Disruption of cholesterol metabolism via sigma-2 ligands induces cell death in pancreatic cancer

Abstract

Presenter: Rony Takchi MD | Washington University, St. Louis Background: The malignant transformation of pancreatic ductal adenocarcinoma (PDAC) is accompanied by alteration of the metabolism to adapt to the new hypoxic and nutrient-deprived micro-environment. Recent studies have shown that PDAC is highly dependent on cholesterol import. Consequently, tumors with a high LDL-receptor (LDL-R) expression phenotype are more aggressive, which is linked with worse patient survival. Like other proliferating tumors, PDAC is characterized by overexpression of the sigma-2 receptor, which is involved in enhanced LDL-R activity and the regulation of cholesterol export from the lysosomes in an NPC1-dependent manner. We thus hypothesized that sigma-2 ligands may facilitate pancreatic cancer cell death via cholesterol trapping in the lysosomes through disruption of the NPC1-mediated export mechanism. Methods: The sigma-2 ligands tested are the azabicyclononane analogs SW43, SW96, SV95, and the benzamide analog, ISO-1. To determine the kinetics and the cellular localization properties of a representative member of the sigma-2 ligand family, SW43 was chemically linked to the fluorophore 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl; SW120). To study the impact on cellular cholesterol trafficking upon sigma-2 ligand treatment, cells were stained with Filipin and analyzed via confocal microscopy. Protein abundance of key players involved in intracellular cholesterol processing/trafficking was determined using state-of-the-art WES technology employing the following antibodies: Sterol-regulatory binding protein 2 (SREBP2), LDL-R, Niemann-Pick C1 protein (NPC1). Cell viability was determined using IncuCyte live-cell analysis system. In order to study the predicted synergism between cholesterol sequestration and blocking of de novo cholesterol synthesis, PDAC cells were treated with sigma-2 ligands in combination with simvastatin, which inhibits the rate-limiting enzyme in cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAr). Drug combination effects (synergy) were determined using CompuSyn software. Results: Utilizing three different human pancreatic cancer cell lines (AsPC1, HPAC, and MiaPaca-2), fluorescently labeled sigma-2 ligand SW120 was shown to be rapidly taken up into the lysosomal compartment within minutes. This rapid uptake mechanism was associated with a moderate cytotoxicity profile of the sigma-2 ligands SW43 and SW96. Of note, the sigma-2 ligand-mediated cytotoxicity correlated with a rapid cholesterol sequestration phenotype in the lysosomes. In contrast, treatment with two other, high-affinity sigma-2 ligands SV95 and ISO1, was not associated with cytotoxicity and did not lead to cholesterol sequestration. Since trapping of cholesterol in the lysosomes renders it unusable for the cell, analogous to the lysosomal storage disease Niemann-Pick type C, actuation of the sterol sensing machinery was predicted. And indeed, shortly (time) after exposure to SW43 or SW96, the cholesterol sensor released SREBP-2 which in turn was activated and induced production of its downstream targets, LDL-R and NPC1. Moreover, the combination of cholesterol sequestration (SW43) and blocking of de novo cholesterol production (simvastatin), demonstrated a synergistic drug activity profile. Conclusion: We identified a cholesterol trapping phenotype as an early event of sigma-2 ligand-mediated cytotoxicity. Targeting both LDL-R-mediated lysosomal cholesterol trapping with sigma-2 ligands in combination with blockage of de-novo cholesterol synthesis lead to synergistic cancer cell death and thus represents an intriguing novel treatment concept for PDAC.