Small‐Molecule Sigma1 Modulator Suppresses PD‐L1‐Mediated Cancer Cell Adaptive Immune Resistance in Response to Interferon‐Gamma

Abstract

BackgroundSigma1 (also known as sigma‐1 receptor) is a unique ligand‐regulated integral membrane protein enriched in the endoplasmic reticulum (ER) of cancer cells. Sigma1 acts as a multi‐functional scaffolding or chaperone protein to maintain protein and lipid homeostasis in the secretory pathway of these cells. Programmed Death‐Ligand 1 (PD‐L1) is a type I integral membrane glycoprotein that is processed through the secretory pathway. Upon reaching the cell surface, PD‐L1 acts as an inhibitory immune checkpoint molecule by binding to its cognate receptor, programmed death‐1 (PD‐1), on activated T cells, resulting in T cell inactivation, thus suppressing the anti‐tumor immune response. PD‐L1 protein expression and transport to the cell surface can be upregulated by interferon‐gamma (IFN‐γ). This mechanism is known as adaptive immune resistance, whereby cancer cells change their phenotype in response to a proinflammatory immune response. PD‐L1 is also incorporated onto cancer cell‐derived extracellular vesicles called exosomes, which can inactivate T cells within the tumor microenvironment and at distant sites. We previously reported that Sigma1 associates with PD‐ L1 and that small molecule Sigma1 inhibitors (Sigma1i) can trigger lysosomal degradation of PD‐L1.ObjectiveThe purpose of our present study is to examine the role of Sigma1 in the PD‐L1 life cycle, including interferon gamma (IFN‐γ) signal transduction, upregulation of PD‐L1 mRNA, post‐translational modification of PD‐L1, transport to the cell surface, and incorporation onto exosomes.MethodsSix cancer cell lines from multiple cancer types were assessed for PD‐L1 protein expression by immunoblot and PD‐L1 mRNA by qRT‐PCR. PD‐L1 surface expression was assessed by flow cytometry and biotinylation assays. Exosomes were isolated by ultracentrifugation and concentrations and particle size profiles were determined using nanoparticle tracking. We used Sigma1i to examine the role of Sigma1 in the PD‐L1 life cycle.ResultsWe found that Sigma1i blocks IFN‐γ induced PD‐L1 at a post‐translational step. Cotreatment of Sigma1i with IFN‐γ results in accumulation of Endoglycosidase H (EndoH) sensitive PD‐L1 in the ER and thus prevents cell‐surface expression of PD‐L1. Furthermore, Sigma1i blocked IFN‐γ‐induced incorporation of PD‐L1 on cancer cell exosomes.ConclusionWe have found that Sigma1i causes nascent PD‐L1 to be retained in the ER, preventing it from progressing through the secretory pathway, reaching the cell surface, and from being packaged onto exosomes. By reducing cell surface PD‐L1 and exosomal PD‐L1, Sigma1 lation could provide a novel approach to enhancing anti‐tumor immunity.