Aberrant expression of junctional adhesion molecule-A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155

Abstract

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule-A (JAM-A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM-A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM-A significantly suppressed cell proliferation and colony-forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM-A reduced cell proliferation ability and that loss of JAM-A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM-A and formed a physical interaction with JAM-A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155-positive cases expressed a high level of JAM-A, and patients with the expression pattern of PVR/CD155 positive/JAM-A high had significantly shorter periods of relapse-free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM-A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM-A is therefore a potential therapeutic target for this malignancy.

Keywords: CD155; junctional adhesion molecule-A; poliovirus receptor; therapeutic target; tight junction protein; uterine cervical adenocarcinoma.

FIGURE 1

Increased expression of junctional adhesion molecule‐A (JAM‐A) in surgical specimens of uterine cervical adenocarcinoma. A, JAM‐A was strongly expressed on the cell membrane in adenocarcinoma cases. Left panels, H&E staining; Right panels, immunohistochemistry of JAM‐A. B, Immunoreactive score (IRS) of JAM‐A in surgical specimens of uterine cervical adenocarcinoma. C, D, Kaplan‐Meier curve analysis. Relapse‐free survival was significantly shorter in the group with high immunoreactive scores (≥27) of JAM‐A. ADC, adenocarcinoma; AIS, adenocarcinoma in situ; CE, cervical epithelia; OS, overall survival, RFS, relapse‐free survival. Bar = 50 µm

FIGURE 2

Junctional adhesion molecule‐A (JAM‐A) contributes to proliferation, colony formation and collective migration of uterine cervical adenocarcinoma cells. A, JAM‐A was expressed in uterine cervical adenocarcinoma cell lines. B, KO of JAM‐A expression was achieved by using the CRISPR/cas9 system in HCA1 cells. C, WST‐8 assay (5000 cells/well). Proliferation of HCA1 cells was significantly inhibited by KO of JAM‐A. D, Colony formation assay (6‐well plate, 1250 cells/well). Number of colonies of HCA1 cells was decreased by KO of JAM‐A. E, F, Immunohistochemistry of Ki‐67 (proliferation marker) and cleaved caspase‐3 (apoptosis marker) in cell block samples of HCA1 cells. KO of JAM‐A significantly decreased the number of Ki‐67‐positive cells and increased the number of cleaved caspase‐3‐positive cells (Ki‐67: n = 5, cleaved caspase‐3: n = 6). The number of positive cells per 100 cells is represented as mean ± SD. G, Wound healing assay. Wound closure was significantly inhibited by KO of JAM‐A. **P < .01 vs control cells. A, B, western blot analysis

FIGURE 3

Junctional adhesion molecule‐A (JAM‐A) contributes to drug resistance and is a potential therapeutic target of uterine cervical adenocarcinoma cells. A, KO of JAM‐A significantly suppressed etoposide resistance of HCA1 cells. WST‐8 assay. B, C, A monoclonal antibody against the extracellular domain of JAM‐A reduced proliferation of HCA1 control cells but not that of JAM‐A‐KO cells. Cell proliferation was measured by BrdU incorporation assay (B) and WST‐8 assay (C) after treatment with the antibody. Graphs represent means ± SD. *P < .05 and **P < .01 vs control cells

FIGURE 4

Comprehensive proteome analysis was carried out to clarify the role of junctional adhesion molecule‐A (JAM‐A) overexpression. A, Volcano plot of identified unique proteins. B, Venn diagram. A total of 2849 unique proteins were identified, 201 proteins being significantly down‐regulated (ratio < 0.67 and P < .05) and 326 proteins being significantly upregulated (ratio > 1.5 and P < .05) by JAM‐A KO. C, GO analysis of significantly downregulated proteins in JAM‐A KO cells. D, Three candidate molecules were selected from downregulated proteins that might promote malignant potentials in association with JAM‐A in uterine cervical adenocarcinoma

FIGURE 5

Poliovirus receptor (PVR)/CD155 was identified as a candidate molecule directly interacting with junctional adhesion molecule‐A (JAM‐A). A, Expression of the candidate JAM‐A‐associated proteins was significantly decreased by JAM‐A KO. B, Expression of PVR/CD155 and ARHGEF1 was increased by transfection of an N‐terminal 3xFLAG‐tagged JAM‐A plasmid in JAM‐A KO cells. C, Immunoprecipitation (IP) assay. PVR/CD155 was detected in immunoprecipitation with an anti‐FLAG antibody from N‐terminal 3xFLAG‐tagged JAM‐A‐expressing JAM‐A KO cells. D, Immunofluorescence of JAM‐A (green) and PVR/CD155 (red) was co‐localized on the plasma membrane of HCA1 cells. E, PVR/CD155 expression was knocked down by PVR/CD155‐specific siRNAs. F, Colony‐forming assay (6‐well plate, 1250 cells/well). The number of colonies of HCA1 cells was significantly decreased by knockdown of PVR/CD155. G, Immunohistochemistry of Ki‐67 in cell block samples. The number of Ki‐67‐positive cells (proliferation marker) was significantly decreased by knockdown of PVR/CD155 (n = 5). The number of positive cells per 100 cells is represented as mean ± SD. **P < .01 vs control cells. A, B, C and E, western blot analysis

FIGURE 6

Positive correlation of overexpression of junctional adhesion molecule‐A (JAM‐A) and poliovirus receptor (PVR)/CD155 in surgical specimens of uterine cervical adenocarcinoma. A, Immunohistochemistry of PVR/CD155 in a surgical specimen of uterine cervical adenocarcinoma (ADC). PVR/CD155 was strongly expressed in that case. B, C, Kaplan‐Meier curve analysis. Relapse‐free survival and overall survival were significantly shorter in the PVR/CD155‐positive group. D, Summary of the expression profiles of JAM‐A and PVR/CD155 in surgical specimens. Percentage of PVR/CD155‐positive cases was significantly higher in the high‐JAM‐A expression group than in the low‐JAM‐A expression group (P = .0179). E, F, Kaplan‐Meier curve analysis. The cases were divided into three groups: PVR/CD155 negative/JAM‐A low (Group I), PVR/CD155 positive/JAM‐A low or PVR/CD155 negative/JAM‐A high (Group II) and PVR/CD155 positive/JAM‐A high (Group III). Kaplan‐Meier analysis showed that patients with the expression pattern of PVR/CD155 positive/JAM‐A high (Group III) had significantly shorter periods of relapse‐free survival (P = .00964) and overall survival (P = .0204) than for the other patients. ADC, adenocarcinoma; CE, cervical epithelia; OS, overall survival; RFS, relapse‐free survival