Ezrin works as a scaffold protein for a macrophage checkpoint molecule CD47, leading to a poor prognosis for patients with uterine cervical squamous cell carcinoma

Abstract

Objectives: Despite recent advances in the immunotherapeutic intervention as the second-line treatment of cervical cancer, including Pembrolizumab and Nivolumab, the advanced stages of the disease are still associated with poor prognosis. CD47 is a macrophage checkpoint molecule overexpressed superficially in nearly all cancer types that binds to its receptor on macrophage surface, leading to a disruption of their phagocytic capacities against cancer cells. Ezrin–Radixin–Moesin (ERM) family member of proteins work as scaffold proteins by crosslinking specific transmembrane proteins to actin filaments, contributing to their plasma membrane localization. This study aimed to investigate the relationship between ERM family and CD47 in the uterine cervical squamous cell carcinoma (UCSCC). Materials and methods: The mRNA expression, intracellular localization, and molecular interaction of CD47 and ERM in BOKU cells derived from human UCSCC were determined using RT-PCR, immunofluorescence, and co-immunoprecipitation, respectively. CD47 plasma membrane expression was measured by flow cytometry three days after transfection with small interfering RNAs against each ERM. CD47 and ERM expression in tumor tissues from patients with uterine cervical cancer was analyzed using a clinical RNA sequencing database. Results: Confocal laser scanning microscopy analysis showed the co-localization of CD47 with all three ERM in the plasma membrane of BOKU cells. RNA interference-mediated knockdown of ezrin but not others reduced the plasma membrane expression of CD47. Furthermore, immunoprecipitation assay demonstrated the molecular interaction of CD47 with ezrin. Notably, bioinformatic analysis indicated that CD47 and ezrin expressions were markedly increased and positively correlated in the clinical uterine cervical tumor tissues and that higher expressions of ezrin correlates with a poor prognosis for the uterine cervical cancers. Conclusion: This study illustrates that in uterine cervical cancers, ezrin may be a dominant scaffold protein responsible for CD47 expression and, therefore, is a potential target for developing a novel macrophage checkpoint blockade therapy.

Keywords

• Uterine cervical squamous cell carcinoma
• Cancer immunotherapy
• Macrophage checkpoint
• CD47
• Ezrin–radixin-moesin family member