Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells

Abstract

Immune checkpoint blockade (ICB) therapies, such as immune checkpoint inhibitors against programmed death ligand-1 (PD-L1), have not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Despite the critical role of PD-L1 in various types of cancers, the regulatory mechanism of PD-L1 expression on the cell surface of PDAC is poorly understood. Therefore, uncovering potential modulators of cell surface localisation of PD-L1 may provide a new strategy to improve ICB therapy in patients with PDAC. Here, we examined the role of ezrin/radixin/moesin (ERM) family scaffold proteins that crosslink transmembrane proteins with the actin cytoskeleton in the surface localisation of PD-L1 in KP-2 cells, a human PDAC cell line. Our results demonstrated the abundant protein expression of PD-L1, ezrin, and radixin, but not moesin, as well as their colocalisation in the plasma membrane. Interestingly, immunoprecipitation analysis detected the molecular interaction of PD-L1 with ezrin and radixin. Moreover, gene silencing of ezrin moderately decreased the mRNA and cell surface expression of PD-L1, while that of radixin greatly decreased the surface expression of PD-L1 without altering the mRNA levels. Thus, radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current ICB therapy in PDAC.

Moesin Serves as Scaffold Protein for PD-L1 in Human Uterine Cervical Squamous Carcinoma Cells

“…Interestingly, our recent studies demonstrated that among the ERM proteins, ezrin predominantly controls the plasma membrane localization of PD-L1, possibly via molecular interaction, in human adenocarcinoma cells derived from uterine cervix (HeLa) and colorectum (LS180) cells, in which ezrin is expressed at the highest level among ERM, based on DepMap global gene expression analysis and RT-PCR data [31,32]. We also found that as a dominant ERM protein, radixin acts as a principal scaffold protein for PD-L1 in KP-2 human pancreatic ductal adenocarcinoma cells [30]. Significantly, siRNA-mediated interference of moesin, but not ezrin and radixin, greatly reduced the cell surface expression of PD-L1, with limited effect on the PD-L1 mRNA level.…”

“…The RNA concentration and purity were evaluated using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). RT-PCR and relative transcript quantification were performed as described previously [30,32]. The sequences of the gene-specific PCR primers used are listed in Table S1.…”

“…Immunoprecipitation assays were conducted as previously described [30][31][32][33], with some modifications. Briefly, 500 µL of whole-cell lysates collected in RIPA buffer containing a protease inhibitor cocktail was incubated with 50 µL of protein A beads (nProtein A Sepharose 4 Fast Flow; Cytiva, Tokyo, Japan) on a rotating wheel at 4 • C for 1 h to eliminate non-specific interactions.…”

“…Flow cytometry analysis was performed as previously described [30][31][32][33], with some modifications. Single-cell suspensions were reacted with an allophycocyanin (APC)conjugated anti-PD-L1 Ab (2.8 µg/tube) in a labeling buffer consisting of D-PBS, 5% normal horse serum, and 1% sodium azide at 4 • C for 1 h. Subsequently, the mean fluorescence intensity of APC-PD-L1 on the cell surface was analyzed using an EC800 Flow Cytometry Analyzer and EC800 software (Sony Imaging Products and Solutions, Tokyo, Japan).…”

“…Furthermore, ERM proteins serve as crosslinkers between several cancer-related transmembrane proteins, including several transmembrane receptor kinases, and the actin cytoskeleton network, contributing to their cell surface localization [27][28][29]. We have recently reported that ERM proteins post-translationally regulate the cell surface localization of PD-L1 by serving as scaffold proteins in different manners in several types of human cancer cells [30][31][32][33][34][35]. However, it remained unclear whether ERM proteins are involved in the cell surface plasma membrane localization of PD-L1 in cervical SCC.…”