The ezrin/radixin/moesin (ERM) family proteins act as linkers between the actin cytoskeleton and P-glycoprotein (P-gp) and regulate the plasma membrane localization and functionality of the latter in various cancer cells. Notably, P-gp overexpression in the plasma membrane of cancer cells is a principal factor responsible for multidrug resistance and drug-induced mutagenesis. However, it remains unknown whether the ERM proteins contribute to the plasma membrane localization and transport function of P-gp in human colorectal cancer cells in which the subcellular localization of ERM has yet to be determined. This study aimed to determine the gene expression patterns and subcellular localization of ERM and P-gp and investigate the role of ERM proteins in the plasma membrane localization and transport function of P-gp using the human colon adenocarcinoma cell line LS180. Using real-time reverse transcription polymerase chain reaction and immunofluorescence analyses, we showed higher levels of ezrin and moesin mRNAs than those of radixin mRNA in these cells and preferential distribution of all three ERM proteins on the plasma membrane. The ERM proteins were highly colocalized with P-gp. Additionally, we show that the knockdown of ezrin, but not of radixin and moesin, by RNA interference significantly decreased the cell surface expression of P-gp in LS180 cells without affecting the mRNA expression of P-gp. Furthermore, gene silencing of ezrin substantially increased the intracellular accumulation of rhodamine123, a typical P-gp substrate, with no alterations in the plasma membrane permeability of Evans blue, a passive transport marker. In conclusion, ezrin may primarily regulate the cell surface localization and transport function of P-gp as a scaffold protein without influencing the transcriptional activity of P-gp in LS180 cells. These findings should be relevant for treating colorectal cancer, which is the second leading cause of cancer-related deaths in males and females combined.
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.
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.
Cancer cells employ programmed cell death ligand-1 (PD-L1), an immune checkpoint protein that binds to programmed cell death-1 (PD-1) and is highly expressed in various cancers, including cervical carcinoma, to abolish T-cell-mediated immunosurveillance. Despite a key role of PD-L1 in various cancer cell types, the regulatory mechanism for PD-L1 expression is largely unknown. Understanding this mechanism could provide a novel strategy for cervical cancer therapy. Here, we investigated the influence of ezrin/radixin/moesin (ERM) family scaffold proteins, crosslinking the actin cytoskeleton and certain plasma membrane proteins, on the expression of PD-L1 in HeLa cells. Our results showed that all proteins were expressed at mRNA and protein levels and that all ERM proteins were highly colocalized with PD-L1 in the plasma membrane. Interestingly, immunoprecipitation assay results demonstrated that PD-L1 interacted with ERM as well as actin cytoskeleton proteins. Furthermore, gene silencing of ezrin, but not radixin and moesin, remarkably decreased the protein expression of PD-L1 without affecting its mRNA expression. In conclusion, ezrin may function as a scaffold protein for PD-L1; regulate PD-L1 protein expression, possibly via post-translational modification in HeLa cells; and serve as a potential therapeutic target for cervical cancer, improving the current immune checkpoint blockade therapy.
Immune checkpoint blockade (ICB) therapy targeting the programmed death ligand-1 (PD-L1)/PD-1 axis has emerged as a promising treatment for uterine cervical cancer; however, only a small subset of patients with uterine cervical squamous cell carcinoma (SCC) derives clinical benefit from ICB therapies. Thus, there is an urgent unmet medical need for novel therapeutic strategies to block the PD-L1/PD-1 axis in patients with uterine cervical SCC. Here, we investigated the involvement of ezrin/radixin/moesin (ERM) family scaffold proteins, which crosslink several plasma membrane proteins with the actin cytoskeleton, on the plasma membrane localization of PD-L1 in BOKU and HCS-2 cells derived from human uterine cervical SCC. Immunofluorescence analysis showed that PD-L1 colocalized with all three ERM proteins in the plasma membrane. Gene knockdown of moesin, but not ezrin and radixin, substantially reduced the plasma membrane expression of PD-L1, with limited effect on mRNA expression. An immunoprecipitation assay demonstrated the molecular interaction between PD-L1 and moesin. Moreover, phosphorylated, i.e., activated, moesin was highly colocalized with PD-L1 in the plasma membrane. In conclusion, moesin may be a scaffold protein responsible for the plasma membrane expression of PD-L1 in human uterine cervical SCC.
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