WBP2 Negatively Regulates Hippo Pathway By Competitively Binding To WWC3 With LATS1 To Promote The Malignant Phenotype Of Non-Small Cell Lung Cancer

Background: WW domain binding protein-2 (WBP2) can function as a YAP/TAZ co-activator and play a vital role in promoting breast cancer progression. However, the expression and potential molecular mechanism of WBP2 in the context of lung cancer are not fully understood. Methods: Expression and subcellular localization of WBP2 in clinical samples and in lung cancer cell lines were analyzed with respect to various clinical pathological parameters using Chi-square tests. We used a series of cell function experiments and tumor formation experiments in nude mice to verify the effect of WBP2 on tumor cell proliferation and invasion. Dual-directional regulation of WBP2 expression, immunoprecipitation, luciferase reporter assays, and quantitative PCR analyses were used to explore the regulatory mechanisms and identify associated molecular markers. Results: We determined that WBP2 was highly expressed in lung cancer specimens and cell lines and that this expression was closely related to the pTNM stage, lymph node metastasis, and poor prognosis of patients. Additionally, gain and loss of function experiments revealed that WBP2 could signicantly promote the proliferation and invasion of lung cancer cells both in vivo and in vitro. To elucidate the underlying molecular mechanism, we determined that wild-type WBP2 could competitively bind to the WW domain of WWC3 with LATS1 through its PPxY motifs to inhibit the formation of the WWC3-LATS1 complex, reduce the phosphorylation level of LATS1, and ultimately promote YAP nuclear translocation to suppress the activity of the Hippo pathway. Conclusions: From the aspect of upstream molecules of Hippo signaling, WBP2 promotes the malignant phenotype of lung cancer cells in a unique manner that is not directly dependent upon YAP, thus providing a corresponding experimental basis for the development of targeted therapeutic drugs for lung cancer. and strong positive (+ + - + + +) in adenocarcinoma (A-d) squamous (A-f) with lymph node metastasis. Magnication: 400×, scale bar: 50 μm. C. D: Western blot analysis revealed that the expression level of WBP2 in lung cancer tissue was signicantly higher than that in adjacent normal lung tissue. GAPDH is used as the loading control. E. F: Kaplan¬–Meier plotter network database analysis indicated that the overall survival (OS, E) and progression free survival (FP, F) of lung cancer patients with high expression of WBP2 were signicantly lower than those of patients with low expression of WBP2. Western blot of expression of WBP2 in the human immortalized bronchial epithelial cell line and in a of non-small cell cancer cell was with YAP was used as a stimulus. TK was used as the internal reference. phosphorylation and YAP phosphorylation through an MST-independent pathway. After overexpression and knockdown of WBP2 in H1299 and A549 cells, respectively, the phosphorylation and total amount of key proteins in the Hippo pathway were analyzed by western blotting. GAPDH was used as the loading control. C (a-b). The results of RT-qPCR analysis demonstrated that the mRNA levels of CTGF and CYR61 were upregulated after WBP2 overexpression (C-a), and the mRNA levels of these two target genes were downregulated (C-b) after silencing of WBP2. P < 0.05 indicates statistical signicance, * P < 0.05, ** P < 0.01. D. Immunouorescence assay results revealed that the level of YAP nuclear translocation increased in response to overexpression of WBP2 in H1299 cells.


