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The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors—rapamycin, temsirolimus, torin 1, and PP242—suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as “stemness-inhibiting drugs” and a promising therapeutic approach to target glioma stem cells.

Glioblastoma (GBM) is the most aggressive type of brain tumors in adults with survival period

Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line - HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers - Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC.

Glioblastoma (GBM), the most malignant of the brain tumors is classified on the basis of molecular signature genes using TCGA data into four subtypes- classical, mesenchymal, proneural and neural. The mesenchymal phenotype is associated with greater aggressiveness and low survival in contrast to GBMs enriched with proneural genes. The proinflammatory cytokines secreted in the microenvironment of gliomas play a key role in tumor progression. The study focused on the role of Oncostatin-M (OSM), an IL-6 family cytokine in inducing mesenchymal properties in GBM. Analysis of TCGA and REMBRANDT data revealed that expression of OSMR but not IL-6R or LIFR is upregulated in GBM and has negative correlation with survival. Amongst the GBM subtypes, OSMR level was in the order of mesenchymal > classical > neural > proneural. TCGA data and RT-PCR analysis in primary cultures of low and high grade gliomas showed a positive correlation between OSMR and mesenchymal signature genes-YKL40/CHI3L1, fibronectin and vimentin and a negative correlation with proneural signature genes-DLL3, Olig2 and BCAN. OSM enhanced transcript and protein level of fibronectin and YKL-40 and reduced the expression of Olig2 and DLL3 in GBM cells. OSM-regulated mesenchymal phenotype was associated with enhanced MMP-9 activity, increased cell migration and invasion. Importantly, OSM induced mesenchymal markers and reduced proneural genes even in primary cultures of grade-III glioma cells. We conclude that OSM-mediated signaling contributes to aggressive nature associated with mesenchymal features via STAT3 signaling in glioma cells. The data suggest that OSMR can be explored as potential target for therapeutic intervention.