3.1 NSE expression was positively correlated with distant metastasis of SCLC.
In order to clarify the biological function of NSE in SCLC, we firstly collected clinical data of 138 cases of SCLC patients, and defined serum NSE concentrations below 25 ng/ml as "low-NSE", while serum NSE concentrations above 25 ng/ml as "high-NSE". Univariate and multivariate analysis results showed that the serum concentration of NSE was significantly correlated with the prognosis of SCLC patients. Moreover, NSE can be used as an independent indicator of the prognosis for SCLC patients (P < 0.05, Fig. 1A). In addition, the serum concentration of NSE was significantly increased in SCLC with advanced stage and distant metastasis (P < 0.05, Fig. 1B-F). The results of immunohistochemistry also showed that the expression level of NSE was higher in SCLC patients with distant metastasis than patients without distant metastasis (Fig. 1G).
3.2 NSE promoted SCLC cell migration and invasion.
To further clarify the phenomenon that NSE promoting the distant metastasis of SCLC, we conducted a series of cell functional experiments to explore the influence of NSE on the migration and invasion of SCLC cells. The differences in the expression of NSE was detected in 3 SCLC cell lines (H446, H69 and H209). The results showed that the expression level of NSE was the lowest in H446 cells and the highest in H69 cells (Fig. 2A). Therefore, H446 and H69 cell lines were choosed for further experiments.
Flag-NSE lentivirus was used to infect H446 cells to establish NSE stably overexpressed cell lines, and Flag-EV was used as an empty control. PLKO-sh1 NSE and PLKO-sh2 NSE lentivirus was used to infect H69 cell lines to establish NSE stablely knockdown cells, PLKO-NTC was used as negative control cells. Western blot and qRT-PCR results showed that the protein and mRNA expression levels of NSE after stably overexpressed or knocked down (P < 0.05, Fig. 2B-D).
Next, a wound healing assay was conducted to test the migration capability. The healing ability of H446-NSE cells was stronger than that of H446-EV cells (P < 0.05, Fig. 2E). Moreover, Transwell migration assay also showed that the number of H446-NSE cells that migrated from the upper chamber to the lower chamber was more than the number of H446-EV cells, while the number of H69-sh1 NSE or H69-sh2 NSE cells that migrated from the upper chamber to the lower chamber was significantly less than the number of H69-NTC cells (P < 0.05, Fig. 2F, G). In addition, Matrigel Invasion Assay was performed to test cell invasion ability. The number of H446-NSE cells invading from the upper chamber to the lower chamber was significantly greater than the number of H446-EV cells (P < 0.05, Fig. 2F). These results indicated that NSE could enhance the migration and invasion capabilities of SCLC cells.
3.3 NSE induced EMT process of SCLC cells.
In order to clarify whether NSE enhanced the migration and invasion ability of SCLC cells by promoting EMT, we tested the expression levels of EMT-related markers. Western blot results showed that compared with H446-EV cells, the expression levels of Snail and N-cadherin were upregulated significantly, while the expression level of E-cadherin was downregulated significantly (P < 0.05, Fig. 3A). qRT-PCR analyses were consistent with the above results (P < 0.05, Fig. 3B). Moreover, compared with H69-NTC cells, the expression levels of Snail and N-cadherin decreased significantly in H69-sh1 NSE and H69-sh2 NSE cells, and the expression level of E-cadherin increased significantly (P < 0.05, Fig. 3C). And the results of qRT-PCR were consistent with the above results (P < 0.05, Fig. 3D). These findings indicated that NSE could promote EMT of SCLC cells.
