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Cancer stem cells promote neoplastic growth, in part by deregulating asymmetric cell division and enhancing self-renewal. To uncover mechanisms and potential therapeutic targets in glioma stem cell (GSC) self-renewal, we performed a genetic suppressor screen for kinases to reverse the tumor phenotype of our Drosophila brain tumor model and identified dCdk5 as a critical regulator. CDK5, the human ortholog of dCdk5 (79% identity), is aberrantly activated in GBMs and tightly aligned with both chromosome 7 gains and stem cell markers affecting tumor-propagation. Our investigation revealed that pharmaceutical inhibition of CDK5 prevents GSC self-renewal in vitro and in xenografted tumors, at least partially by suppressing CREB1 activation independently of PKA/cAMP. Finally, our TCGA GBM data analysis revealed that CDK5, stem cell, and asymmetric cell division markers segregate within non-mesenchymal patient clusters, which may indicate preferential dependence on CDK5 signaling and sensitivity to its inhibition in this group., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

We investigated the intersection of epidermal growth factor receptor (EGFR) and CCAAT enhancer binding protein (C/EBP)-β signaling in glioblastoma (GBM), given that both gene products strongly influence neoplastic behavior. C/EBP-β is known to drive the mesenchymal transcriptional signature in GBM, likely through strong microenvironmental influences, whereas the genetic contributions to its up-regulation in this disease are not well described. We demonstrated that stable overexpression and activation of WT EGFR (U87MG-WT) led to elevated C/EBP-β expression, as well as enhanced nuclear translocation and DNA-binding activity, leading to up-regulation of C/EBP-β transcription and translation. Deeper investigation identified bidirectional regulation, with C/EBP-β also causing up-regulation of EGFR that was at least partially dependent on the STAT3. Based on ChIP-based studies, we also found that that the translational isoforms of C/EBP-β [liver-enriched transcription-activating protein (LAP)-1/2 and liver inhibitory protein (LIP)] have differential occupancy on STAT3 promoter and opposing roles in transcriptional regulation of STAT3 and EGFR. We further demonstrated that the shorter C/EBP-β isoform, LIP, promoted proliferation and migration of U87MG glioma cells, potentially via induction of cytokine IL-6. Our molecular dissection of EGFR and C/EBP-β pathway interactions uncovered a complex signaling network in which increased activity of either EGFR or C/EBP-β leads to the up-regulation of the other, enhancing oncogenic signaling. Disrupting the EGFR-C/EBP-β signaling axis could attenuate malignant behavior of glioblastoma.-Selagea, L., Mishra, A., Anand, M., Ross, J., Tucker-Burden, C., Kong, J., Brat, D. J. EGFR and C/EBP-β oncogenic signaling is bidirectional in human glioma and varies with the C/EBP-β isoform., (© FASEB.)

How necrotic areas develop in tumors is incompletely understood but can impact progression. Recent findings suggest that the formation of vascular microthrombi contributes to tumor necrosis, prompting investigation of coagulation cascades. Here, we report that loss of tumor suppressor P14ARF can contribute to activating the clotting cascade in glioblastoma. P14ARF transcriptionally upregulated TFPI2, a Kunitz-type serine protease in the tissue factor pathway that inhibits the initiation of thrombosis reactions. P14ARF activation in tumor cells delayed their ability to activate plasma clotting. Mechanistically, P14ARF activated the TFPI2 promoter in a p53-independent manner that relied upon c-JUN, SP1, and JNK activity. Taken together, our results identify the critical signaling pathways activated by P14ARF to prevent vascular microthrombosis triggered by glioma cells. Stimulation of this pathway might be used as a therapeutic strategy to reduce aggressive phenotypes associated with necrotic tumors, including glioblastoma., (©2014 AACR)

The gene that encodes the epidermal growth factor receptor (EGFR) is frequently overexpressed or mutated in human cancers, including glioblastoma. However, the efficacy of EGFR-targeted small-molecule inhibitors or monoclonal antibodies in glioblastomas that also have mutation or deletion of the gene encoding phosphatase and tensin homolog (PTEN) has been modest. We found that EGFR signaling was blocked by a small molecule (G5-7) that selectively inhibited Janus kinase 2 (JAK2)-mediated phosphorylation and activation of EGFR and STAT3 (signal transducer and activator of transcription 3) by binding to JAK2, thereby decreasing the activity of downstream signaling by mTOR (mammalian target of rapamycin) and inducing cell cycle arrest. G5-7 inhibited the proliferation of PTEN-deficient glioblastoma cell lines harboring a constitutively active variant of EGFR (U87MG/EGFRvIII) and human glioblastoma explant neurosphere cultures, but the drug only weakly inhibited the proliferation of either glioblastoma cell lines that were wild type for EGFR and stably transfected with PTEN (U87MG/PTEN) or normal neural progenitor cells and astrocytes. Additionally, G5-7 reduced vascular endothelial growth factor (VEGF) secretion and endothelial cell migration and induced apoptosis in glioblastoma xenografts, thereby suppressing glioblastoma growth in vivo. Furthermore, G5-7 was more potent than EGFR or JAK2 inhibitors that interfere with either ligand or adenosine 5'-triphosphate (ATP) binding at impeding glioblastoma cell proliferation, demonstrating that this allosteric JAK2 inhibitor may be an effective clinical strategy.

