DMRTA2 supports glioma stem-cell mediated neovascularization in glioblastoma.

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Due to its fast proliferation, diffusive growth and therapy resistance survival times are less than two years for patients with IDH-wildtype GBM. GBM is noted for the considerable cellular heterogeneity, high stemness indices and abundance of the glioma stem-like cells known to support tumor progression, therapeutic resistance and recurrence. Doublesex- and mab-3-related transcription factor a2 (DMRTA2) is involved in maintaining neural progenitor cells (NPC) in the cell cycle and its overexpression suppresses NPC differentiation. Despite the reports showing that primary GBM originates from transformed neural stem/progenitors cells, the role of DMRTA2 in gliomagenesis has not been elucidated so far. Here we show the upregulation of DMRTA2 expression in malignant gliomas. Immunohistochemical staining showed the protein concentrated in small cells with high proliferative potential and cells localized around blood vessels, where it colocalizes with pericyte-specific markers. Knock-down of DMRTA2 in human glioma cells impairs proliferation but not viability of the cells, and affects the formation of the tumor spheres, as evidenced by strong decrease in the number and size of spheres in in vitro cultures. Moreover, the knockdown of DMRTA2 in glioma spheres affects the stabilization of the glioma stem-like cell-dependent tube formation in an in vitro angiogenesis assay. We conclude that DMRTA2 is a new player in gliomagenesis and tumor neovascularization and due to its high expression in malignant gliomas could be a biomarker and potential target for new therapeutic strategies in glioblastoma.

The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.

Osteopontin (SPP1, a secreted phosphoprotein 1) is primarily involved in immune responses, tissue remodelling and biomineralization. However, it is also overexpressed in many cancers and regulates tumour progression by increasing migration, invasion and cancer stem cell self-renewal. Mechanisms of SPP1 overexpression in gliomas are poorly understood. We demonstrate overexpression of two out of five SPP1 isoforms in glioblastoma (GBM) and differential isoform expression in glioma cell lines. Up-regulated SPP1 expression is associated with binding of the GLI1 transcription factor to the promoter and OCT4 (octamer-binding transcription factor 4) to the first SPP1 intron of the SPP1 gene in human glioma cells but not in non-transformed astrocytes. GLI1 knockdown reduced SPP1 mRNA and protein levels in glioma cells. GLI1 and OCT4 are known regulators of stem cell pluripotency. GBMs contain rare cells that express stem cell markers and display ability to self-renew. We reveal that SPP1 is overexpressed in glioma initiating cells defined by high rhodamine 123 efflux, sphere forming capacity and stemness marker expression. Forced differentiation of human glioma spheres reduced SPP1 expression. Knockdown of SPP1, GLI1 or CD44 with siRNAs diminished sphere formation. C6 glioma cells stably depleted of Spp1 displayed reduced sphere forming capacity and downregulated stemness marker expression. Overexpression of the wild type Spp1, but not Spp1 lacking a Cd44 binding domain, rescued cell ability to form spheres. Our findings show re-activation of the embryonic-type transcriptional regulation of SPP1 in malignant gliomas and point to the importance of SPP1-CD44 interactions in self-renewal and pluripotency glioma initiating cells.

Down-regulation of IKKß expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma.

Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKß, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKß levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKß expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.

TGF beta signaling and its role in glioma pathogenesis.

Transforming growth factor beta (TGF-ß) signaling is involved in the regulation of proliferation, differentiation and survival/or apoptosis of many cells, including glioma cells. TGF-ß acts via specific receptors activating multiple intracellular pathways resulting in phosphorylation of receptor-regulated Smad2/3 proteins that associate with the common mediator, Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of many genes. Furthermore, TGF-ß-activated kinase-1 (TAK1) is a component of TGF-ß signaling and activates mitogen-activated protein kinase cascades. Negative regulation of TGF-ß/Smad signaling may occur through the inhibitory Smad6/7. Increased expression of TGF-ß1-3 correlates with a degree of malignancy of human gliomas. TGF-ß may contribute to tumor pathogenesis by direct support of tumor growth, self-renewal of glioma initiating stem cells and inhibiting of anti-tumor immunity. TGF-ß1,2 stimulate expression of the vascular endothelial growth factor as well as the plasminogen activator inhibitor and some metalloproteinases that are involved in vascular remodeling, angiogenesis and degradation of the extracellular matrix. Inhibitors of TGF-ß signaling reduce viability and invasion of gliomas in animal models and show promises as novel, potential anti-tumor therapeutics.