Takuro Kobori

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

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

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.

Radixin modulates the plasma membrane localization of CD47 in human uterine cervical adenocarcinoma cells

Despite the dramatic success of immune checkpoint blockers in treating numerous cancer cell types, current therapeutic modalities provide clinical benefits to a subset of patients with cervical cancers. CD47 is commonly overexpressed in a broad variety of cancer cells, correlates with poor clinical prognosis, and acts as a dominant macrophage checkpoint by interacting with receptors expressed on macrophages. It allows cancer cells to escape from the innate immune system and hence is a potential therapeutic target for developing novel macrophage checkpoint blockade immunotherapies. As the intracellular scaffold proteins, ezrin/radixin/moesin (ERM) family proteins post-translationally regulate the cellular membrane localization of numerous transmembrane proteins, by crosslinking them with the actin cytoskeleton. We demonstrated that radixin modulates the plasma membrane localization and functionality of CD47 in HeLa cells. Immunofluorescence analysis and co-immunoprecipitation assay using anti-CD47 antibody showed the colocalization of CD47 and all three ERM families in the plasma membrane, and the molecular interactions between CD47 and all three ERM. Interestingly, gene silencing of only radixin, reduced the CD47 plasma membrane localization and functionality by means of flow cytometry and phagocytosis assay but had little influence on its mRNA expression. Together, in HeLa cells radixin may function as a principal scaffold protein responsible for the CD47 plasma membrane localization.