Endoglin and TGF-ß signaling in glioblastoma.

Microvascular proliferation is a key feature of glioblastoma and neovascularization has been implicated in tumor progression. Glioblastomas use pro-angiogenic factors such as vascular endothelial growth factor (VEGF) for new blood vessel formation. Yet, anti-VEGF therapy does not prolong overall survival so that alternative angiogenic pathways may need to be explored as drug targets. Both glioma cells and glioma-associated endothelial cells produce TGF-ß superfamily ligands which bind TGF-ß receptors (TGF-ßR). The TGF-ßR type III endoglin (CD105), is a marker of proliferating endothelium that has already been studied as a potential therapeutic target. We studied endoglin expression in glioblastoma tissue and in glioma-associated endothelial cells in a cohort of 52 newly diagnosed and 10 recurrent glioblastoma patients by immunohistochemistry and by ex vivo single-cell gene expression profiling of 6 tumors. Endoglin protein levels were similar in tumor stroma and endothelium and correlated within tumors. Similarly, endoglin mRNA determined by ex vivo single-cell gene expression profiling was expressed in both compartments. There was positive correlation between endoglin and proteins of TGF-ß superfamily signaling. No prognostic role of endoglin expression in either compartment was identified. Endoglin gene silencing in T98G glioma cells and in human cerebral microvascular endothelial cells (hCMEC) did not affect constitutive or exogenous TGF-ß superfamily ligand-dependent signaling, except for a minor facilitation of pSmad1/5 signaling in hCMEC. These observations challenge the notion that endoglin might become a promising therapeutic target in glioblastoma.

A tumor-promoting role for soluble TßRIII in glioblastoma.

PURPOSE: Members of the transforming growth factor (TGF)-ß superfamily play a key role in the regulation of the malignant phenotype of glioblastoma by promoting invasiveness, angiogenesis, immunosuppression, and maintaining stem cell-like properties. Betaglycan, a TGF-ß coreceptor also known as TGF-ß receptor III (TßRIII), interacts with members of the TGF-ß superfamily and acts as membrane-associated or shed molecule. Shed, soluble TßRIII (sTßRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TßRIII may improve our understanding of TGF-ß pathway activity in glioblastoma METHODS: Protein levels of TßRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TßRIII levels were assessed investigating long-term glioma cell lines (LTCs), cultured human brain-derived microvascular endothelial cells (hCMECs), glioblastoma-derived microvascular endothelial cells, and glioma-initiating cell lines (GICs). The impact of TßRIII on TGF-ß signaling was investigated, and results were validated in a xenograft mouse glioma model RESULTS: Immunohistochemistry and ex vivo single-cell gene expression profiling of glioblastoma tissue showed that TßRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TßRIII expression in vitro. Specifically, we detected sTßRIII in glioblastoma-derived microvascular endothelial cells. STßRIII facilitated TGF-ß-induced Smad2 phosphorylation in vitro and overexpression of sTßRIII in a xenograft mouse glioma model led to increased levels of Smad2 phosphorylation, increased tumor volume, and decreased survival CONCLUSIONS: These data shed light on the potential tumor-promoting role of extracellular shed TßRIII which may be released by glioblastoma endothelium with high sTßRIII levels.

TGF-ß induces oncofetal fibronectin that, in turn, modulates TGF-ß superfamily signaling in endothelial cells.

Gene splicing profiles are frequently altered in cancer, and the splice variants of fibronectin (FN) that contain the extra-domains A (EDA) or B (EDB), referred to as EDA+FN or EDB+FN, are highly upregulated in tumor vasculature. Transforming growth factor ß (TGF-ß) signaling has been attributed a pivotal role in glioblastoma, with TGF-ß promoting angiogenesis and vessel remodeling. By using immunohistochemistry staining, we observed that the oncofetal FN isoforms EDA+FN and EDB+FN are expressed in glioblastoma vasculature. Ex vivo single-cell gene expression profiling of tumors by using CD31 and α-smooth muscle actin (αSMA) as markers for endothelial cells, and pericytes and vascular smooth muscle cells (VSMCs), respectively, confirmed the predominant expression of FN, EDA+FN and EDB+FN in the vascular compartment of glioblastoma. Specifically, within the CD31-positive cell population, we identified a positive correlation between the expression of EDA+FN and EDB+FN, and of molecules associated with TGF-ß signaling. Further, TGF-ß induced EDA+FN and EDB+FN in human cerebral microvascular endothelial cells and glioblastoma-derived endothelial cells in a SMAD3- and SMAD4-dependent manner. In turn, we found that FN modulated TGF-ß superfamily signaling in endothelial cells via the EDA and EDB, pointing towards a bidirectional influence of oncofetal FN and TGF-ß superfamily signaling.

