MTA2 overexpression in ESCC tissues correlates with ESCC aggressiveness
We firstly performed an IHC and western blotting analysis to investigate the MTA2 protein expression in normal esophageal squamous epithelium, precancerous lesion tissues and esophageal squamous cell carcinoma tissues. The result showed that the expression of MTA2 was increased in ESCC, compared with precancerous lesion tissue or the corresponding non-tumor tissues (Fig.S1A-B), consistent with our previous result [25].
To investigate the expression level of MTA2 gene in ESCC malignancy, we measured MTA2 mRNA expression in 98 human ESCC tissues using reverse transcription and quantitative PCR (RT-qPCR). The results revealed that MTA2 was highly expressed in ESCC tissues when compared with that in the corresponding non-tumor tissues from the same donor or compared with that in normal esophageal epithelial tissue samples (Fig. 1A and Fig. S1C). Further analysis revealed that the primary tumor invasion depth (Fig. 1B), advanced TNM stage (Fig. 1C), and lymph node metastasis (Figure.1D) were all positively correlated with MTA2 expression. On the other hand, we divided the samples into an MTA2 low-expression group and an MTA2 high-expression group according to the median MTA2 expression in the ESCC tissues to detect the clinical pathological features of ESCC patients and MTA2 expression levels. As expected, there were positive correlations between the MTA2 expression levels and the primary tumor invasion depth, lymph node metastasis, distant metastasis, and TNM stage, while the tumor differentiation grade and tumor size displayed no correlation (Table.1). Furthermore, we examined MTA2 expression in primary tumor tissues and the corresponding lymph–node metastatic tumor tissues from three patients. The results indicated that the expression of MTA2 was much higher in lymph–node metastatic tumor tissues compared to that in primary tumor tissues (Fig. 1E), which strongly suggested that MTA2 might contribute to ESCC malignancy, particularly to metastasis.
Additionally, a prognostic analysis using 79 ESCC tissues with complete follow-up information revealed that high MTA2 expression in ESCC tissues was associated with reduced overall survival (Fig. 1F). We also evaluate the effect of the MTA2 expression level on the clinical prognosis of different cancers using the data from the GEPIA (http://gepia.cancer-pku.cn/index.html) and found that high MTA2 expression was correlated with poor survival probability in mesothelioma (Fig.S1D), adrenocortical carcinoma (Fig.S1E), and liver hepatocellular carcinoma (Fig.S1F). Therefore, we concluded that MTA2 over- expression was associated with ESCC malignancy and poor prognosis.
MTA2 promotes proliferation, migration and invasion of ESCC cells in vitro.
In order to clarify the function of MTA2 in the ESCC cells, we firstly analyzed MTA2 expression in eight ESCC cell lines and a normal esophageal epithelial cell line. Compared with normal esophageal epithelial cell line, MTA2 expression was significantly higher in ESCC cell lines (Fig. S2A). Then MTA2 expression was knocked down in KYSE30 and KYSE510 cells through the transfection of siRNA or shRNA, or was overexpressed through the transfection of the pCDH-MTA2 plasmid (Fig.S2B). Wound healing and transwell assays were performed to explore the impact of MTA2 on the migration and invasion of ESCC cells. As shown in the Fig. 2A, after transfection with siRNA to knockdown the expression of MTA2, the wound healing process was delayed. Moreover, the number of cells penetrating the membrane of the chambers in both the migration and invasion assays was significantly lower in the MTA2 knockdown group compared with that in the control group (Fig. 2B and C). In contrast, the exogenous overexpression of MTA2 facilitated the wound closure of both the KYSE30 and KYSE510 cell lines (Fig. 2D). Transwell migration and Matrigel invasion assays also implied that the overexpression of MTA2 prominently enhanced the migratory and invasive capabilities of both ESCC cell lines compared to those of the controls (Fig. 2E and F).
Moreover, we also found that compared to the controls, MTA2 depletion inhibited the proliferation of KYSE30 and KYSE510 cells based on a MTS assay (Fig.S2C), while MTA2 overexpression promoted the viability of both cell lines (Fig.S2D). The colony formation assay also demonstrated the similar function of MTA2 (Fig.S2E).
Taken together, these data clearly demonstrate that MTA2 plays a carcinogenic role in ESCC by promoting the viability, migration and invasion of ESCC cell lines.
MTA2 promotes the growth and metastasis of transplanted tumors in vivo.
