Effect of Cytokines and Sulfated Polysaccharides on Expression of Programmed Death Ligand-1 in Cancer Cells

Abstract

Cancer is an important health burden, and its incidence is increasing in the world. Recently, the role of immune system in carcinogenesis has been highlighted and cancer immune evasion is regarded as one of the hallmarks of cancer. Cancers adapt themselves to hide and resist the attack from immune system of the host using a variety of mechanisms, in which upregulation of negative immune regulatory checkpoint receptors and ligands is one of strategies (Tumor immune escape). Programmed death ligand 1 (PDL1) is the major ligand of the immune checkpoint receptor, programmed death receptor (PD-1) and its expression is reported to be upregulated in many types of cancers including liver and breast cancers. Binding of PD-L1 on cancer cells to PD1 on T cells leads to the inhibition of T cells functions resulting in T cells exhaustion which eventually allows cancer cells evade from the host’s immune defense. Many factors in tumor microenvironment such as cytokines, chemokines, hypoxia or components of the extracellular matrix influence the expression of PD-L1. The aim of this study was to explore the effect of tumor-related cytokines and sulfated or non-sulfated polysaccharides, glycosaminoglycans of the ECM, on the induction or expression of PDL1 in the hepatocellular carcinoma cell line, HepG2, with little or no constitutive expression of PD-L1 or PD-L1 highly expressing breast cancer cell line, MDAMB-231, respectively. Changes in expression of PD-L1 gene and PD-L1 protein were detected using real-time RT-PCR and Western blot analysis. Cell membrane PD-L1 level was also observed using immunocytochemistry. Liver cancer is sixth most common cancer in 2020 and its carcinogenesis is related to inflammation. The pro-inflammatory cytokines such as TNFα, IL1β or oncostatin M (OSM) were investigated in HepG2 for their effect on the PD-L1 expression induced by IFNγ, which is the most prominent cytokine reported to induce PD-L1 in many cancers. While both TNFα and IL1β showed synergistic upregulation of PD-L1 expression when combined with IFNγ, the effect was most significant and dose-dependent in case of IFNγ and TNFα combination. When underlying signaling mechanisms for this synergism between IFNγ and TNFα were sought, activation of STAT1, JNK and p65 were found responsible for the enhanced expression of PD-L1. Among these pathways, JAK/STAT1 pathway was the major pathway and inhibition of STAT1 alone was adequate to prevent PD-L1 induction in HepG2 cells. Furthermore, effect of Atorvastatin, a common cholesterol-lowering medicine, which inhibits STAT1 activation on cytokines-induced PD-L1 expression was explored. Both of mRNA and protein levels of PD-L1 in HepG2 induced by either IFNγ alone or IFNγ-TNFα combination was significantly diminished by the cotreatment with atorvastatin, prompting the potential of statin to be used in combination with checkpoint-blocking antibodies-therapy in cancers. Breast cancer is the leading cancer-related death in women and triple-negative breast cancer (TNBC) making up 10-20% of them. TNBC is known for its aggressive and has high expression of PD-L1. The effect of sulfated glycosaminoglycan (GAG) on PDL1 expression was investigated in comparison with non-sulfated GAG or semi-synthetic sulfated polysaccharide in one of TNBC cell line, MDAMB-231, to understand the role of extracellular matrix (ECM) components in immune evasion of cancer. Among sulfated and non-sulfated polysaccharides examined: chondroitin sulfate-A (CS-A), chondroitin sulfate-C (CS-C), heparan sulfate (HS), pentosan polysulfate (PPS) and Hyaluronan (HA), only CS-C demonstrated the significant decreasing effect on PD-L1 protein expression in MDAMB-231, speculating the role of different sulfation pattern or sugar composition of GAG in TME on immune escape of cancer. Results of this study, either relating to cytokines or sulfated polysaccharides, highlighted that the immune escape mechanism in cancers is strongly affected by local tumor microenvironment and suggested the potential use in combination with immune checkpoint inhibitor therapy.