Cutting Edge: ERK1 Mediates the Autocrine Positive Feedback Loop of TGF-ß and Furin in Glioma-Initiating Cells.

Glioblastoma is the most common and aggressive intrinsic brain tumor in adults. Self-renewing, highly tumorigenic glioma-initiating cells (GIC) have been linked to glioma invasive properties, immunomodulation, and increased angiogenesis, leading to resistance to therapy. TGF-ß signaling has been associated with the tumorigenic activity of GIC. TGF-ß is synthesized as a precursor molecule and proteolytically processed to the mature form by members of the family of the proprotein convertases subtilisin/kexin. In this study we report that furin is unique among the proprotein convertases subtilisin/kexin in being highly expressed in human GIC. Furin cleaves and promotes activation of pro-TGF-ß1 and pro-TGF-ß2, and TGF-ß2 in turn increases furin levels. Notably, TGF-ß2 controls furin activity in an ALK-5-dependent manner involving the ERK/MAPK pathway. We thus uncover a role of ERK1 in the regulation of furin activity by supporting a self-sustaining loop for high TGF-ß activity in GIC.

Prognostic Relevance of Transforming Growth Factor-ß Receptor Expression and Signaling in Glioblastoma, Isocitrate Dehydrogenase-Wildtype.

The transforming growth factor (TGF)-ß signaling pathway has been recognized as a major factor in promoting the aggressive behavior of glioblastoma, isocitrate dehydrogenase-wildtype. However, there is little knowledge about the expression of TGF-ß receptors in glioblastoma. Here, we studied the expression patterns of TGF-ß receptor II (TGFßRII), type I receptors activin receptor-like kinase (ALK)-5, and ALK-1, as well as of the transcriptional regulators inhibitor of differentiation (Id) 2, Id3, and Id4 in human glioblastoma. The expression of TGFßRII, ALK-5, and ALK-1 varied greatly, with TGFßRII and ALK-5 being the most abundant and ALK-1 being the least expressed receptor. None of the 3 receptors was preferentially expressed by tumor vasculature as opposed to the tumor bulk, indicating tumor bulk-governed mechanisms of TGF-ß signaling with regard to glioblastoma-associated angiogenesis. A positive correlation was found between ALK-1 and Id2, suggesting that Id2, broadly expressed in the tumor cells, is a downstream target of this receptor-dependent pathway. Furthermore, there was a trend for high expression of ALK-5 or Id2 to be associated with inferior overall survival. Hence, we propose that ALK-5 may be used for patient stratification in future anti-TGF-ß treatment trials and that Id2 might be a potential target for anti-TGF-ß interventions.

FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma.

BACKGROUND: Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. METHODS: We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions ("RT-PCR cohort") and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples ("TMA cohort") and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples ("NGS cohort") to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. RESULTS: In the "RT-PCR cohort," we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the "NGS cohort," we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. CONCLUSIONS: FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

Negative allosteric modulators of metabotropic glutamate receptor 3 target the stem-like phenotype of glioblastoma.

Glioblastoma is an invariably deadly disease. A subpopulation of glioma stem-like cells (GSCs) drives tumor progression and treatment resistance. Two recent studies demonstrated that neurons form oncogenic glutamatergic electrochemical synapses with post-synaptic GSCs. This led us to explore whether glutamate signaling through G protein-coupled metabotropic receptors would also contribute to the malignancy of glioblastoma. We found that glutamate metabotropic receptor (Grm)3 is the predominantly expressed Grm in glioblastoma. Associations of GRM3 gene expression levels with survival are confined to the proneural gene expression subtype, which is associated with enrichment of GSCs. Using multiplexed single-cell qRT-PCR, GSC marker-based cell sorting, database interrogations, and functional assays in GSCs derived from patients' tumors, we establish Grm3 as a novel marker and potential therapeutic target in GSCs. We confirm that Grm3 inhibits adenylyl cyclase and regulates extracellular signal-regulated kinase. Targeting Grm3 disrupts self-renewal and promotes differentiation of GSCs. Thus, we hypothesize that Grm3 signaling may complement oncogenic functions of glutamatergic ionotropic receptor activity in neuroglial synapses, supporting a link between neuronal activity and the GSC phenotype. The novel class of highly specific Grm3 inhibitors that we characterize herein have been clinically tested as cognitive enhancers in humans with a favorable safety profile.

Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization.

The current clinical care of glioblastomas leaves behind invasive, radio- and chemo-resistant cells. We recently identified mammary-derived growth inhibitor (MDGI/FABP3) as a biomarker for invasive gliomas. Here, we demonstrate a novel function for MDGI in the maintenance of lysosomal membrane integrity, thus rendering invasive glioma cells unexpectedly vulnerable to lysosomal membrane destabilization. MDGI silencing impaired trafficking of polyunsaturated fatty acids into cells resulting in significant alterations in the lipid composition of lysosomal membranes, and subsequent death of the patient-derived glioma cells via lysosomal membrane permeabilization (LMP). In a preclinical model, treatment of glioma-bearing mice with an antihistaminergic LMP-inducing drug efficiently eradicated invasive glioma cells and secondary tumours within the brain. This unexpected fragility of the aggressive infiltrating cells to LMP provides new opportunities for clinical interventions, such as re-positioning of an established antihistamine drug, to eradicate the inoperable, invasive, and chemo-resistant glioma cells from sustaining disease progression and recurrence.

Corrigendum: Computer-assisted quantification of motile and invasive capabilities of cancer cells.

This corrects the article DOI: 10.1038/srep15338.

Negative control of the HGF/c-MET pathway by TGF-ß: a new look at the regulation of stemness in glioblastoma.

Multiple target inhibition has gained considerable interest in combating drug resistance in glioblastoma, however, understanding the molecular mechanisms of crosstalk between signaling pathways and predicting responses of cancer cells to targeted interventions has remained challenging. Despite the significant role attributed to transforming growth factor (TGF)-ß family and hepatocyte growth factor (HGF)/c-MET signaling in glioblastoma pathogenesis, their functional interactions have not been well characterized. Using genetic and pharmacological approaches to stimulate or antagonize the TGF-ß pathway in human glioma-initiating cells (GIC), we observed that TGF-ß exerts an inhibitory effect on c-MET phosphorylation. Inhibition of either mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway attenuated this effect. A comparison of c-MET-driven and c-MET independent GIC models revealed that TGF-ß inhibits stemness in GIC at least in part via its negative regulation of c-MET activity, suggesting that stem cell (SC) maintenance may be controlled by the balance between these two oncogenic pathways. Importantly, immunohistochemical analyses of human glioblastoma and ex vivo single-cell gene expression profiling of TGF-ß and HGF confirm the negative interaction between both pathways. These novel insights into the crosstalk of two major pathogenic pathways in glioblastoma may explain some of the disappointing results when targeting either pathway alone in human glioblastoma patients and inform on potential future designs on targeted pharmacological or genetic intervention.

Biological Role and Therapeutic Targeting of TGF-ß3 in Glioblastoma.

Transforming growth factor (TGF)-ß contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-ß1 and TGF-ß2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-ß3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-ß3 in glioblastoma models. TGF-ß3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-ß1 or TGF-ß2 in most cell lines. Inhibition of TGF-ß3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-ß1 or TGF-ß2 mRNA expression or protein levels. Moreover, inhibition of TGF-ß3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-ß3 also attenuates signaling evoked by TGF-ß1 or TGF-ß2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-ß3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-ß1 and TGF-ß2 levels were unaffected following systemic treatment with TGF-ß3 -specific antisense oligonucleotides. We conclude that TGF-ß3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-ß1 and TGF-ß2 isoforms. Targeting TGF-ß3in vivo may represent a promising strategy interfering with aberrant TGF-ß signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR.

Durable Control of Metastatic AKT1-Mutant WHO Grade 1 Meningothelial Meningioma by the AKT Inhibitor, AZD5363.