To further examine the oncogenic activity of MTA2 in tumor progression in vivo, we generated animal models by subcutaneously injecting KYSE30/shMTA2 or KYSE30/MTA2 cells into nude mice. Both the control and KYSE30/MTA2 groups formed tumors after injection, and the tumor formation rate in the KYSE30/shMTA2 group was 80% (4/5). The growth rate and average tumor weight of the xenografts were both lower in the MTA2 knockdown group than in the control group. (Fig. 3A). Moreover, the expression levels of both the cell proliferation marker Ki-67 and the tumor angiogenesis marker CD31 were significantly reduced in the MTA2 knockdown group (Fig. 3B). However, the tumors that were derived from MTA2 overexpressed cells were significantly larger than those in the control group (Fig. 3C).As expected, MTA2 overexpression promoted the expression of Ki-67 and CD31 in the xenografts (Fig. 3D).
On the other hand, the influence of MTA2 expression on ESCC metastasis was evaluated. KYSE30/shNC and KYSE30/shMTA2 cells were injected into the tail vein of nude mice. As shown in the figure, though no visible metastasis was found in either group, histologic analysis showed that many pulmonary metastatic foci was formed in the lung of mice in the control group, while no pulmonary tumor nodules were found in the MTA2 knockdown group (Fig. 3E). These results strongly suggest that MTA2 promoted the growth and metastasis of esophageal carcinoma in vivo.
MTA2 Promotes Epithelial-mesenchymal Transition In ESCC Cells
There is strong evidence that EMT is involved in the different stages of tumor metastasis and promotes a malignant phenotype in tumors. To further investigate the mechanism behind the MTA2-mediated promotion of metastasis in ESCC cells, the expression of molecules that are associated with the transition from an epithelial to a mesenchymal phenotype was examined. The results indicated that compared to the controls, the expression of N-cadherin, Vimentin, MMP2,MMP9 and ZEB1 was suppressed, and the expression of E-cadherin and ZO-1 was
upregulated when the expression of MTA2 was silenced in both KYSE30 and KYSE510 cells (Fig. 4A). Likewise, compared to the controls, in MTA2-overexpressing cells, the expression levels of N-cadherin, Vimentin, MMP2, MMP9 and ZEB1 was significantly increased, whereas those of E-cadherin and ZO-1was decreased (Fig. 4B).
Furthermore, this change was further confirmed by examination the subcellular presence of proteins using IF staining. As shown in the Fig. 4C, compare to the controls, MTA2 deficiency repressed the expression of N-cadherin and Vimentin, while the MTA2-knockdown group exhibited increased E-cadherin staining at the cell membrane.
We also analyzed E-cadherin, N-cadherin and Vimentin expression in xenograft tumor tissues to explore whether MTA2 promoted EMT transformation in vivo. The result was consistent with the findings in vitro (Fig. 4D). All these data prompted us to hypothesize that the MTA2-mediated metastasis of ESCC might be linked to the promotion of EMT.
EIF4E Is A Target Of MTA2
Moreover, to further examine the molecular mechanism that underlies the pro-tumorigenic role of MTA2, we conducted a gene expression microarray analysis of KYSE30 cells that were depleted of MTA2 and of control cells and 93 genes with significant changes in expression (fold change > 2, P < 0.05, GSE112495) were identified (Fig. 5A). For microarray analysis result of MTA2 knockdown, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the most significantly overrepresented pathways involved in RNA transport, Rap1 signaling pathway and TGF-β signaling pathway(Fig. 5B).The mRNA levels of six representative genes (three upregulated and three downregulated) were confirmed by qRT-PCR, as shown in Fig. 5C. Among these genes, we found that EIF4E expression was consistently and significantly decreased in ESCC cells with depletion of MTA2 compared to that in controls. Then we detect the expression of EIF4E in ESCC tissues from 60 patients in our study, and the results showed that EIF4E expression was significantly increased in ESCC tissues as compared to that in the matched para-cancerous tissues (Fig. 5D). Further analysis revealed that EIF4E expression was positively correlated with primary tumor invasion depth (Fig. 5E), lymph node metastasis (Fig. 5F) and TNM stage (Fig. 5G). Moreover,the Kaplan- Meier survival analysis was then conducted and the result demonstrated that higher EIF4E levels in patients were correlated with shorter overall survival than that in patients with low EIF4E levels (Fig. 5H). According to the GEPIA analysis (http://gepia.cancer-pku.cn/index.html), high EIF4E expression was correlated with poor survival probability in glioma patients (Fig.S3A), liver hepatocellular carcinoma patients (Fig.S3B) and lung adenocarcinoma patients (Fig.S3C).These results suggested that EIF4E overexpression was an important prognosis factor of various types of cancer, including ESCC.
