Contribution of Ezrin on the Cell Surface Plasma Membrane Localization of Programmed Cell Death Ligand-1 in Human Choriocarcinoma JEG-3 Cells

Abstract

Immune checkpoint blockade (ICB) antibodies targeting programmed cell death ligand-1 (PD-L1) and programmed cell death-1 (PD-1) have improved survival in patients with conventional single agent chemotherapy-resistant gestational trophoblastic neoplasia (GTN). However, many patients are resistant to ICB therapy, the mechanisms of which are poorly understood. Unraveling the regulatory mechanism for PD-L1 expression may provide a new strategy to improve ICB therapy in patients with GTN. Here, we investigated whether the ezrin/radixin/moesin (ERM) family, i.e., a group of scaffold proteins that crosslink actin cytoskeletons with several plasma membrane proteins, plays a role in the regulation of PD-L1 expression using JEG-3 cells, a representative human choriocarcinoma cell line. Our results demonstrate mRNA and protein expressions of ezrin, radixin, and PD-L1, as well as their colocalization in the plasma membrane. Intriguingly, immunoprecipitation experiments revealed that PD-L1 interacted with both ezrin and radixin and the actin cytoskeleton. Moreover, gene silencing of ezrin but not radixin strongly diminished the cell surface expression of PD-L1 without altering the mRNA level. These results indicate that ezrin may contribute to the cell surface localization of PD-L1 as a scaffold protein in JEG-3 cells, highlighting a potential therapeutic target to improve the current ICB therapy in GTN.

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

“…Accumulating evidence suggests that radixin principally regulates the surface plasma membrane localisation of numerous drug transporters including multidrug resistance protein 2, presumably because radixin is dominantly expressed in the hepatic tissues and cells among ERM proteins [48][49][50][51][52][53]. Therefore, radixin may primarily contribute to the cell surface localisation of PD-L1 in KP-2 cells as a predominant ERM protein, as is the case with ezrin in HeLa, JEG-3, and LS180 cells we have recently reported [29][30][31]. This issue has yet to be unravelled and should be addressed in future studies.…”

“…ERM proteins play a pivotal role in the cell surface localisation and functional activity of several cancer-related transmembrane proteins by anchoring them to the plasma membrane via post-translational modification [21,23,25,28]. We recently reported that ezrin functions as a scaffold protein for PD-L1, which leads to the cell surface localisation of PD-L1 in human uterine cervical adenocarcinoma HeLa cells and human choriocarcinoma JEG-3 cells, both of which have the highest expression level of ezrin among ERM protein based on the database analysis utilizing DepMap and/or our experimental data [29,30]. Furthermore, we have found that in human colon adenocarcinoma LS180 cells, ezrin and radixin contributed equally to the cell surface localisation of PD-L1 via physiological interaction and colocalisation with PD-L1, despite extremely low expression level of radixin in LS180 cells [31].…”

“…In fact, a growing body of evidence suggests that ERM proteins post-translationally regulate the plasma membrane localisation and functional activity of cancer-related proteins, including several transmembrane receptor kinases, cluster of differentiation (CD) 20, and P-glycoprotein (P-gp) through direct molecular interaction [21][22][23][24][25][26][27][28]. Interestingly, we have recently found that in some types of human cancer cell lines, the ERM family crosslinks PD-L1 with the actin cytoskeleton in a different fashion, contributing to the plasma membrane localisation of PD-L1, possibly via posttranslational modification rendering them as scaffold proteins [29][30][31]. However, it remains to be elucidated whether the ERM family also regulates the plasma membrane localisation of PD-L1 as a scaffold protein in PDAC cells.…”

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

“…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.…”

“…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.…”