WBP2 is highly expressed in NSCLC and is associated with poor prognosis
To explore if WBP2 plays a specific role in NSCLC, we first detected the expression of WBP2 in samples of lung tissue from lung cancer patients and investigated the association of this expression with the survival and prognosis of patients with lung cancer via immunohistochemistry staining. Our results indicated that in terms of localization, WBP2 was localized in the cytoplasm of lung cancer cells, whereas with regard to expression, WBP2 was poorly or even negatively expressed in normal cells (71.9%, 23/32) (low expression in normal bronchial epithelial cells, negative expression in normal alveolar epithelium) but highly expressed in lung adenocarcinoma and squamous cell carcinoma (55.2%, 70/127, Fig. 1-A) Additionally, the expression difference between para-cancerous and cancerous tissues was significant (P < 0.05, Fig. 1-B). Notable, the expression of WBP2 in patients with lymph node metastasis was significantly higher than that observed in patients without lymph node metastasis (44.7% vs. 70.6%, P < 0.05, Fig. 1-A, B). The chi-square test revealed that high expression of WBP2 was closely related to advanced pTNM stage (P = 0.001) and positive lymph node metastasis (P = 0.006) in patients with NSCLC (Table 2). Cox univariate and multivariate analyses indicated that a high TNM stage, adenocarcinoma histological type, and high WBP2 expression (P = 0.038, P = 0.017 and P = 0.030, respectively; Table 3) were all independent prognostic factors in NSCLC. Accordingly, western blot analyses showed that the expression level of WBP2 in lung cancer tissues was also significantly higher than that in adjacent tissues (14/16, Fig. 1-C, D). The online network database (http://www.kmplot.com) suggested that high WBP2 expression negatively correlated with overall survival (OS) and progression-free survival (FP) (P = 0.0066, and P = 0.015, respectively; Fig. 1-E, F); our Kaplan–Meier survival analysis also verified these results (P < 0.05, Supplementary Figure S1). When the lung cancer cell lines were compared to the normal bronchial epithelial cell line HBE, we determined that WBP2 was also highly expressed in the four lung cancer cell lines (n = 5), and immunofluorescence staining revealed that WBP2 was located in the cytoplasm of lung cancer cells (Supplementary Figure S2). This finding was consistent with the results obtained using clinical tissue specimens. Based on the above results, we can conclude that WBP2 may play a role in promoting malignancy by functioning as an oncogene.
Table 2
Association of WBP2 expression with clinical and pathological characteristics in NSCLC
Clinicopathological | N | Positive | Negative | χ2 | P(two-side) |
characteristics |
Age (years) | |
<60 | 37 | 25 | 12 | 3.271 | 0.080 |
≥60 | 90 | 45 | 45 |
Gender | |
Male | 79 | 43 | 36 | 0.040 | 0.856 |
Female | 48 | 27 | 21 |
Histological type | |
Squamous cell carcinoma | 54 | 32 | 22 | 0.651 | 0.473 |
Adenocarcinoma | 73 | 38 | 35 |
Differentiation | |
Well | 40 | 22 | 18 | 0.000 | 1.000 |
Moderate & Poor | 87 | 48 | 39 |
TNM classification | |
Ⅰ + Ⅱ | 99 | 47 | 52 | 10.604 | 0.001* |
Ⅲ | 28 | 23 | 5 |
Lymph node metastasis | |
Positive | 51 | 36 | 15 | 8.245 | 0.006* |
Negative | 76 | 34 | 42 |
*: P < 0.05, statistically significant |
Table 3
Summary of Cox univariate and multivariate regression analysis of the association between clinicopathological characteristics and overall survival in 93 cases of non-small cell lung cancer
Clinicopathological | Hazard ratio | P |
characteristics | (95% CI) | |
Univariate analysis | | |
Age older than 60 | 0.534 (0.286–1.032) | 0.062 |
Gender: male | 0.973 (0.515–1.839) | 0.934 |
Histological type: Adenocarcinoma | 0.450 (0.237–0.855) | 0.015* |
Poor differentiation | 1.906 (0.968–3.754) | 0.062 |
High TNM classification | 3.768 (1.908–7.444) | 0.000* |
Positive lymph node metastasis | 2.713 (1.445–5.096) | 0.002* |
Positive WBP2 expression | 3.274 (1.599–6.701) | 0.001* |
Multivariate analysis | | |
Histological type : Adenocarcinoma | 0.431 (0.216–0.860) | 0.017* |
High TNM classification | 2.756 (1.058–7.179) | 0.038* |
Positive WBP2 expression | 2.294 (1.082–4.867) | 0.030* |
Ectopic expression of WBP2 promotes the proliferation, migration, and invasion of NSCLC cells both in vitro and in vivo