High-throughput analyses have revealed the presence of activating mutations in the AKT1 gene in a subpopulation of meningiomas. We report a female patient with multiple intracranial tumor manifestations and histologically verified meningotheliomatous meningioma in the lung. The tumor was continuously growing at multiple sites despite six surgical resections, radiotherapy, and two lines of systemic therapy. Following detection of an AKT1E17K mutation in three independent tumor samples by sequencing, treatment with AZD5363, a selective AKT inhibitor, was initiated. Ex vivo cultured meningioma cells exhibited sensitivity to the drug as shown by pAKT accumulation on immunoblots. Treatment with AZD5363 resulted, for the first time, in stable disease and minor radiographic response. The patient has been on that treatment for more than one year with ongoing clinical and radiographic response. This is the first report of an AKT1-mutant meningioma responding to AKT inhibition, suggesting that molecular screening may result in clinical benefit.

Modulation of cerebral endothelial cell function by TGF-ß in glioblastoma: VEGF-dependent angiogenesis versus endothelial mesenchymal transition.

Glioblastoma are among the most angiogenic tumors. The molecular mechanisms that control blood vessel formation by endothelial cells (EC) in glioblastoma remain incompletely understood. Transforming growth factor-ß (TGF-ß) is a key regulatory cytokine that has proinvasive and stemness-maintaining autocrine properties in glioblastoma and confers immunosuppression to the tumor microenvironment. Here we characterize potential pro- and anti-angiogenic activities of TGF-ß in the context of glioblastoma in vitro, using human brain-derived microvascular endothelial cells (hCMEC/D3) and glioblastoma-derived endothelial cells (GMEC) as model systems. We find that TGF-ß induces vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) mRNA expression and protein release in a TGF-ß receptor (TßR) II / activin-like kinase (ALK)-5-dependent manner under normoxia and hypoxia, defining potential indirect proangiogenic activity of TGF-ß in glioblastoma. In parallel, exogenous TGF-ß has also inhibitory effects on EC properties and induces endothelial-mesenchymal transition (EndMT) in hCMEC and GMEC. Accordingly, direct inhibition of endogenous TGF-ß/ALK-5 signalling increases EC properties such as tube formation, von-Willebrand factor (vWF) and claudin (CLDN) 5 expression. Yet, the supernatant of TGF-ß-stimulated hCMEC and GMEC strongly promotes EC-related gene expression and tube formation in a cediranib-sensitive manner. These observations shed light on the complex pro- and anti-angiogenic pathways involving the cross-talk between TGF-ß and VEGF/PLGF signalling in glioblastoma which may involve parallel stimulation of angiogenesis and EndMT in distinct target cell populations.

Computer-assisted quantification of motile and invasive capabilities of cancer cells.

High-throughput analysis of cancer cell dissemination and its control by extrinsic and intrinsic cellular factors is hampered by the lack of adequate and efficient analytical tools for quantifying cell motility. Oncology research would greatly benefit from such a methodology that allows to rapidly determine the motile behaviour of cancer cells under different environmental conditions, including inside three-dimensional matrices. We combined automated microscopy imaging of two- and three-dimensional cell cultures with computational image analysis into a single assay platform for studying cell dissemination in high-throughput. We have validated this new approach for medulloblastoma, a metastatic paediatric brain tumour, in combination with the activation of growth factor signalling pathways with established pro-migratory functions. The platform enabled the detection of primary tumour and patient-derived xenograft cell sensitivity to growth factor-dependent motility and dissemination and identified tumour subgroup-specific responses to selected growth factors of excellent diagnostic value.

Pirfenidone inhibits TGF-beta expression in malignant glioma cells.

Due to its immunosuppressive properties, the cytokine transforming growth factor (TGF)-beta has become a promising target in the experimental treatment of human malignant gliomas. Here, we report that the antifibrotic drug 5-methyl-1-phenyl-2-(1H)-pyridone (pirfenidone, PFD) elicits growth-inhibitory effects and reduces TGF-beta2 protein levels in human glioma cell lines. This reduction in TGF-beta2 is biologically relevant since PFD treatment reduces the growth inhibition of TGF-beta-sensitive CCL-64 cells mediated by conditioned media of glioma cells. The downregulation of TGF-beta is mediated at multiple levels. PFD leads to a reduction of TGF-beta2 mRNA levels and of the mature TGF-beta2 protein due to decreased expression and direct inhibition of the TGF-beta pro-protein convertase furin. In addition, PFD reduces the protein levels of the matrix metalloproteinase (MMP)-11, a TGF-beta target gene and furin substrate involved in carcinogenesis. These data define PFD or PFD-related agents as promising agents for human cancers associated with enhanced TGF-beta activity.