Gene Expression In Glioblastoma Cells Research Articles

SRSF2 promotes glioblastoma cell proliferation by inducing alternative splicing of FSP1 and inhibiting ferroptosis

Objective: To investigate the effect of serine/arginine-rich splicing factor 2 (SRSF2) on ferroptosis and its possible mechanism in glioblastoma cells. Methods: The online database of gene expression profiling interactive analysis 2 (GEPIA 2) and Chinese Glioma Genome Atlas were used to analyze the expression of SRSF2 in glioblastoma tissue and its association with patients prognosis. To validate the findings of the online databases, the pathological sections of glioblastoma and non-tumor brain tissues from Tianjin Medical University General Hospital, Tianjin, China were collected and analyzed by using immunohistochemistry. Silencing SRSF2 gene expression in glioblastoma cells by siRNA was analyzed with Western blot. The proliferation index was detected by using CCK8 assay. The rescued experiment was conducted by using expression plasmid of pcDNA3.1(+)-SRSF2. The activity of ferroptosis was assessed by using the levels of iron ions and malondialdehyde in glioblastoma cells and the changes in the ratio of glutathione to oxidized glutathione. The changes of gene expression and differential pre-mRNA alternative splicing (PMAS) induced by SRSF2 were monitored by using the third-generation sequencing technology analysis, namely Oxford nanopore technologies (ONT) sequencing analysis. Results: SRSF2 expression was higher in glioblastoma tissues than non-tumor brain tissues. Immunohistochemistry also showed a positive rate of 88.48%±4.60% in glioblastoma tissue which was much higher than the 9.97%±4.57% in non-tumor brain tissue. The expression of SRSF2 was inversely correlated with overall and disease-free disease survivals (P<0.01). The proliferation index of glioblastoma cells was significantly reduced by silencing with SRSF2 siRNA (P<0.01) and could be reversed with transfection of exogenous SRSF2. The levels of intracellulariron ions and malondialdehyde increased (P<0.05), but the glutathione/oxidized glutathione ratio and the expression of key proteins in the glutathione pathway remained unchanged (P>0.05). ONT sequencing results showed that silencing SRSF2 in glioblastoma cells could induce a significant alternative 3' splice site change on ferroptosis suppressor protein 1 (FSP1). Conclusion: SRSF2 inhibits the ferroptosis in glioblastoma cells and promotes their proliferation, which may be achieved by regulating FSP1 PMAS.

TAMI-51. HORIZONTAL MITOCHONDRIAL TRANSFER FROM THE TUMOR MICROENVIRONMENT TO GLIOBLASTOMA INCREASES TUMORIGENICITY

Abstract Communication between glioblastoma (GBM) and its microenvironment facilitates tumor growth and therapeutic resistance, and is facilitated through a variety of mechanisms. Organelle transfer between cells was recently observed, including mitochondria transfer from astrocytes to neurons after ischemic stroke. Given the dependence of GBM on microenvironmental interactions, we hypothesized that mitochondria transfer from tumor microenvironment to GBM cells could occur and affect metabolism and tumorigenicity. We interrogated this in vivo by establishing intracranial GBM tumors in mito::mKate2 mice (with trackable fluorescent mitochondria) using syngeneic GFP-expressing tumor cells (SB28 and GL261 models). We also cultured stromal cell types from mito::mKate2 mice with tumor cells, enabling sorting of tumor cells with and without exogenous mitochondria. Confocal microscopy revealed horizontal transfer of mKate2+ mitochondria from mouse cells to implanted GBM cells in vivo and was confirmed by flow cytometry where 20-40% of GBM cells acquired exogenous mitochondria. Transfer was negligible in wildtype mice transplanted with mito::mKate2 bone marrow cells, suggesting that brain-resident cells were the main donors. In vitro, astrocytes and microglia exhibited 5 to 10-fold higher mitochondrial transfer rate than bone-marrow derived macrophages. Seahorse metabolic profiling revealed that GBM cells with mKate2+ mitochondria had 40% lower respiratory reserve compared to cells without exogenous mitochondria. Median survival of mice implanted with SB28 that acquired mitochondria was significantly shorter and in vivo limiting dilution confirmed the frequency of tumor-initiating cells was 3-fold higher in SB28 cells with exogenous mitochondria. Our data indicate that horizontal mitochondrial transfer from brain-resident glia to mouse GBM tumors alters tumor cell metabolism and increases their tumorigenicity. Ongoing studies are assessing gene expression in GBM cells acquiring exogenous mitochondria; validating findings in human specimens; and screening for transfer inhibitor drugs. Horizontal mitochondrial transfer represents a foundational tumor microenvironment interaction contributing to glioblastoma plasticity, and is likely to inform next-generation treatment strategies.