Background
The Hippo pathway was initially identi ed in Drosophila, and this pathway is highly conserved in the evolution of species, where it plays a crucial role in maintaining homeostasis and regulating cell proliferation, differentiation, apoptosis, and other physiological processes. Increasing numbers of studies have con rmed that disorders in Hippo activity lead to tumor formation and progression [1,2]. In the classical Hippo pathway, the molecular composition usually includes upstream molecules, such as Fatcadherin, Merlin, WWCs, and AMOT protein, a central kinase complex (MST1/2-SAV-LATS1/2-MOB), and downstream effective molecules (YAP protein and its homolog TAZ). The target genes of the Hippo pathway typically include CTGF, CYR61, and other related genes. When the Hippo pathway is activated, the upstream molecules induce cascade phosphorylation of the central kinase MST-LATS complex. Increases in LATS phosphorylation levels can promote the phosphorylation of YAP, and this protein remains in the cytoplasm, binds to the 14-3-3 protein, and is then degraded by the ubiquitin proteasome pathway. However, when the Hippo pathway is inhibited, YAP escapes from the protease and accumulates within the cytoplasm, after which, YAP translocates into the nucleus to bind to the transcription factor TEADs to activate the transcription of Hippo target genes [3][4][5].
WW domain binding protein-2 (WBP2) was initially identi ed as a homologous ligand protein of the YAP-WW domain and was demonstrated to interact with the pair box gene (PAX8) transcription factor with unknown function [6][7][8][9]. Currently, a number of studies have focused on the molecular function of WBP2 in human solid tumors by assessing the impact of this protein on the biological phenotype of breast cancer. For example, in breast cancer, WBP2 is highly expressed and is signi cantly associated with poor prognosis [10,11]. WBP2 can bind to estrogen and progesterone receptors and can act as a co-activator of estrogen receptor (ER) and progesterone receptor (PR) hormone signaling transduction [12][13][14][15]. Additionally, the synergetic effect of WBP2 and YAP/TAZ in the nucleus plays an important role in promoting the proliferation and invasion of breast cancer cells by regulating the activities of classical signal pathways in both tumor tissues and normal tissues [16][17][18][19][20]. However, the expression pattern of WBP2 in lung cancer and the ability of this protein to regulate the activity of the Hippo pathway in an indirect manner that is dependent upon YAP both remain unreported.
WW and C2 domain-containing protein-3 (WWC3) belongs to the WWC family (KIBRA/WWC1, WWC2, and WWC3) that serves as the classic upstream protein molecules in the Hippo pathway. Joachim et al.
observed that WWCs could activate Hippo pathway activity by binding to the PPxY motif of LATS kinase via the WW domain to promote LATS1 auto-phosphorylation [21,22]. Our previous studies further demonstrated that WWC3 also regulates the activities of both the Hippo and Wnt pathways primarily through interactions with LATS and DVLs that is facilitated by the WW domain and that WWC3 plays an important role in inhibiting the malignant phenotype of lung cancer [23,24]. Based on literature reviews and the results of our previous studies, we speculate that the PPxY motif within the structure of WBP2 may also bind to the WW domain of WWC3 to regulate the activity of Hippo pathways at the upstream level through a non-direct YAP/TAZ-dependent pathway.
In this study, we detected the expression and subcellular localization of WBP2 in clinical samples and in lung cancer cell lines and then analyzed the relationship of this expression to various clinical pathological parameters. Second, we veri ed the effect of WBP2 on tumor cell proliferation and invasion using a series of cell function experiments and tumor formation experiments in nude mice. Dualdirectional regulation of WBP2 expression, immunoprecipitation, luciferase reporter assays, and quantitative PCR analyses were applied to explore the speci c regulatory mechanism of WBP2 in the context of the Hippo pathway, and this provided an experimental basis for the identi cation of molecular markers of lung cancer and the development of targeted therapeutic drugs.

Patient information and specimens
This study was approved by the ethics committee of China Medical University and was performed according to the requirements of the Declaration of Helsinki. All lung cancer patients who participated in this study were aware of the study and signed an informed consent form. A total of 127 lung cancer specimens and 32 normal lung tissue samples were collected from the Department of Pathology of the First A liated Clinical Hospital of China Medical University. None of the patients received chemotherapy or radiotherapy prior to the operation. There were 79 men and 48 women included in this study, and the average age of the participants was 60 years (range, 45-83 years). According to the 2015 lung cancer classi cation standard [25], 99 cases were classi ed as stage I-II and 28 cases were classi ed as stage III. Among the 127 lung cancer patients examined, complete follow-up data were obtained for 93 patients.

