ヒト肺腺癌細胞A549におけるエンドセリン-2欠失による抗がん効果

概要

A. Background
Lung cancer is the leading cause of death in both sexes, according to the estimated data GLOBOCAN 2020. Based on its histological type, lung cancer is divided into small cell lung cancer (SCLC, 20% of all lung cancers) and non-small lung cancer (NSCLC, 80% of all lung cancers). The major types of NSCLC include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma is the most common subtype of lung cancer, accounting for 40% of lung cancers and 60% of NSCLCs. Despite the development of numerous novel treatments, lung cancer remains a serious problem. Therefore, more effective treatment strategies are required.

Endothelin (ET) consists of three isoforms (ET-1, ET-2, and ET-3), which act on two G-protein coupled receptors, ET receptor type A and type B (ETA and ETB). Among these three isoforms, ET-1 has been the most extensively studied and is involved in the pathophysiology of various diseases, including cancer. Unlike ET1, only a few studies have reported the role of ET-2 in cancer. ET-2 is upregulated in human breast cancer, and its expression of ET-2 mRNA is three times higher in basal cell carcinoma than in normal skin. Additionally, ET-2 reduces apoptosis under hypoxia and potentiates the invasiveness of cancer. It is hypothesized that ET-2 may have distinct pathophysiological roles in cancer.

In the present study, analysis from public online databases of human cancer identified that ET-2 mRNA was significantly higher in lung adenocarcinoma (LUAD) tissues compared to normal lung tissues. In addition, high ET-2 expression was associated with poor overall survival (OS) in patients with LUAD. Therefore, the aim of this study was to explore the role of ET-2 in human adenocarcinoma cells A549 by silencing ET-2 and further investigate the underlying mechanisms.

B. Methods
Bioinformatics Analysis. Measurement of ET-1 and ET-2 mRNA levels was conducted from Gene Expression of Normal and Tumor tissues 2 (GENT2) (http://gent2.appex.kr/gent2/) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) (http://gepia2.cancer-pku.cn/#analysis), interactive web-based tools that comprise normal and tumor samples. Overall survivals (OS) of patients with LUAD cancer (all stages) from the datasets were analyzed using the Kaplan– Meier plotter web tool.

For in vitro studies, ET-2 was silenced in A549 human lung adenocarcinoma cells. Several functional assays were performed to evaluate the proliferation, migration, and invasion ability of ET-2 depleted A549 cells compared with control. Apoptosis was examined by caspase and PARP protein analysis. The cells were then subjected to mRNA and protein analysis.

C. Results
1. High Endothelin-2 (ET-2) Expression in Lung Adenocarcinoma
Analysis from the GEPIA2 (LUAD sample, 483; normal, 59) and GENT2 (LUAD sample, 254; normal, 509) databases identified significantly higher ET-2 expression in LUAD tissues than in normal lung tissues. In contrast, ET-1 expression was significantly lower in LUAD tissues than in normal lung tissues.

2. High ET-2 mRNA Levels Were Associated with a Poor Prognosis of Lung Adenocarcinoma The Kaplan–Meier analysis and log-rank test evaluating the median expression values of ET-2 and ET-1 mRNA demonstrated that the survival rate of patients with LUAD with high ET-2 expression, in terms of OS, was significantly worse than that of patients with low expression. The survival rate of patients with LUAD with low ET-1 expression in terms of OS was significantly worse than that in the high-expression group. Accordingly, these findings suggest that the role of ET-2 in LUAD progression needs to be further elucidated.

3. ET-2 Downregulation Decreased A549 Cell Proliferation
ET-2 silenced A549 cells showed a decreased cell growth rate compared to the negative control.

4. ET-2 Downregulation Retarded A549 Cell Migration and Invasion
Migration of A549 cells was performed using wound healing and transwell migration assays. While for invasion assay, transwell insert membranes coated with Matrigel were used. A549 cells transfected with ET2 siRNA exhibited significantly decreased migration and invasion abilities.

5. ET-2 Silencing Facilitated A549 Cell Apoptosis
During apoptosis, cleavage of PARP-1 is a useful hallmark of this type of cell death. This cleavage is well studied and is generated by caspase-3 and caspase-7, proteases activated during apoptosis. The levels of cleaved PARP, caspase-3, and caspase-7 increased in the ET-2 silencing A549 cells. Taken together, these data showed that ET-2 plays a role in proliferation, migration, invasion, and apoptosis.

6. ET-2 Silencing Reduced X-linked Inhibitor of Apoptosis–Survivin and Epithelial-Mesenchymal Transition Marker in A549 cells
Survivin and XIAP form a complex that binds directly to caspase-9 and plays a role in cancer cell migration. ET-2 ablation reduced the mRNA and protein levels of survivin and XIAP. Epithelial-mesenchymal transition (EMT) was important for A549 cells migration. E-cadherin, an epithelial marker, was increased by silencing of ET-2. Vimentin, SM22, and Twist mRNA, mesenchymal markers, were reduced by silencing ET-2.

D. Discussion
In the present study, ET-2 mRNA expression was significantly elevated in LUAD tissues of patients compared to that in normal lung tissues, according to two bioinformatics human cancer databases. In addition, high ET-2 mRNA levels are associated with poor OS in patients with LUAD. Moreover, ET-2 knockdown in human LUAD A549 cells markedly reduced proliferation, migration, invasion, and enhanced apoptosis. These anticancer effects were mediated by the dual inhibition of XIAP–survivin mRNA and protein levels and suppression of EMT.

XIAP and survivin are inhibitors of the apoptosis protein (IAP) family members, a group of proteins that modulate the balance between proliferation and apoptosis. In addition, IAPs play an important role in cancer cell migration. XIAP and survivin are significantly increased in NSCLC and LUAD according to bioinformatics analyses. Survivin is a well-known regulator of cell proliferation and division. In particular, XIAP is the most explored and potent member of the IAP family. XIAP interacts with and inhibits the activation of caspase-3, caspase7, and caspase-9.

EMT is an important phenomenon involved in the metastasis, recurrence, and drug resistance of lung cancer. In addition, EMT progression is associated with a significantly poor prognosis in patients with LUAD (Sowa et al., 2015). XIAP has been reported to play a role in the migration of cancer cells, partly via the inhibition of EMT (Jin et al., 2019). Eventually, ET-2 depletion reduced the migration and invasion of A549 LUAD cells, partly through the inhibition of EMT.

More in vitro experiments using other LUAD cell lines, in vivo studies, and associated molecular mechanisms are necessary to further explore the pathophysiological functions of ET-2 in cancer. Nevertheless, these findings provide insights into the participation of ET-2 in LUAD and suggest a plausible therapeutic target for LUAD.