Role of Ezrin/Radixin/Moesin in the Surface Localization of Programmed Cell Death Ligand-1 in Human Colon Adenocarcinoma LS180 Cells

Abstract

Programmed cell death ligand-1 (PD-L1), an immune checkpoint protein highly expressed on the cell surface in various cancer cell types, binds to programmed cell death-1 (PD-1), leading to T-cell dysfunction and tumor survival. Despite clinical successes of PD-1/PD-L1 blockade therapies, patients with colorectal cancer (CRC) receive little benefit because most cases respond poorly. Because high PD-L1 expression is associated with immune evasion and poor prognosis in CRC patients, identifying potential modulators for the plasma membrane localization of PD-L1 may represent a novel therapeutic strategy for enhancing the efficacy of PD-1/PD-L1 blockade therapies. Here, we investigated whether PD-L1 expression in human colorectal adenocarcinoma cells (LS180) is affected by ezrin/radixin/moesin (ERM), functioning as scaffold proteins that crosslink plasma membrane proteins with the actin cytoskeleton. We observed colocalization of PD-L1 with all three ERM proteins in the plasma membrane and detected interactions involving PD-L1, the three ERM proteins, and the actin cytoskeleton. Furthermore, gene silencing of ezrin and radixin, but not of moesin, substantially decreased the expression of PD-L1 on the cell surface without affecting its mRNA level. Thus, in LS180 cells, ezrin and radixin may function as scaffold proteins mediating the plasma membrane localization of PD-L1, possibly by post-translational modification.

Ezrin Modulates the Cell Surface Expression of Programmed Cell Death Ligand-1 in Human Cervical Adenocarcinoma Cells

“…Our present immunoprecipitation analysis demonstrated that not only moesin but also ezrin and radixin physiologically interacted with PD-L1 and the triple complex of PD-L1, ERM, and actin cytoskeleton. This is in agreement with our recent publication that all three ERM proteins interact with PD-L1 as determined by co-immunoprecipitation assays in human colorectal cancer cell line [57]. Collectively, the present and previous findings raise the possibility that ERM function as crosslinkers to regulate the plasma membrane localization of PD-L1 via post-translational modifications in HeLa cells.…”

“…Meng et al have also reported that cell-surface PD-L1 levels are decreased by treatment with moesin siRNA without any impact on its mRNA expression levels, resulting in T-cell activation in in vitro cell culture model, although the effect of gene silencing of ezrin and radixin has not yet been determined [40]. In contrast, our recent study demonstrated that gene silencing of ezrin and radixin equally decreased the cell-surface PD-L1 levels but not its mRNA expression levels [57]. Given the fact that ERM proteins involved in the plasma membrane localization of P-gp, a typical partner protein for ERM, vary according to the types of cancer, organ, and animal species [33,[59][60][61][62][63], this discrepancy among the present and previous results may be attributed, at least in part, to the different expression profiles of ERM in cancer cell types.…”

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

“…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]. By contrast, another group has shown that moesin interacts with and stabilises PD-L1 in the cell surface membrane by preventing the proteasomal degradation of PD-L1 in human breast cancer adenocarcinoma, MDA-MB-231, though the influence of ezrin and radixin gene suppression remains to be determined [47].…”

“…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.…”

“…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.…”