3.4 Wnt/β-catenin signaling pathway was activated in NSE-overexpressing SCLC cells.
To explore the mechanism of NSE in promoting distant metastasis, we performed GSEA to identify the potential signaling pathway. There were several pathways enriched in high NSE groups, among them the wnt/β-catenin pathway was mostly related to metastasis (Fig. 4A). Based on this, we further studied whether this pathway played a key role in NSE-mediated metastasis. The expression of β-catenin and its downstream target genes were detected at protein level and mRNA level. As shown in Fig. 4B, the expression levels of β-catenin, C-myc, and Slug were significantly higher in H446-NSE cells than those in H446-EV cells (P < 0.05, Fig. 4B). And the mRNA expression levels of C-myc and Slug were consistent with the western blot results (P < 0.05, Fig. 4C). Similarly, the expression levels of β-catenin, C-myc, Slug were much lower in H69-sh1 NSE and H69-sh2 NSE cells than those in H69-NTC cells (P < 0.05, Fig. 4D). The mRNA expression levels of C-myc and Slug were consistent with the western blot results (P < 0.05, Fig. 4E). However, modulation of NSE failed to change the mRNA expression level of β-catenin (Fig. 4C, E). These findings indicated that NSE could activate the wnt/β-catenin signaling pathway. Moreover, NSE may only change the protein level of β-catenin but not its mRNA expression level.
3.5 β-catenin played a key role in NSE promoting metastasis.
To further verify that NSE promotes the EMT, migration and invasion of SCLC cells by activating the Wnt/β-catenin signaling pathway, we conducted the following replenishment experiments.
We used PLKO-NTC and PLKO-shβ-catenin lentivirus to infect H446-EV and H446-NSE cells to establish stable transgenic cells (H446-NSE + shβ-catenin, H446-NSE + NTC, H446-EV + NTC). Similarly, H69-NTC and H69-sh1 NSE cells were infected with Flag-EV and Flag-β-catenin lentivirus to establish stably transfected cells (H69-sh NSE + β-catenin, H69-shNSE + EV, H69-NTC + EV).
As shown in Fig. 5A and B, when β-catenin was suppressed by shRNA, the effect that overexpression of NSE activated the wnt/β-catenin pathway and upregulated the expression EMT process was eliminated (P < 0.05). As expected, when β-catenin was overexpressed, the effect that knockdown NSE inhibited the wnt/β-catenin pathway and downregulated the expression EMT process was abolished (P < 0.05, Fig. 5C, D). Furthermore, knocking down β-catenin abolished the enhancing migration and invasion of overexpression NSE in H446 cell (P < 0.05, Fig. 5E, F). Consistently, overexpression β-catenin eliminated the attenuating migration of knockdown NSE in H69 cell (P < 0.05, Fig. 5G).
In summary, these findings indicated that β-catenin played an essential role in NSE's promotion of EMT, migration, and invasion of SCLC cells.
3.6 NSE interacted with β-catenin and inhibited its degradation.
As mentioned earlier, NSE mainly regulates the protein level of β-catenin. We verified the interaction between NSE and β-catenin through CO-IP experiments (Fig. 6A, B). Moreover, overexpression of NSE can inhibited the degradation of β-catenin in SCLC cells (P < 0.05, Fig. 6C). This indicated that NSE inhibited β-catenin degradation by interacting with β-catenin, thereby activating the Wnt/β-catenin pathway.
3.7 NSE promoted EMT process and tumor metastasis by activating the wnt/β-catenin pathway in vivo.
Our above studies have confirmed that NSE can promote the migration, invasion and EMT process of SCLC cells by activating Wnt/β-catenin pathway in vitro. Therefore, we constructed a metastasis mouse model through cardiac injection to verify whether NSE can promoted EMT process and distant metastasis of SCLC cells by activating the wnt/β-catenin pathway in vivo. As shown in Fig. 7A, the distant metastasis rate was higher in H446-NSE group, compared with H446-EV group. The distant metastasis rate was lower in H69-sh1NSE and H69-sh2 group, compared with H69-NTC group (Fig. 7B). Meanwhile, overexpression NSE accelerated body weight loss (Fig. 7C) and shown disbenefit in animal survival (Fig. 7E), while knockdown NSE decelerated body weight loss (Fig. 7D) and shown benefit in animal survival (Fig. 7F). In addition, extracting RNA from nude mice tumor, the qRT-PCR analyses shown that Wnt/β-catenin pathway was activated and promoted EMT process in NSE-overexpressing H446 cells (Fig. 7G), while knockdown NSE suppressed these effects (Fig. 7H).
Taken together, our findings indicated that NSE could promote EMT process and tumor distant metastasis of SCLC cells by activating Wnt/β-catenin pathway in vivo.