Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

High density is a property of slow-cycling and treatment-resistant human glioblastoma cells.

Slow-cycling and treatment-resistant cancer cells escape therapy, providing a rationale for regrowth and recurrence in patients. Much interest has focused on identifying the properties of slow-cycling tumor cells in glioblastoma (GBM), the most common and lethal primary brain tumor. Despite aggressive ionizing radiation (IR) and treatment with the alkylating agent temozolomide (TMZ), GBM patients invariably relapse and ultimately succumb to the disease. In patient biopsies, we demonstrated that GBM cells expressing the proliferation markers Ki67 and MCM2 displayed a larger cell volume compared to rare slow-cycling tumor cells. In optimized density gradients, we isolated a minor fraction of slow-cycling GBM cells in patient biopsies and tumorsphere cultures. Transcriptional profiling, self-renewal, and tumorigenicity assays reflected the slow-cycling state of high-density GBM cells (HDGCs) compared to the tumor bulk of low-density GBM cells (LDGCs). Slow-cycling HDGCs enriched for stem cell antigens proliferated a few days after isolation to generate LDGCs. Both in vitro and in vivo, we demonstrated that HDGCs show increased treatment-resistance to IR and TMZ treatment compared to LDGCs. In conclusion, density gradients represent a non-marker based approach to isolate slow-cycling and treatment-resistant GBM cells across GBM subgroups.

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.