Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells

Abstract

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.

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

“…By contrast, the gene expression of radixin in LS180 cells was relatively lower than that in Caco-2 cells ( Figure 1 c). The gene expression of moesin was detected in LS180 cells but not in Caco-2 cells ( Figure 1 d), the result of which is consistent with previous reports indicating a lack of moesin in Caco-2 cells [ 46 , 47 ]. Thus, we adopted LS180 cells in the subsequent experiments to investigate the involvement of each ERM proteins in the regulatory mechanism of gene and protein expression of PD-L1.…”

“…In this study, we initially determined the gene and protein expression levels of ezrin, radixin, and moesin in LS180 cells, which were in line with our recent study [ 47 ]. Moreover, the present CLSM analysis data clearly demonstrated that all three ERM proteins were preferentially and highly localized to the plasma membrane.…”

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

“…Furthermore, ezrin also contributes to the cancer progression because of its role for the maintenance of migratory and the invasive capacity of tumor cells through the specific interaction with some tumor-associated plasma membrane proteins, leading to the metastatic behavior of tumor cells [ 66 , 67 , 68 ]. Accumulated evidence suggests that ezrin serves as a scaffold protein for some drug transporters [ 33 , 34 , 35 , 63 ] and certain cancer-related proteins, including EGFR2 and several receptor kinases [ 37 , 39 ]. In addition to these features, our study provides new molecular insight into the function of ezrin as a regulator for the plasma membrane localization of the immune checkpoint molecule, PD-L1.…”

“…CLSM analysis was carried out as described previously, with some modifications [ 63 , 71 , 72 ]. Single- and double-immunofluorescence staining were performed to confirm the intracellular localization of ezrin, radixin, moesin, and PD-L1, or the colocalization of PD-L1 with ezrin, radixin, and moesin, respectively.…”

“…Flow cytometry analysis was carried out as described previously with some modifications [ 63 , 71 , 72 ]. After treatment of HeLa cells with siRNAs for 3 days, the cells were detached using 500 μL of Accutase (Nacalai Tesque) and transferred into 5 mL tubes filled with 2 mL of a labeling buffer consisting of D-PBS supplemented with 5% normal horse serum (Biowest) and 1% sodium azide (FUJIFILM Wako Pure Chemical) and centrifuged (260× g for 5 min at 4 °C).…”