Pharmacological Characterization Of Sigma-2 Receptor Ligand-Induced Metabolic Stimulation In Human Sk-N-Sh Neuroblastoma Cells

We have previously reported a novel metabolically stimulative function of the sigma-2 receptor, with stimulation of glycolytic hallmarks; effects consistent with a pro-survival role and receptor upregulation in cancer cells. These include increased reductive capacity as indicated by stimulation of MTT reduction, increase in cellular ATP level, reduction in basal ROS level, and stabilization of HIF-1α, in human SK-N-SH neuroblastoma cells. The stimulation of MTT reduction was blocked by sigma-2 receptor antagonists, supporting mediation by sigma-2 receptors. Here we further characterize this effect using analogs of the canonical sigma-2 antagonist, SN79. CM764, CM571, and WA504 (sigma-2 Ki = 3.5, 21.7, and 2.5 nM, respectively) all induced dose-dependent stimulation of MTT reduction. At the highest dose examined (30 μM) CM764, CM571, and WA504 induced 45%, 33%, and 75% stimulation of MTT reduction after a 24 h treatment period. Seahorse analysis showed that all three compounds increased both basal and compensatory glycolytic rate as assessed by ECAR. Since these active compounds all have significant sigma-1 receptor affinity (Ki = 8.1-86.6 nM), we examined the effect of structurally similar compounds that lack sigma-1 activity to help rule out its involvement. CM350 (sigma-1 Ki = 1,202 nM; sigma-2 Ki = 83.3 nM) retained stimulative activity, while CM179 (sigma-1 Ki = 1,426 nM; sigma-2 Ki = 2,260 nM) had no activity, suggesting a selective role of the sigma-2 receptor. Our previous study showed that 10 μM CM764 caused a time-dependent increase in HIF-1α level that became prominent at 6 h and continued to increase up to 24 h. HIF-1α is a global regulator of several components of the glycolytic pathway. An examination of the time course revealed that it takes 3 to 6 hours of treatment for ligand-induced increase in MTT reduction to fully develop, consistent with the time course for HIF-1α stabilization. There is evidence that calcium signals can induce expression of HIF-1α. CM764 and CM571 (10 and 30 μM) were found to induce an immediate, transient, and dose-dependent increase in cytosolic calcium in Fura-2 loaded cells. Thapsigargin (150 nM) pretreatment completely eliminated the calcium response, indicating that calcium release derives from endoplasmic reticulum stores. Carbachol also produces a robust thapsigargin-sensitive calcium signal via muscarinic cholinergic receptors, but fails to induce stimulation of MTT reduction. This indicates that if a calcium transient is involved in the mechanism, it must be produced or accompanied by sigma-2 receptor engagement. Taken together, the data suggests that these sigma-2 receptor ligands impact glycolysis via immediate ER calcium release which then induces a latent downstream upregulation of HIF-1α, subsequently resulting in upregulation of glycolytic enzymes and transporters. This will need further investigation to confirm. Citation Format: Bridget M. McVeigh, Alissa K. Oakes, Cheri Z. Liu, Christophe Mesangeau, Walid F. Alsharif, Christopher R. McCurdy, Wayne D. Bowen. Pharmacological characterization of sigma-2 receptor ligand-induced metabolic stimulation in human SK-N-SH neuroblastoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2559.