Abstract
Background/Aim: Annexin A10 (ANXA10) is a member of the annexin family and a calcium-dependent phospholipid-binding protein. The aim of this study was to clarify the clinical significance of ANXA10 expression in lung adenocarcinoma. Materials and Methods: ANXA10 expression was immunohistochemically examined in surgical specimens of lung adenocarcinoma obtained from 74 consecutive patients who underwent complete resection from January 2014 to December 2014. Expression of ANXA10 was down-regulated in A549 cells via siRNA transfection and the effect of ANXA10 on cell migration was assessed by the wound healing assay. Expression of ANXA10 was examined by immunocytochemistry and polymerase chain reaction. Results: High ANXA10 expression was significantly correlated with poor overall survival (p=0.00705). Multivariate analysis with the Cox proportional hazard model demonstrated that ANXA10 expression was an independent prognostic factor. Cell migration was suppressed in ANXA10-down-regulated A549 cell lines. Conclusion: ANXA10 has a role in cancer cell migration and high ANXA10 expression is a novel prognostic marker in lung adenocarcinoma.
For patients with clinical stage I-II non-small-cell lung cancer (NSCLC), lobectomy or pneumonectomy with lymph node excision is the first-choice treatment (1). The pooled analysis by the Lung Adjuvant Cisplatin Evaluation (LACE) Collaborative Group showed that the additional effect of postoperative platinum-based chemotherapy on the 5-year overall survival (OS) was 5.4% (2). To improve the prognosis of patients with NSCLC, it is important to properly select patients who need to undergo adjuvant chemotherapy because the estimated 5-year OS for patients with stage IA and IB lung adenocarcinoma is 81%-87% and 72%, respectively (3), which is insufficient.
Annexin A members comprise a subgroup of the annexin family, including 12 members (4). Annexin A10 (ANXA10) is a member of the annexin family of calcium-dependent phospholipid-binding proteins. ANXA10 has a crucial role in cellular and physiological processes (5, 6). The organs that express ANXA10 are the stomach, bladder, kidney, urinary bladder and duodenum (7). On the other hand, ANXA10 is less expressed in normal lungs (8). Several studies demonstrated that a high expression level of ANXA10 was associated with poor prognosis in small bowel adenocarcinoma, papillary thyroid cancer, serous epithelial ovarian cancer and colorectal cancer patients (7, 9, 10). On the other hand, a low expression level of ANXA10 was reported to be associated with poor prognosis of gastric carcinoma and hepatocellular carcinoma (11-13). However, the role of ANXA10 in lung adenocarcinoma has not been clarified.
In this study, we examined the expression of ANXA10 in 74 consecutive lung adenocarcinoma patients to determine the clinical significance of ANXA10 expression in lung adenocarcinoma.
Materials and Methods
Patients. Seventy-eight consecutive patients with lung adenocarcinoma treated by complete surgical resection at Kobe University Hospital, Kobe, Japan, between January 2014 and December 2014 were reviewed in this study. Patients with positive surgical margins were excluded from this study, and the remaining 74 patients were analyzed. The methods of data collection and analysis were approved by the institutional review board (permission number: 160117), and written informed consent was obtained from all the patients.
Spiral array block construction and pathological studies. The paraffin embedded blocks were processed into a spiral array blocks by Pathology Institute (14, 15).
All histologic specimens were diagnosed based on the 2015 WHO classification (16). Pathological stage was determined based on the TNM classification of the International Union Against Cancer (UICC). The expression levels of ANXA10 were assessed independently by two pathologists (NJ and TN) who were unaware of the clinical data.
Immunohistochemical staining. Anti-human ANXA10 antibody (lot no. C114395, Sigma, St. Louis, MO, USA) was used as the primary antibody (1:1,000). The polymer method was employed for immunohistochemical staining (17). Samples with high ANXA10 expression were defined as those in which at least 50% of cancer cells were stained.
