate DNA damage repair and therapeutic sensitivity. Here, we irradiated glioblastoma cells with different p53 status and explored the effects of Rho pathway inhibition to elucidate how actin cytoskeleton disruption affects the DNA damage response and repair pathways. p53-wild-type and p53-mutant cells were subjected to Rho GTPase pathway modulation by treatment with C3 toxin; knockdown of mDia-1, PFN1 and MYPT1; or treatment with F-actin polymerization inhibitors. Rho inhibition increased the sensitivity of glioma cells to IR by increasing the number of DNA double-strand breaks and delaying DNA repair by nonhomologous end-joining in p53-wild-type cells. p53 knockdown reversed this phenotype by reducing p21 expression and Rho signaling activity, whereas reactivation of p53 in p53-mutant cells by treatment with PRIMA-1 reversed these effects. The interdependence between p53 and Rho is based on nuclear p53 translocation facilitated by G-actin and enhanced by IR. Isolated IR-resistant p53-wild-type cells showed an altered morphology and increased stress fiber formation: inhibition of Rho or actin polymerization decreased cell viability in a p53-dependent manner and reversed the resistance phenotype. p53 silencing reversed the Rho inhibition-induced sensitization of IR-resistant cells. Rho inhibition also impaired the repair of IR-damaged DNA in 3D spheroid models. Rho GTPase activity and actin cytoskeleton dynamics are sensitive targets for the reversal of acquired resistance in GBM tumors with wild-type p53.