The MTA2-mediated promotion of malignancy in ESCC is mediated by EIF4E
Next we will evaluate whether EIF4E are important factor of MTA2 function. Firstly, our result showed that knockdown of MTA2 decreased the expression of EIF4E, while overexpression of MTA2 exerts contrast function (Fig. 6A). Consistently, decreased EIF4E was detected in tumor tissues in knockdown MTA2 group (Fig. 6B). Furthermore, a positive correlation between the expression of EIF4E and MTA2 was found in the human ESCC tissues in our study (Fig. 6C), which was consistent with the data from the GEPIA (http://gepia.cancer-pku.cn/index.html)(Fig.S3D). Thus, we focused on the role of EIF4E in the promotion of malignancy in ESCC. Transwell assays were performed to investigate whether the cell migration and invasion ability was influenced by EIF4E.The downregulation of EIF4E with siRNA inhibited both the migration and invasion capacities of the cell lines (Fig. 6D). After EIF4E overexpression, the migration and invasion ability of both cell lines was dramatically increased (Fig. 6E). We next conducted a rescue experiment by reintroducing EIF4E expression in stable MTA2-silenced KYSE30 cells. As expected, the inhibition of the migration and invasion that was caused by MTA2 knockdown was abrogated as a result of the forced expression of EIF4E (Fig. 6F). Therefore, our findings firstly revealed that EIF4E was a major downstream mediator of MTA2-induced metastatic activity.
MTA2 promotes epithelial-mesenchymal transition in ESCC via the regulation of EIF4E
The aforementioned results indicated that the interaction of MTA2 and EIF4E promotes ESCC metastasis. Therefore, we were interested in determining whether EIF4E was involved in the MTA2-mediated regulation of EMT in ESCC. Firstly, we examined the effect of EIF4E on EMT transformation in ESCC. The expression of E-cadherin and ZO-1 evaluated by western blotting was significantly reduced, and the expression of N-cadherin, Vimentin, MMP2 and MMP9 was elevated after EIF4E overexpression in both ESCC cell lines (Fig. 7A). Accordingly, knocking down EIF4E with siRNA demonstrated the opposite result (Fig. 7B). Furthermore, we found that the overexpression of EIF4E was able to rescue the loss of N-cadherin and Vimentin induced by MTA2 absence. In contrast, the expression of E-cadherin was decreased after EIF4E reintroduction compared with that in the MTA2 knockdown group (Fig. 7C). However, knocking down EIF4E with siRNA in both cell lines, which were ectopically overexpressing MTA2, generated the opposite result (Fig. 7D). These data indicated that the inducible expression of EIF4E mediated, at least in part, the promotion of EMT by MTA2 in ESCC.
MTA2 and EIF4E-Twist formed a positive feedback to repress E-cadherin expression
Twist, a basic helix–loop–helix (bHLH) transcription factor, which recognizes the canonical E-box (CANNTG) to regulate gene transcription, plays a critical role in metastasis. MTA2 is the vital component of proteins complex Twist/Mi2/NuRD, in which Twist is the most important transcription factor that regulated EMT [24]. In our study, the forced expression of Twist prominently enhanced the migratory and invasive capabilities of KYSE30 and KYSE510 cells (Fig. 8A). Furthermore, compared to those of the controls, cells with enhanced expression of Twist inhibited the E-cadherin and ZO-1 levels significantly, whereas N-cadherin and Vimentin exhibited the opposite trend (Fig. 8B). Thus, we were interested in determining whether Twist participates in the MTA2-mediated regulation of EMT in ESCC. Co-IP with the extraction from MTA2-overexpressing KYSE30 cells followed by immunoblotting with a Twist antibody verified that Twist was co-precipitated with MTA2 (Fig. 8C). Additionally, this interaction was confirmed with endogenously expressed proteins in KYSE30 cells (Fig. 8D). To investigate whether E-cadherin is a transcriptional target of Twist, we analyzed by bioinformatics the promoter of the E-cadherin gene and found several putative Twist consensus binding sites (Fig. 8E). Furthermore, ChIP assay with anti-Twist followed by RT-PCR and qPCR validated the occupancy of Twist at the E-cadherin promoter. Furthermore, the immuno- precipitated materials associated with the anti-Twist complex were subjected to re-ChIP using an antibody against MTA2. The results showed that MTA2 was also enriched at the promoter of E-cadherin, with IgG as a negative control (Fig. 8F). Taken together,above results all showed that MTA2 was recruited by Twist to the promoter of E-cadherin and repress its transcription.
To further investigate the effect of the association between MTA2 and Twist on the EMT in ESCC, we also reintroduced Twist to stable MTA2- silenced KYSE30 cells. The rescue experiment showed that the ectopic overexpression of Twist reversed the increase in E-cadherin and ZO-1 levels caused by MTA2 depletion, while the expression of N-cadherin and Vimentin was elevated after Twist reintroduction compared with that in the MTA2 knockdown group (Fig. 8G). We also found that compared to the controls, the expression of Twist was suppressed when the expression of EIF4E was silenced. On the contrary, the expression level of Twist was significantly increased in the EIF4E- overexpressing cells (Fig. 8H), which was consistent with previous report [26]. Collectively, these results indicated that MTA2 and EIF4E-Twist formed a positive feedback to regulate the development of ESCC.