Cell culture. The lung adenocarcinoma cell line A549 was obtained from American Type Culture Collection (Manassas, VA, USA) and was cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Wako, Osaka, Japan) under 5% CO2 at 37°C.
ANXA10 knockdown by siRNAs. ANXA10 siRNAs (#1: s22116 and #2 s22117) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Control siRNA (sc37007) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were treated with ANXA10 siRNAs or control siRNA duplexes, which were mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in serum-free RPMI-1640 medium as described by the manufacturer’s protocol, and added to the plated cells. The cells were assessed 48 h later.
Quantitative reverse transcription PCR (qRT-PCR). RNA was isolated from lung cancer cell line (A549) according to the protocol of the manufacturer of Sepasol®-RNAI Super G (Nacalai Tesque, Inc. Kyoto, Japan). Extracted and purified RNA was reverse transcribed to to generate cDNA using the a PrimeScript RT Reagent Kit (Takara, Kusatsu, Shiga, Japan). Real-time PCR was performed using Thermal CyclerDice® Real Time System II (Takara). Relative mRNA levels were calculated with the ∆∆Ct method using GAPDH mRNA as an internal control. The primers used in this study were as follows: 5’-AACTTGGTCCAGGCAAACAGG-3’ and 5’-ACGAACAATAGCA ACCAGCAGC-3’ for ANXA10 and 5’-CCACTCCTCCACCT TTGAC-3’ and 5’-ACCCTGTTGCTGTAGCCA-3’ for GAPDH.
Wound healing assay. To determine the effect of ANXA10 knockdown on cell migration, we performed a wound healing assay. Confluent A549 cells in culture plates were scratched with 200 μl pipette tips (WATSON Bio Lab, Aliso Viejo, CA, USA), and the width of the gap was measured up to 12 h later.
Statistical analysis. All statistical analyses were performed with EZR version 1.37 (Saitama Medical Center Jichi Medical University; Kanda, 2018), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.4.1) (18). The Pearson χ2 test or Fisher exact test were used toevaluate the differences in patient characteristics between the two groups. Cumulative survival was estimated by the Kaplan–Meier method, and differences in variables were calculated by the log-rank test. A multivariate regression analysis was performed by using the Cox proportional hazard model. All p-values reported are 2-sided, and a p-value of less than 0.05 was considered significant.
Results
A total of 74 consecutive specimens of adenocarcinoma of the lung were examined for ANXA10 expression (Figure 1). High ANXA10 expression in the nucleus of cancer cells was observed in 28 of the 74 specimens (38%). The relationships between ANXA10 expression and clinicopathologic factors are summarized in Table I. The median age of the patients in the high ANXA10 group was 71.5 years (range=55-83 years); 18 patients were male, and 10 were female. Twenty-one patients in the ANXA10-positive group were current or former smokers. No significant differences in any of these characteristics were found between the high ANXA10 group and low ANXA10 group.
Differences in immunostaining for ANXA10. The upper column shows representative images of samples with low ANXA10 expression; the lower column shows representative images of samples with high ANXA10 expression. The left columns show low-magnification images (bars, 250 μm). The right columns show high-magnification images (bars, 100 μm). All sections were made from spiral array blocks and stained with DAB.
Patient characteristics.
The overall survival curve obtained by the Kaplan–Meier method is shown in Figure 2. High ANXA10 expression was significantly correlated with poor overall survival (p=0.00705), suggesting that the lung adenocarcinomas that express ANXA10 show aggressive features. The Human Protein Atlas also showed that high ANXA10 expression was significantly correlated with poor overall survival in 500 patients with lung adenocarcinoma (p=0.00011).
Kaplan–Meier survival curves of patients with lung adenocarcinoma with or without ANXA10 expression. The overall survival of patients with high expression of ANXA10 was worse than that of patients with low expression of ANXA10 (log-rank test, p=0.00705).
