Justin Vareecal Joseph

TGF-β as a therapeutic target in high grade gliomas – Promises and challenges ☆

Transforming growth factor-β (TGF-β) is a cytokine with a key role in tissue homeostasis and cancer. TGF-β elicits both tumor suppressive and tumor promoting functions during cancer progression, in a wide range of cancers. Here, we review the tumor promoting function of TGF-β and its possible promise as a therapeutic target in high grade gliomas, including glioblastoma multiforme (GBM), a disease with very poor prognosis. TGF-β signaling is highly active in high grade gliomas and elevated TGF-β activity has been associated with poor clinical outcome in this deadly disease. Common features of GBMs include fast cell proliferation, invasion into normal brain parenchyma, hypoxia, high angiogenic - and immunosuppressive activity, characteristics that all have been linked to activation of the TGF-β pathway. TGF-β signaling has also been connected with the cancer stem cell (CSC) phenotype in GBM. CSCs represent a subset of GBM cells thought to be responsible for tumor initiation, progression and relapse of disease. Following the description of these different properties of TGF-β signaling and the underlying mechanisms identified thus far, the promise of TGF-β targeted therapy in malignant gliomas is discussed. Several drugs targeting TGF-β signaling have been developed that showed potent antitumor activity in preclinical models. A number of agents are currently evaluated in early clinical studies in glioma patients. Available results of these studies are highlighted and a perspective on the promise of TGF-β-targeted therapy is given.

Hypoxia enhances migration and invasion in glioblastoma by promoting a mesenchymal shift mediated by the HIF1α–ZEB1 axis

Glioblastoma (GBM) is the most common brain tumor in adults and the mesenchymal GBM subtype was reported to be the most malignant, presenting severe hypoxia and necrosis. Here, we investigated the possible role of a hypoxic microenvironment for inducing a mesenchymal and invasive phenotype. The exposure of non-mesenchymal SNB75 and U87 cells to hypoxia induced a strong change in cell morphology that was accompanied by enhanced invasive capacity and the acquisition of mesenchymal marker expression. Further analyses showed the induction of HIF1α and HIF2α by hypoxia and exposure to digoxin, a cardiac glycoside known to inhibit HIF1/2 expression, was able to prevent hypoxia-induced mesenchymal transition. ShRNA-mediated knockdown of HIF1α, and not HIF2α, prevented this transition, as well as the knockdown of the EMT transcription factor ZEB1. We provide further evidence for a hypoxia-induced mesenchymal shift in GBM primary material by showing co-localization of GLUT1, ZEB1 and the mesenchymal marker YKL40 in hypoxic regions of the tumor. Collectively, our results identify a HIF1α-ZEB1 signaling axis that promotes hypoxia induced mesenchymal shift and invasion in GBM in a cell line dependent fashion.

Subclassification of newly diagnosed glioblastomas through an immunohistochemical approach

Molecular signatures in Glioblastoma (GBM) have been described that correlate with clinical outcome and response to therapy. The Proneural (PN) and Mesenchymal (MES) signatures have been identified most consistently, but others including Classical (CLAS) have also been reported. The molecular signatures have been detected by array techniques at RNA and DNA level, but these methods are costly and cannot take into account individual contributions of different cells within a tumor. Therefore, the aim of this study was to investigate whether subclasses of newly diagnosed GBMs could be assessed and assigned by application of standard pathology laboratory procedures. 123 newly diagnosed GBMs were analyzed for the tumor cell expression of 23 pre-identified proteins and EGFR amplification, together allowing for the subclassification of 65% of the tumors. Immunohistochemistry (IHC)-based profiling was found to be analogous to transcription-based profiling using a 9-gene transcriptional signature for PN and MES subclasses. Based on these data a novel, minimal IHC-based scheme for subclass assignment for GBMs is proposed. Positive staining for IDH1R132H can be used for PN subclass assignment, high EGFR expression for the CLAS subtype and a combined high expression of PTEN, VIM and/or YKL40 for the MES subclass. The application of the proposed scheme was evaluated in an independent tumor set, which resulted in similar subclass assignment rates as those observed in the training set. The IHC-based subclassification scheme proposed in this study therefore could provide very useful in future studies for stratification of individual patient samples.