Tumor associated coagulopathy is a complex phenomenon that has been the subject of study for more than a century. Many mechanisms have been implicated in this process including tumor-host interactions, growth factor-, and cytokine-mediated signaling in the tumor microenvironment. Tissue factor (TF), a potent procoagulant, is considered to be critical for physiologic and pathological coagulation. The genetic and microenvironmental features of cancer appear to strongly influence the expression of TF and lead to a procoagulant state both locally within the neoplasm as well as systemically. In association with cytokines and other procoagulants, upregulated TF and its downstream effectors such as thrombin not only lead to abnormal blood clotting in cancer patients but also stimulate a wide number of oncogenic signaling mechanisms that may prove important in cancer progression. In glioblastoma multiforme (GBM), EGFR expression and PTEN loss both lead to increased TF expression and thrombosis in a manner that appears to cause hypoxia-induced tumor progression. This review consolidates aspects of aberrant coagulation in cancer, the relevance of TF and its regulation by oncogenic alterations, with specific reference to GBM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Interleukin-8 (IL-8, or CXCL8), which is a chemokine with a defining CXC amino acid motif that was initially characterized for its leukocyte chemotactic activity, is now known to possess tumorigenic and proangiogenic properties as well. In human gliomas, IL-8 is expressed and secreted at high levels both in vitro and in vivo, and recent experiments suggest it is critical to glial tumor neovascularity and progression. Levels of IL-8 correlate with histologic grade in glial neoplasms, and the most malignant form, glioblastoma, shows the highest expression in pseudopalisading cells around necrosis, suggesting that hypoxia/anoxia may stimulate expression. In addition to hypoxia/anoxia stimulation, increased IL-8 in gliomas occurs in response to Fas ligation, death receptor activation, cytosolic Ca(2+), TNF-alpha, IL-1, and other cytokines and various cellular stresses. The IL-8 promoter contains binding sites for the transcription factors NF-kappaB, AP-1, and C-EBP/NF-IL-6, among others. AP-1 has been shown to mediate IL-8 upregulation by anoxia in gliomas. The potential tumor suppressor ING4 was recently shown to be a critical regulator of NF-kappaB-mediated IL-8 transcription and subsequent angiogenesis in gliomas. The IL-8 receptors that could contribute to IL-8-mediated tumorigenic and angiogenic responses include CXCR1 and CXCR2, both of which are G-protein coupled, and the Duffy antigen receptor for cytokines, which has no defined intracellular signaling capabilities. The proangiogenic activity of IL-8 occurs predominantly following binding to CXCR2, but CXCR1 appears to contribute as well through independent, small-GTPase activity. A precise definition of the mechanisms by which IL-8 exerts its proangiogenic functions requires further study for the development of effective IL-8-targeted therapies.

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

Infiltrative astrocytic neoplasms are by far the most common malignant brain tumors in adults. Clinically, they are highly problematic due to their widely invasive nature which makes a complete resection almost impossible. Biologic progression of these tumors is inevitable and adjuvant therapies are only moderately effective in prolonging survival. Glioblastoma multiforme (GBM; WHO grade IV), the most malignant form of infiltrating astrocytoma, can evolve from a lower grade precursor tumor (secondary GBM) or can present as high grade lesion from the outset, so-called de novo GBM. Molecular genetic investigations suggest that GBMs are comprised of multiple molecular genetic subsets. Notwithstanding the diversity of genetic alterations leading to the GBM phenotype, the vascular changes that evolve in this disease, presumably favoring further growth, are remarkably similar. Underlying genetic alterations in GBM may tilt the balance in favor of an angiogenic phenotype by upregulation of pro-angiogenic factors and down-regulation of angiogenesis inhibitors. Increased vascularity and endothelial cell proliferation in GBMs are also driven by hypoxia-induced expression of pro-angiogenic cytokines, such vascular endothelial growth factor (VEGF). Understanding the contribution of genetic alterations and hypoxia in angiogenic dysregulation in astrocytic neoplasms will lead to the development of better anti-angiogenic therapies for this disease. This review will summarize the properties of angiogenic dysregulation that lead to the highly vascularized nature of these tumors.