In addition, univariate analyses showed that pT stage <2 (p=0.00000113), pN stage <1 (p=0.00000537), pleural infiltration <1 (p=0.000000855), vascular invasion (p=0.00679), and lymphatic invasion (p=0.0153) were correlated with short overall survival (Table II). Table III shows the impact of potential predictive factors on the overall survival of patients with adenocarcinoma expressing ANXA10 based on the results of the multivariate Cox proportional hazard analysis. All significant univariate factors were included in the multivariate analysis. ANXA10 was shown to be an independent predictor of overall survival.
Univariate analysis.
Multivariate analysis.
To examine the effect of decreased ANXA10 expression on aggressive cell behavior in vitro, the migration ability of A549 cells treated with ANXA10 siRNA or negative control was examined by using a wound healing assay. First, the interference efficiency of the ANXA10 siRNA was confirmed by qRT–PCR (Figure 3A). Then, the cell migration ability was monitored at 0, 6, and 12 h. Cell migration was suppressed in ANXA10-down-regulated cancer cells (Figure 3B). This result suggests that ANXA10 has a role in cell migration.
The role of ANXA10. (A) Knockdown efficiency of ANXA10 siRNA in A549 cells. (B) Wound healing assay. Knockdown of ANXA10 delayed migration of cells into the gap. The upper columns show the negative control, and the lower columns show cells treated with ANXA10 siRNA (0, 6, and 12 h). Bars, 100 μm.
Discussion
In this study, we showed for the first time that high expression levels of ANXA10 were associated with poor overall survival in lung adenocarcinoma patients. In addition, multivariate analysis showed that ANXA10 was an independent prognostic marker in lung adenocarcinoma. Although the role of ANXA10 in cancer is controversial, analysis of data from the Human Protein Atlas, a large-scale database, also revealed that high expression levels of ANXA10 were associated with short overall survival in lung adenocarcinoma patients.
A previous study showed that ANXA10 promotes cell proliferation via phosphorylation of AKT and ERK 1/2 in esophageal cancer (19). In the present study, we showed that ANXA10 was associated with cell migration. A study revealed that ANXA10 promoted the progression of perihilar cholangiocarcinoma and facilitated metastasis by promoting the epithelial-mesenchymal transition (EMT) process via the PLA2G4A/prostaglandin E2 (PGE2)/signal transducer and activator of transcription 3 (STAT3) pathway (20). A recent study also revealed that ANXA10 knockout reduced cell migration and that ANXA10 promoted the metastatic activity of melanoma by suppressing E3 ligase TRIM41-directed protein kinase D1 (PKD1) degradation (21). However, PKD1, which is a key signaling molecule, has different biological properties depending on the tissue and cell type. In HeLa cells and breast cancer cells, PKD1 has been reported to block actin remodeling through the phosphorylation of phosphatase slingshot 1L and p21-activated kinase 4 (PAK4) and to inhibit cell migration (22-24). These differences in the functions of PKD1 might explain the differences in the function of ANXA10 in cancers.
In conclusion, ANXA10 has a role in cancer cell migration, and ANXA10 expression is a novel prognostic marker for adenocarcinoma of the lung.
Acknowledgements
The Authors thank all the members of the Division of Respiratory Medicine, Kobe University Graduate School of Medicine, for their helpful discussions.
Footnotes
Authors’ Contributions
MY and TN wrote the manuscript. MY and RD performed immunostaining. TK and MT collected the data. TN and NJ performed pathological analysis. YT, TI and YM collected the samples. KK and YN provided technical assistance. MY and TN conducted the statistical analysis. All Authors analyzed the data, conceived the study, and read and approved the final manuscript.
Conflicts of Interest
This work was supported by JSPS KAKENHI 17K09614 and Takeda Science Foundation funding (to Tatsuya Nagano).
- Received December 18, 2021.
- Revision received January 10, 2022.
- Accepted January 11, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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