Glioblastomas within the Subventricular Zone Are Region-Specific Enriched for Mesenchymal Transition Markers: An Intratumoral Gene Expression Analysis.

Simple SummaryInvolvement of the subventricular zone (SVZ) in glioblastoma is associated with poor prognosis and is associated with specific tumor-biological characteristics. In this study, we demonstrate that patient-derived glioblastoma samples from within the SVZ region show increased (epithelial-)mesenchymal transition and angiogenesis/hypoxia signaling as compared to glioblastoma samples from the same patient from outside the SVZ. These results suggest that intratumoral alterations in oncogenic signaling could be mediated by the SVZ microenvironment. Our findings offer rationale for specific targeting of the SVZ in the development of glioblastoma therapy.Background: Involvement of the subventricular zone (SVZ) in glioblastoma is associated with poor prognosis and is associated with specific tumor-biological characteristics. The SVZ microenvironment can influence gene expression in glioblastoma cells in preclinical models. We aimed to investigate whether the SVZ microenvironment has any influence on intratumoral gene expression patterns in glioblastoma patients. Methods: The publicly available Ivy Glioblastoma database contains clinical, radiological and whole exome sequencing data from multiple regions from resected glioblastomas. SVZ involvement of the various tissue samples was evaluated on MRI scans. In tumors that contacted the SVZ, we performed gene expression analyses and gene set enrichment analyses to compare gene (set) expression in tumor regions within the SVZ to tumor regions outside the SVZ. We also compared these samples to glioblastomas that did not contact the SVZ. Results: Within glioblastomas that contacted the SVZ, tissue samples within the SVZ showed enrichment of gene sets involved in (epithelial-)mesenchymal transition, NF-κB and STAT3 signaling, angiogenesis and hypoxia, compared to the samples outside of the SVZ region from the same tumors (p < 0.05, FDR < 0.25). Comparison of glioblastoma samples within the SVZ region to samples from tumors that did not contact the SVZ yielded similar results. In contrast, we observed no differences when comparing the samples outside of the SVZ from SVZ-contacting glioblastomas with samples from glioblastomas that did not contact the SVZ at all. Conclusion: Glioblastoma samples in the SVZ region are enriched for increased (epithelial-)mesenchymal transition and angiogenesis/hypoxia signaling, possibly mediated by the SVZ microenvironment.

MiR-125a-5p inhibits cancer stem cells phenotype and epithelial to mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is a common type of cancer with high mortality. Epithelial to mesenchymal transition (EMT) plays a vital role in the development of glioblastoma. The aim of this study is to evaluate the role of miR-125a-5p in glioblastoma and in the tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma. The role of miR-125a-5p in the regulation of CSCs, EMT, migration, and invasion in glioblastoma was measured in this study. We showed the roles of miR-125a-5p in the regulation of CSCs, EMT, migration, and invasion in glioblastoma. miR-125a-5p can inhibit the CSCs phenotype and EMT in glioblastoma cells. In addition, its over-expression can significantly regulate CSCs-associated genes and EMT-associated gene expression in glioblastoma cells. We concluded that miR-125a-5p is one of the key microRNAs regulating CSCs and EMT programs in glioblastoma. The results suggested that miR-125a-5p might be a novel therapy target for glioblastoma.

Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25µM) and/or 0.5mM SAS for 1, 3, or 5days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

Expression of the reporter genes lacZ and EGFP in human glioblastoma cells using Listeria monocytogenes as vector for gene transfer

Objective: Glioblastoma has become a model target for viral gene therapy approaches. To circumvent some of the inherent problems of viral vectors, we examined the feasibility of Listeria monocytogenes for foreign gene expression in glioblastoma cells.Methods: The internalin-dependent uptake of L. monocytogenes into human glioma cell lines and into 22 cell cultures obtained during neurosurgery was studied by in vitro infection assays. Internalization rates of wild-type L. monocytogenes were compared with a deletion mutant and to four strains with plasmid-based recomplementation of the internalin A/B operon. For gene transfer experiments, plasmids encoding lacZ or EGFP under control of the cytomegalovirus promotor were transfected into Listeriae. Incubation times in cell culture were studied regarding infection of cells, intracellular listerial replication and reporter gene translation. Gene transfer efficiency was determined by β-galactosidase activity and by fluorescence microscopy.Results: The internalin-defective strain (ΔinlAB2) showed less uptake compared with the wild type. An increased invasion rate was observed in strain ΔinlAB2 + inlBPinlA overexpressing internalin B; complementation of the entire operon (ΔinlAB2 + inlABPinlA) revealed a synergistic effect. For gene transfer experiments into glioblastoma cells, the pathogenic strain 4bL312 was employed, and efficiencies up to 5% were achieved.Discussion: Internalins A and B are major determinants for listerial uptake into neuroepithelial tumors. The L. monocytogenes vector system enables foreign gene expression in glioblastoma cells. Ongoing research deals with optimization of transfection efficiencies, with the use of less pathogenic substrains and the cloning of suicide gene vectors.

Autocrine Platelet-Derived Growth Factor–Dependent Gene Expression in Glioblastoma Cells Is Mediated Largely by Activation of the Transcription Factor Sterol Regulatory Element Binding Protein and Is Associated with Altered Genotype and Patient Survival in Human Brain Tumors

A complex profile of gene expression elicited by autocrine platelet-derived growth factor (PDGF) signaling was identified in U87 MG glioblastoma cells by microarray analysis. The most striking pattern observed was a PDGF-dependent activation of at least 25 genes involved with biosynthesis and/or uptake of cholesterol and isoprenoids, including mevalonate pyrophosphate decarboxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and low-density lipoprotein receptor. Activity of the HMG-CoA synthase promoter was induced by autocrine PDGF activity as indicated by significant reductions following forced expression of dominant-negative PDGF-A (88%) or treatment with the PDGF receptor antagonist CT52923 (50%). Induction of the HMG-CoA synthase promoter required a binding site for sterol regulatory element binding proteins (SRE-BP), consistent with a key role for these transcription factors in the induction of this gene network. Neither proteolytic activation nor nuclear localization of SRE-BP was affected by disruption of the PDGF autocrine loop, indicating that PDGF signaling is required for other signaling events involved in activation of SRE-BP target genes. Analysis of an expression databank derived from human glial tumors (n = 77) identified a subgroup exhibiting a profile consistent with PDGF dependence, including increased expression of SRE-BP target genes. This subgroup displayed an absence of epidermal growth factor receptor gene amplification, decreased incidence of allelic loss of 10q, increased frequency of TP53 mutations and allelic losses of 1p and 19q, and longer patient survival. This study identifies genes associated with oncogenic activity of PDGF and provides important insights into biomarkers and therapeutic targets in malignant gliomas.

Vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenesis and vascular permeability factor which is expressed in high amounts in perinecrotic palisading cells in human glioblastomas. In vitro VEGF gene expression is enhanced approximately ten times by hypoxia. Current evidence suggests, that hypoxia is also the driving force for VEGF gene expression in glioblastoma cells in vivo and represents the most important trigger for tumor angiogenesis and edema. Our approaches to inhibit tumor angiogenesis and edema formation in glioblastoma patients will concentrate on the disruption of VEGF/VEGF receptor signal transduction pathway in vivo.