As numerous studies have indicated that WBP2 plays an essential role in promoting tumor progression in breast cancer, we attempted to explore if WBP2 can exert an underlying impact on the malignant phenotype of tumor cells in NSCLC. As shown in Figure-1E, WBP2 expression was the highest in A549 cells lines but was relatively low in H1299 cells. Therefore, we selected the H1299 cell line for the overexpression experiments. The colony formation assay showed that in comparison with that of the control group, the proliferative abilities of H1299 cells were significantly enhanced after stable transfection of WBP2 (Fig. 2-A). Additionally, we found that WBP2 overexpression promoted the migration and invasiveness of lung cancer cells (Fig. 2-B, C). These experimental results indicated that WBP2 possessed the ability to promote the malignant phenotype of tumors in vitro. To further verify if WBP2 exerts similar effects in vivo, we performed subcutaneous tumor transplantation experiments and lung metastasis experiments based on tail vein injections in nude mice, and found that in comparison with those of control group, the volumes and weights of the subcutaneous tumors (Fig. 2-D,E) and the number of metastatic foci (Fig. 2-F,G) in the lungs of mice stably overexpressing WBP2 were significantly increased, which was consistent with the results obtained in vitro.
WBP2 knockdown weakens the malignant phenotype of lung cancer cells both in vivo and in vitro
We further attempted to detect any changes in the biological functions of the lung cancer cells after WBP2 knockdown. For these experiments, we used lentivirus-coated shRNA-WBP2 to transfect the A549 cell line with high WBP2 expression. Using colony formation, cell migration, and matrix invasion assays, we found that in contrast to the results of the functional experiments, weakened proliferative, migratory, and invasive abilities were observed in A549 cells after WBP2 knockdown (Fig. 3-A, B, C). In vivo, we found that the volumes and weights of subcutaneous transplanted tumors derived from A549 cells transfected with lentivirus-shRNA-WBP2 in nude mice were significantly lower than those derived from control mice (Fig. 3-D,E) and that the number of lung metastases induced by caudal vein metastasis was also significantly reduced (Fig. 3-F,G). Therefore, combined with the results of the in vivo and in vitro experiments, we can conclude that WBP2 functions as a tumor promoting factor when exerting its potential biological functions in lung cancer cells.
WBP2 is a negative regulator of the Hippo signaling pathway in lung cancer cells
It is currently unclear exactly how WBP2 affects the biological function of lung cancer cells. In breast cancer, WBP2 has been reported to inhibit the occurrence and development of breast cancer by inhibiting the activity of the Hippo pathway. Based on this, we first examined the effect of WBP2 on Hippo pathway activity in the context of lung cancer cells. Initially, a dual-luciferase reporter assay was used to demonstrate that WBP2 overexpression in H1299 could significantly upregulate YAP-induced transcriptional activity of the TEAD reporter assay. Specifically, the activity of the Hippo pathway was inhibited (Fig. 4A-a). In contrast, the transcriptional activity of the TEAD reporter gene was downregulated by siRNA-WBP2 transfection in A549, indicating that the Hippo pathway was activated (Fig. 4A-b). Cascade phosphorylation of the MST-LATS complex in the Hippo pathway plays a central role in classical Hippo activation. However, the impact of WBP2 on MST and LATS phosphorylation has not been previously elucidated. To address this, we performed western blot analyses to explore the effect of WBP2 on the phosphorylation of these two kinases. The results indicated that the phosphorylation levels of LATS1 and YAP were significantly downregulated in response to WBP2 overexpression; however, the phosphorylation levels and total amounts of MST did not exhibit any significant changes (Fig. 4B), suggesting that WBP2 affected the phosphorylation of LATS1 and regulated the activity of the Hippo pathway in an MST-independent manner. We further assessed the changes in the transcription levels of CTGF and CYR61 in Hippo pathway target genes using RT-qPCR. We found that the transcription levels of CTGF and CYR61 were significantly upregulated after transfection with WBP2 (Fig. 4C-a), and the results were the opposite in response to silencing of WBP2 (Fig. 4C-b). The GEPIA online correlation database (gepia.cancer-pku.cn) also revealed a positive association between WBP2 and YAP target genes, including CTGF, CYR61, and AREG (Supplementary Figure S3). The import of YAP into the nucleus is a direct indicator of Hippo pathway inactivation, and laser confocal detection assays revealed that the level of YAP within the nucleus was increased after transfection with WBP2 (Fig. 4D). These results indicate that WBP2 promotes YAP translocation into the nucleus by inhibiting LATS1 phosphorylation, and this ultimately inhibits Hippo pathway activity.
