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.
Introduction
Cervical cancer is one of the most common gynecological cancers, ranking third in incidence and second in mortality worldwide in adolescents and young adults (AYA) women aged 15–39 years, with an estimated 112,287 new cases and 31,743 deaths in 2020 [ ]. The prognosis of patients with recurrent and/or metastatic cervical cancers is still poor, although the immunotherapeutic intervention including Pembrolizumab and Nivolumab, Cemiplimab, and Tisotumab Vedotin has recently showed promising results in the second-line treatment of cervical cancers [ ]. Despite the recent clinical success of immune checkpoint blockers such as antibodies (Abs) against programmed death-1 and the programmed death-ligand 1 (PD-L1) axis in a wide range of cancer cell types [ , ], these drugs provide clinical benefits to only a small subset of patients with cervical cancers [ , ]. Thus, developing a novel immunotherapeutic strategy is imminent for women with cervical cancer.
Cluster of differentiation (CD) 47 is a dominant macrophage checkpoint molecule superficially expressed in nearly all cancer types [ , ]. CD47 binds to signal regulatory protein α, an inhibitory innate immune receptor that is primarily present on myeloid cells, including macrophages, resulting in a failure of macrophages to phagocyte cancer cells by means of the anti-phagocytic “don’t eat me” signal that allows cancer cells to evade the immune surveillance by the innate immune system [ ]. The expression level of CD47 is ordinally enhanced in a wide variety of cancer cells [ , ] and correlates with poor clinical prognosis [ ]. Therefore, CD47 is a candidate target molecule for developing novel macrophage checkpoint blockade immunotherapies.
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 and functionality [ ]. We recently demonstrated that the ERM family acts as a scaffold protein for PD-L1, belonging to the immunoglobulin superfamily, similar to CD47, which leads to the plasma membrane localization of PD-L1 in several human cancer cell types, including cervical cancers [ ]. However, whether the ERM family functions as a scaffold for CD47 in cancer cells remains unclear. In this study, we determined the role of ERM family members in the plasma membrane expression of CD47 and their relationship using a human uterine cervical squamous cell carcinoma (UCSCC), which accounts for approximately 80 % of all the histological classification of cervical cancers [ ] and clinical RNA sequencing datasets derived from patients with uterine cervical cancer.
Materials and methods
Cell culture
BOKU cells (IFO50323; the Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan), a cell line derived from human UCSCC established by Nozawa S. et al., were cultured according to the conventional method as previously described [ ].
Real-time reverse transcription (RT)- polymerase chain reaction (PCR)
Total RNA was extracted from BOKU cells, and real-time RT-PCR was performed as previously described [ , ]. All RT-PCR primer sequences (TaKaRa Bio, Shiga, Japan) are listed in Supplementary Table S1 .
Immunoblotting
The protein expression of CD47 and ERM in whole-cell lysates extracted from BOKU cells was measured by western blotting, as previously described by our team [ , ], with some modifications. The sources and dilution ratios of all primary and secondary Abs and the original immunoblots are shown in Supplementary Table S2 and Supplementary Fig.S1 , respectively.
Double immunofluorescence staining
Intracellular colocalization of CD47 with ERM was determined by double immunofluorescence staining followed by confocal laser scanning microscopy (CLSM), as previously described by our group [ , ], with some modifications. To determine the colocalization ratio of CD47 with all three ERM, the coefficients of Pearson’s correlation and Mander’s overlap were calculated from the three-dimensional reconstructed images using the NIS-Elements Ar Analysis software (Nikon Instrument, Tokyo, Japan). The sources and dilution ratios of all primary and secondary Abs are summarized in Supplementary Table S2 .
Small interfering (si) RNA treatment
BOKU cells were transfected with siRNAs to transiently knock down the gene and protein expressions of interest, as previously described by our group [ , ]. Briefly, siRNAs ( Supplementary Table S3 ) (Thermo Fisher Scientific, Waltham, MA, USA) at a final concentration of 5 nM were delivered by lipofection into BOKU cells, followed by continuous culture for three days.
Flow cytometry
The cell surface expression levels of CD47 in BOKU cells were analyzed by flow cytometry, as previously described [ , ], with some modifications, using allophycocyanin (APC)-conjugated anti-human CD47 Ab.
Co-immunoprecipitation
The protein–protein interaction between CD47 and each ERM was assessed by the co-immunoprecipitation assays using the whole-cell lysates from BOKU cells containing 700 μg of the crude proteins as previously described [ , ], with some modifications. The sources and dilution ratios of all primary and secondary Abs and the original immunoblots are shown in Supplementary Table S2 and Supplementary Fig.S1 , respectively.
Bioinformatic analysis
Relative gene expression levels of CD47 and ERM in tumor tissues derived from patients with uterine cervical cancer and those in corresponding normal tissues were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) [ ]. GEPIA is a freely available interactive web server for analyzing RNA sequencing (RNA-seq) expression data of 9736 tumors and 8587 normal samples from The Cancer Genome Atlas (TCGA) [ ] and Genotype-Tissue Expression (GTEx) projects [ ].
Statistical analysis
For in vitro experiments, the results were expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s test using Prism version 3 (GraphPad Software, La Jolla, CA, USA). Differences were considered statistically significant at P < 0.05.
For GEPIA database analysis, gene expression levels of interest were expressed as box-and-whisker plots representing the median values and interquartile ranges, and the minimum and maximum values with jitter. Statistical comparisons were performed using the Mann–Whitney U test. The cutoff for P value was set at 0.001. Correlation analysis was performed using Pearson’s correlation coefficient, which was automatically calculated using the GEPIA database tool [ ]. The Kaplan–Meier method was used for the overall survival analysis, and statistical differences were determined by the log-rank test automatically calculated using the GEPIA database tool [ ].
Ethical statement
Because all the data analyzed in this article are from the public database of TCGA, GTEx, and GEPIA, there are no ethical statement needed to be declared for this manuscript. Similarly, there are no informed consent form documents are needed to be signed by any patients, because all the participants had been giving informed consent before taking part in the TCGA, GTEx, and GEPIA public databases.
Results
Expression profiles of CD47 and the ERM family in the human UCSCC cells
In 12 human UCSCC cell lines registered in the public databases of the Cancer Cell Line Encyclopedia and Cancer Dependency Map (Depmap) portal data explore [ , ], RNA-seq expression data showed that the mRNA expressions of CD47, and ezrin, radixin, and moesin, were all detected in BOKU cells ( Fig. 1 A). Similarly, we confirmed that CD47, ezrin, radixin, and moesin were present in BOKU cells at both the mRNA and protein levels ( Fig. 1 B and C).
Co-localization of CD47 with the ERM family in BOKU cells
Double immunofluorescence staining indicated that CD47 was co-localized with all three ERM family members in the plasma membrane of BOKU cells ( Fig. 2 A–C). In contrast, no fluorescence signals were observed in BOKU cells incubated with each secondary Ab conjugated with the fluorochromes used without the primary Abs ( Supplementary Fig.S2 ).
siRNA-mediated knockdown of the ERM family in BOKU cells
We checked the knockdown activity of siRNAs against each ERM on the mRNA and protein expression of the respective targets in BOKU cells. Transfection of BOKU cells with siRNAs targeting each ERM family member substantially decreased the mRNA ( Fig. 3 A) and protein ( Fig. 3 B) expression levels of the respective targets, with little impact on cell viability ( Supplementary Fig.S3 ).
