MTA1-upregulated EpCAM is associated with metastatic behaviors and poor prognosis in lung cancer

Abstract

Background: Overexpression of Metastasis-associated protein 1 (MTA1) in various cancer cells promotes tumor invasion and migration and predicts cancer patients' poor prognosis. The pilot RNA-Seq data from our laboratory indicated that Epithelial cell adhesion molecule (EpCAM) was statistically reduced in MTA1-silencing cells. EpCAM has been recognized as more than a mere cell adhesion molecule and recent findings have revealed its causal role in mediating migratory and invasive capacity. Thus, this study was aimed to explore whether MTA1 was able to upregulate EpCAM expression and, consequently, modulate its effects on invasion and migration of the lung cancer cells as well as patients' prognosis.

Methods: We checked the EpCAM expression by overexpressing or silencing MTA1 in lung cancer cells. Furthermore, these lung cancer cells with stably overexpressed or silenced MTA1 were transfected with siEpCAM or EpCAM-expressing plasmids and then subjected to western blot, invasion and migration assays. In addition, patients (n = 118) with early-stage lung cancer were enrolled in this study to confirm the correlations between MTA1 and EpCAM and pathoclinical parameters by using immunohistochemistry (IHC). All statistical analyses were performed with SPSS 20.0 statistical software.

Results: MTA1 upregulated EpCAM expression in lung cancer cell lines, and EpCAM overexpression rescued the inhibitory effects by silencing MTA1 on cell invasion and migration in vitro. What's more, both MTA1 and EpCAM, correlated to each other, were overexpressed in lung cancer tissues and significantly correlated with their clinical stages, tumor diameters, lymph node metastasis. Multivariate analysis indicated that local advancement (p = 0.03), MTA1 overexpression (p = 0.001) and EpCAM overexpression (p = 0.045) of the lung cancer tissues remained significant in predicting unfavorable overall survival.

Conclusions: We revealed a new molecular mechanism of MTA1-mediated invasion and metastasis in lung cancer through downstream target EpCAM, and interfering with EpCAM function may be a novel therapeutic strategy for treatment of MTA1-overexpressing lung carcinoma.

Fig. 1

MTA1 could upregulate EpCAM expression and promote invasion and migration via upregulation of EpCAM expression in lung cancer cells. a Western blot analysis of the expression level of EpCAM in MTA1-overexpressing or MTA1-silencing cells. shNC: A549, PC-9, NCI-H446 cells transfected with Lenti-shNC-GFP; shMTA1: A549, PC-9, NCI-H446 cells transfected with Lenti-shMTA1; NC: A549, PC-9, NCI-H446 cells transfected with Lenti-NC-GFP. MTA1: A549, PC-9, NCI-H446 cells transfected with Lenti-MTA1. b Images of the transwell invasion assay in NC cells, MTA1-overepressed cells transfected with Scramble siRNA or EpCAMsiRNA and MTA1-silenced cells transfected with vector control or EpCAM. c Quantitative analysis of the number of invasion cells in the five groups of cells. The invaded cells were counted under × 200 magnification (five randomly selected fields). First, the number of invasive shMTA1 cells was significantly reduced (*p < 0.05, shMTA1 vs. shNC), and the number of invasive MTA1 cells was dramatically increased (*p < 0.05, MTA1 vs. NC). Second, invasive response of MTA1-overexpression cells was impaired significantly after downregulatingEpCAM (*p < 0.05, EpCAMsiRNA vs. Scramble siRNA in MTA1-overexpression cells), and invasion could be rescued by overexpressing EpCAM (*p < 0.05, EpCAM vs. Vector control in MTA1-silencing cells). d Representative images of the five cell groups in the wound healing assay (magnification,×200). e Quantitative analysis of the migration ability of lung cancer cells. First, the MTA1-silencing could reduce cells motility when compared with the negative controls cells (*p < 0.05, shMTA1 vs. shNC), and the MTA1-overexpression could increase cells motility when compared with the negative controls cells (*p < 0.05, MTA1 vs. NC). Second, a knockdown of EpCAM expression in MTA1-overexpression cells could dramatically attenuate cell migration (*p < 0.05, EpCAMsiRNA vs. Scramble siRNA in MTA1-overexpression cells), and the upregulation of EpCAM in MTA1-silencing cells could regain cell migration (*p < 0.05, EpCAM vs. Vector control in MTA1-silencing cells). All experiments were repeated three times

Fig. 2

Expression of MTA1 and EpCAM in tissue microarray samples. a The immunohistochemistry results showed that MTA1 were overexpressed in lung cancer tissues when compared with nonneoplastic tissues (*p < 0.05).b The immunohistochemistry results showed that EpCAM were overexpressed in lung cancer tissues when compared with nonneoplastic tissues (*p < 0.05). c Representative images showing immunohistochemical expression in lung cancer tissue specimens of MTA1 and EpCAM. Cases with strong and weak expression are represented. Original magnification, ×200

Fig. 3

MTA1 expression positively correlates with EpCAM expression in lung cancer patients. Statistics were calculated using Spearman testing. *, p < 0.01

Fig. 4

Kaplan-Meier analysis of overall survival in all lung cancer patients. a Patients with MTA1 overexpression had a poorer prognosis. b Patients with EpCAM overexpression had a poorer prognosis. The log-rank test was used to calculate p value. *, p < 0.01