Immunohistochemistry
All tissues were xed using neutral formaldehyde, embedded in para n, and then sectioned to 4 µm thickness. S-P immunohistochemical staining was used. Slides were incubated with polyclonal rabbitderived WBP2 antibody (HPA065682, 1:50, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. Phosphate buffered-saline (PBS) was used as a negative control. After incubation at 37 °C for 30 min, the sections were DAB stained. Five visual elds were randomly selected from each tissue section, and 100 tumor cells were counted in each eld. According to the percentage of cells stained, the expression of WBP2 was divided into ve grades that included 0 (no staining), 1 (1-25%), 2 (26-50%), 3 (51-75%), and 4 (more than 75%). According to the intensity of cell staining, the expression of WBP2 was divided into three grades that included 0 (no staining), 1 (light yellow particles), and 2 (dark yellow or yellow brown particles). Each tissue section corresponds to a percentage fraction and a color fraction. When these two parameters are multiplied, the product is the nal score of the slice. We evaluated the expression of WBP2 in normal lung tissue located adjacent to the tumor using this scoring standard. We observed that the expression intensity of WBP2 in most cases was greater than 4. Therefore, we de ned expression of WBP2 that was less than 4 as negative expression (-), while 4-5 was considered weakly positive (+), 6-7 was moderately positive (++), and ≥ 7 was strongly positive (+++).

Cell lines
The immortalized human bronchial epithelial cell line (HBE) was purchased from the American Type Culture Collection (
Approximately 3000 transfected cells were cultured in 10% serum in a 96-well plate, and 20 µL of 5 mg/mL MTT (thiazolyl blue) solution was added to each well. After incubation at 37 °C for 4 h, the solution was removed and the resulting MTT crystals were dissolved in 150 µL of DMSO. The absorbance peak at 490 nm was detected using a spectrophotometer.

Cell migration and matrix invasion assays
For the matrix invasion assay, the matrix adhesive (BD Biosciences) was diluted in the 24-well plate at a 1:3 ratio in DMEM medium that was free of fetal serum and antibiotics. The lung cancer cells were inoculated in the upper chamber at a density of 5 × 10 5 cells in 100 µL of medium without fetal serum, and culture medium containing 10% fetal serum (Hyclone) was placed in the lower chamber. After 24 h of cultivation, cells were xed for 15 min using methanol, and hematoxylin staining was subsequently performed. Ten elds were randomly selected to count the number of invading cells. The experiment was repeated in triplicate, and the average value was acquired accordingly.

Colony formation experiment
The cells were transfected for 48 h and then inoculated into a 6-cm cell culture dish (1000 cells/plate) and incubated for 12 days. The cells were then washed with PBS (three times for ve minutes) and subsequently stained using hematoxylin for 10 minutes before counts were performed; the experiments were repeated in triplicate to acquire an average value.

Co-immunoprecipitation
The cells were washed twice with 5 mL of PBS and then placed into an ice bath where they were lysed for 2 min using NP-40 buffer (Beyotime Biotechnology, Shanghai, China). Lysates were transferred from the culture plate to a new 1.5 ml Eppendorf tube. The supernatant was centrifuged for 15 min at 16,000 g and 4 °C. Protein concentrations were quanti ed using the BCA method (Beyotime Biotechnology), and the immune complex was collected for western blot analysis.

RNA extraction and quantitative PCR
The RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) was used to extract total RNA from the cells. Quantitative PCR was performed using an ABI-7900 system (Applied Biosystems, Foster City, CA) with 20 µL SYBR Green PCR Master Mix (Takara, Tokyo, Japan). The PCR parameters were 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. GAPDH mRNA is used to normalize the relative expression of the target mRNAs. A dissociation step was applied to generate a melting curve to con rm primer speci city. The relative levels of gene expression were represented as ΔCT = CT gene -CT reference , and the fold change in gene expression was calculated using the 2 −ΔΔCT method. The experiments were repeated in triplicates. The primer sequences used for this study are listed in Table 1. 5′-CAAGAGCGCATGTTGAAGGAA-3′ GST-pulldown The WWC3 protein coupled to a GST label was induced in E. coli BL21 (30 °C, 3 h, 200 rpm/min) and puri ed according to standard steps. The puri ed protein was recombined with glutathione sepharose (GE Healthcare, Waukesha, WI, USA) magnetic beads and then incubated with H1299 cell lysate transfected with Myc-WBP2 plasmid at 4 °C overnight. Finally, the complexes were detected by western blot and Coomassie brilliant blue staining.