The upstream protein WWC3 of the Hippo pathway was identified as WBP2 binding protein
Previous studies suggested that WBP2 localizes within the nucleus of breast cancer cells and acts as a co-activator of YAP to promote the transcription of downstream genes [17]. Interestingly, we found that WBP2 was localized within the cytoplasm of lung cancer cells, and based on this, we speculated that WBP2 may modulate the Hippo pathway in a YAP-indirect-dependent manner. This knowledge combined with our previous results indicated that WBP2 can downregulate LATS1 phosphorylation levels, and we therefore focused our studies on the upstream protein molecules that can cause changes in LATS1 phosphorylation levels. The WWCs protein family contains the classical upstream molecules of the Hippo pathway that can interact with LATS1 and promote LATS1 phosphorylation. Our previous studies also confirmed the important role of WWC3 in lung cancer [22, 23]. In this study, immune-co-precipitation and GST-pulldown assays were performed to verify that WBP2 can dramatically interact with the upstream protein WWC3, and our results indicate that these proteins can directly interact (Figure. 5A, B). Confocal laser scanning revealed the co-localization of WBP2 and WWC3 in the cytoplasm of A549 cells that possess high expression levels of both proteins (Fig. 5C). To further explore the structural basis underlying the binding of WBP2 and WWC3 [23], we constructed a series of WBP2 (deletion of PPxY motifs) and WWC3 mutants (deletion of double WW domains) and then transfected them into H1299 cells, as these cells exhibited high transfection efficiency. Additionally, immunoprecipitation studies indicated that WBP2 was bound to the WW domain of WWC3 through its PPxY motifs (Fig. 5D).
WBP2 inhibits the activity of the Hippo pathway by inhibiting the phosphorylation of LATS1 via interaction with WWC3
WWC3 can activate the Hippo pathway by binding of the WW domain in competition with LATS1 kinase to promote LATS1 phosphorylation; WBP2 can also combine with the WW domain of WWC3 via PPxY motifs. Therefore, the binding of the PPxY motifs of WBP2 to the WW domain of WWC3 led us to speculate that WBP2 may competitively bind to the WW domain of WWC3 with LATS1 to weaken the activity of the WWC3-LATS1 complex. To verify this possibility, we first increased the expression of WBP2 in H1299 cells that exhibit low WWC3 and WBP2 expression and high LATS1 expression [26]. In these cells, immunoprecipitation experiments showed that the binding of WWC3 and LATS1 gradually decreased in response to WBP2 overexpression in a dose-dependent manner (Fig. 6A). Conversely, the binding ability of LATS1 to WWC3 was dramatically increased after WBP2 knockdown in A549 cells (low expression of LATS1 and high expression of WWC3 and WBP2) (Fig. 6B). Conversely, we overexpressed LATS1 in A549 cells and found that the binding of WBP2 and WWC3 gradually decreased (Fig. 6C); however, after LATS1 was knocked down in H1299, the binding of WBP2-WWC3 gradually increased in a dose-dependent manner (Fig. 6D). These results clearly demonstrate that WBP2 competitively binds to WWC3 with LATS1. Next, we explored the underlying impact of this competitive interaction between these three factors on the Hippo pathway. First, by using a dual-luciferase reporter assay, we found that the ectopic expression of WBP2 in H1299 cells significantly rescued the decrease in YAP-TEAD transcriptional activity caused by WWC3 (Fig. 6E). However, after knockdown of WBP2 in A549 cells, the inhibition of Hippo induced by LATS1 was further promoted (Fig. 6F). In contrast, overexpression of LATS1 could dramatically reduce the increase in YAP-TEAD activity that was induced by WBP2 (Fig. 6G), and this effect was reversed after LATS1 knockdown (Fig. 6H). Accordingly, western blot analyses also revealed that WBP2 overexpression significantly reversed the upregulation of LATS1 and YAP phosphorylation that was induced by WWC3 in H1299 cells, and this effect was abrogated after WBP2 knockdown (Fig. 6I, J). Based on the above results, we believe that WBP2 is competitive with LATS1 for binding to WWC3, and this competitive binding results in a decrease in WWC3-LATS binding and eventually leads to downregulation of LATS1 phosphorylation to inhibit the activity of the Hippo pathway.