Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas

Background Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors. Methods GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7. Results Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD. Conclusions These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

CBMT-37. OSMR CONTROLS CELLULAR RESPIRATION AND RESISTANCE TO THERAPY

Cytokines and their receptors play important roles in the regulation of cell fate, inflammation and immunity. The cytokine receptor for Oncostatin M (OSMR) drives brain tumor stem cell (BTSC) proliferation and tumorigenesis. Here, we report a novel role for OSMR in regulation of cellular respiration. Genetic knockdown of OSMR in BTSCs impairs mitochondrial biogenesis and oxidative phosphorylation. Importantly, blockade of OSMR signalling in BTSCs sensitizes them to ionizing radiation and Temozolomide in vitro and in vivo. This mechanism whereby OSMR regulates mitochondrial respiration and metabolism is conserved in post-mitotic neutrons. Thus, while OSMR is required for BTSC proliferation, OSMR confers resistance to therapy via enhancing mitochondrial respiration, independent of its role in proliferation. Our data suggest that therapeutic targeting of OSMR may be beneficial to eradicate quiescent tumour stem cells and tumour relapse.