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.Peer reviewe

Role of the Amygdala in Antidepressant Effects on Hippocampal Cell Proliferation and Survival and on Depression-like Behavior in the Rat

The stimulation of adult hippocampal neurogenesis by antidepressants has been associated with multiple molecular pathways, but the potential influence exerted by other brain areas has received much less attention. The basolateral complex of the amygdala (BLA), a region involved in anxiety and a site of action of antidepressants, has been implicated in both basal and stress-induced changes in neural plasticity in the dentate gyrus. We investigated here whether the BLA modulates the effects of the SSRI antidepressant fluoxetine on hippocampal cell proliferation and survival in relation to a behavioral index of depression-like behavior (forced swim test). We used a lesion approach targeting the BLA along with a chronic treatment with fluoxetine, and monitored basal anxiety levels given the important role of this behavioral trait in the progress of depression. Chronic fluoxetine treatment had a positive effect on hippocampal cell survival only when the BLA was lesioned. Anxiety was related to hippocampal cell survival in opposite ways in sham- and BLA-lesioned animals (i.e., negatively in sham- and positively in BLA-lesioned animals). Both BLA lesions and low anxiety were critical factors to enable a negative relationship between cell proliferation and depression-like behavior. Therefore, our study highlights a role for the amygdala on fluoxetine-stimulated cell survival and on the establishment of a link between cell proliferation and depression-like behavior. It also reveals an important modulatory role for anxiety on cell proliferation involving both BLA-dependent and –independent mechanisms. Our findings underscore the amygdala as a potential target to modulate antidepressants' action in hippocampal neurogenesis and in their link to depression-like behaviors

Cutting edge: ERK1 mediates the autocrine positive feedback loop of TGF-β 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-β signaling has been associated with the tumorigenic activity of GIC. TGF-β 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-β1 and pro-TGF-β2, and TGF-β2 in turn increases furin levels. Notably, TGF-β2 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-β activity in GIC

A tumor-promoting role for soluble TβRIII in glioblastoma

PURPOSE Members of the transforming growth factor (TGF)-β 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-β coreceptor also known as TGF-β receptor III (TβRIII), interacts with members of the TGF-β superfamily and acts as membrane-associated or shed molecule. Shed, soluble TβRIII (sTβRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TβRIII may improve our understanding of TGF-β pathway activity in glioblastoma METHODS: Protein levels of TβRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TβRIII 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 TβRIII on TGF-β 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 TβRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TβRIII expression in vitro. Specifically, we detected sTβRIII in glioblastoma-derived microvascular endothelial cells. STβRIII facilitated TGF-β-induced Smad2 phosphorylation in vitro and overexpression of sTβRIII 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 TβRIII which may be released by glioblastoma endothelium with high sTβRIII levels

Negative control of the HGF/c-MET pathway by TGF-β: 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)-β 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-β pathway in human glioma-initiating cells (GIC), we observed that TGF-β 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-β 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-β 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-β3 in Glioblastoma

Abstract Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β1 and TGF-β2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β3 in glioblastoma models. TGF-β3 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-β1 or TGF-β2 in most cell lines. Inhibition of TGF-β3 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-β1 or TGF-β2 mRNA expression or protein levels. Moreover, inhibition of TGF-β3 expression reduces invasiveness in vitro. Interestingly, depletion of TGF-β3 also attenuates signaling evoked by TGF-β1 or TGF-β2. In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-β1 and TGF-β2 levels were unaffected following systemic treatment with TGF-β3-specific antisense oligonucleotides. We conclude that TGF-β3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β1 and TGF-β2 isoforms. Targeting TGF-β3 in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177–86. ©2017 AACR.</jats:p

Endoglin and TGF-β 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-β superfamily ligands which bind TGF-β receptors (TGF-βR). The TGF-βR 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-β 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-β 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

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