EMT‐ and MET‐related processes in nonepithelial tumors: importance for disease progression, prognosis, and therapeutic opportunities

The epithelial‐to mesenchymal (EMT) process is increasingly recognized for playing a key role in the progression, dissemination, and therapy resistance of epithelial tumors. Accumulating evidence suggests that EMT inducers also lead to a gain in mesenchymal properties and promote malignancy of nonepithelial tumors. In this review, we present and discuss current findings, illustrating the importance of EMT inducers in tumors originating from nonepithelial/mesenchymal tissues, including brain tumors, hematopoietic malignancies, and sarcomas. Among these tumors, the involvement of mesenchymal transition has been most extensively investigated in glioblastoma, providing proof for cell autonomous and microenvironment‐derived stimuli that provoke EMT‐like processes that regulate stem cell, invasive, and immunogenic properties as well as therapy resistance. The involvement of prominent EMT transcription factor families, such as TWIST, SNAI, and ZEB, in promoting therapy resistance and tumor aggressiveness has also been reported in lymphomas, leukemias, and sarcomas. A reverse process, resembling mesenchymal‐to‐epithelial transition (MET), seems particularly relevant for sarcomas, where (partial) epithelial differentiation is linked to less aggressive tumors and a better patient prognosis. Overall, a hybrid model in which more stable epithelial and mesenchymal intermediates exist likely extends to the biology of tumors originating from sources other than the epithelium. Deeper investigation and understanding of the EMT/MET machinery in nonepithelial tumors will shed light on the pathogenesis of these tumors, potentially paving the way toward the identification of clinically relevant biomarkers for prognosis and future therapeutic targets.

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.

Microenvironment involved in FPR1 expression by human glioblastomas

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.

Glioblastoma Stem-Like Cells Are More Susceptible Than Differentiated Cells to Natural Killer Cell Lysis Mediated Through Killer Immunoglobulin-Like Receptors–Human Leukocyte Antigen Ligand Mismatch and Activation Receptor–Ligand Interactions

Glioblastoma (GBM) is the most aggressive brain malignancy in adults, where survival is approximately 14.6 months. Novel therapies are urgently needed and immunotherapy has hailed a new dawn for treatment of solid tumors. Natural killer (NK) cells may be amenable therapeutic effectors against heterogeneous GBM, since they also do not require co-stimulation and antigen specificity. However, it is unclear how culture media routinely used in pre-clinical studies affect GBM cell responses to NK-mediated cytotoxicity. We hypothesized that the culture medium would affect GBM cell phenotype, proliferation, and responses to NK cytotoxicity. We investigated in paired analyses n = 6 patient-derived primary GBM cells propagated in stem cell or serum-containing medium for morphology, proliferation, as well as susceptibility to NK cytolysis and related this to expression of surface and intracellular lineage markers, as well as ligands for NK cell activating and inhibitory receptors. We genotyped the GBM cells for human leukocyte antigen (HLA) as well as the killer immunoglobulin-like receptors (KIR) of the n = 6 allogeneic NK cells used as effector cells. Culture in serum-containing medium induced a switch in GBM cell morphology from suspension neuropsheres to adherent epithelial–mesenchymal-like phenotypes, which was partially reversible. The differentiated cells diminished expression of nestin, CD133 (prominin-1), and A2B5 putative glioma stem-cell markers, attenuated growth, diminished expression of ligands for activating NK cell receptors, while upregulating class I HLA ligands for NK cell inhibitory receptors. When maintained in serum-containing medium, fewer GBM cells expressed intercellular cell adhesion molecule-1 (ICAM-1) and were less susceptible to lysis by NK cells expressing αLβ2 integrin receptor (LFA-1), mediated through combination of inhibitory KIR–HLA ligand mismatch and diminished activation receptor–ligand interactions compared to cells maintained in stem cell media. We conclude that development of preclinical immunotherapy strategies against GBM should not use cells propagated in serum-containing media to avoid misinterpretation of potential therapeutic responses.

Correction: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

Erratum to: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

[This corrects the article DOI: 10.1186/2193-1801-3-495.].