Dual-luciferase reporter genes assay
Cells were co-transfected with either WBP2 plasmid or siRNA-WBP2 and with the YAP-associated transcription factor TEAD reporter pGL3b_8xGTIIC-luciferase (#34615, Addgene) in 24-well plates. Renilla luciferase was used as a signal normalization control. The assays were performed according to the manufacturer's protocol (Promega). Six independent transfections were performed for each experiment. The data were normalized to those for the empty vector control and are presented as average ± S.D.

Animal experiments
For the subcutaneous tumor formation experiment in nude mice, a total of 12 BALB/c nude mice (4 weeks old, female, 16-20 g, Speci c Pathogen Free, SPF-standard) were purchased from Beijing Charles River company (Beijing, China). The food and drinking water were sterilized using a semi-barrier system at constant temperature and humidity. All animal experiments were performed in accordance with the ethical regulations of animal experiments at China Medical University. The tumor formation experiment was divided into four groups (three rats per group) that included H1299 cells stably transfected with empty plasmid, H1299 cells stably expressing WBP2, A549 cells stably transfected with shRNA-WBP2, and cells stably transfected with control shRNA. The cell concentration for each group was adjusted to 5 × 10 6 cells/mL, and 0.2 mL of cells was injected subcutaneously into the backs of nude mice. After 4 weeks of observation beginning from the day of injection, the mice were euthanized, and the weights and volumes of the subcutaneous tumors were recorded accordingly.
For the intrapulmonary metastasis experiment, the mice were divided into four groups (eight rats per group) that were the same as the groups used for the subcutaneous tumor formation experiment. The numbers and sizes of the metastatic foci in the lungs were recorded. After six weeks, the lungs were removed and xed using 10% neutral formalin. Para n-embedded sections were prepared and stained with hematoxylin and eosin (H&E).

Statistical analysis
All data were analyzed using SPSS22.0. Chi-square tests were used to test the correlation between WBP2 expression and clinicopathological factors. The student's t-test was used to analyze differences between groups. A P < 0.05 was considered as statistically signi cant.

WBP2 is highly expressed in NSCLC and is associated with poor prognosis
To explore if WBP2 plays a speci c role in NSCLC, we rst 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 signi cant (P < 0.05, Fig. 1-B). Notable, the expression of WBP2 in patients with lymph node metastasis was signi cantly 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 signi cantly 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 progressionfree survival (FP) (P = 0.0066, and P = 0.015, respectively; Fig. 1-E, F); our Kaplan-Meier survival analysis also veri ed 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 immuno uorescence staining revealed that WBP2 was located in the cytoplasm of lung cancer cells (Supplementary Figure S2). This nding 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.  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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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 rst 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 signi cantly upregulate YAP-induced transcriptional activity of the TEAD reporter assay. Speci cally, 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 signi cantly downregulated in response to WBP2 overexpression; however, the phosphorylation levels and total amounts of MST did not exhibit any signi cant 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 signi cantly 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 identi ed 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 con rmed 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 e ciency. Additionally, immunoprecipitation studies indicated that WBP2 was bound to the WW domain of WWC3 through its PPxY motifs (Fig. 5D). 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 signi cantly 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 signi cantly 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.

Discussion
Currently, increasing numbers of studies have reported the carcinogenic effect of WBP2 in human solid tumors, particularly from breast cancer. For example, WBP2 can promote G1/S phase transformation of breast cancer cells and can accelerate cell cycle progression [27]. WBP2 can also promote the proliferation and invasion of triple negative and non-triple negative breast cancer cells through ER/PRdependent or independent mechanisms, and adverse prognosis for breast cancer patients and their treatment sensitivity to chemotherapy drugs have also been closely correlated to WBP2 status [11-15, 28, 29]. Additionally, WBP2 can interact with α-enolase (ENO-1) and Homer protein homolog-3 (homer-3) in glioma and can promote the malignant phenotype of glioma cells through the ENO-1-PI3K-AKT signaling pathway and the glycolysis pathway [30]. However, the expression of WBP2 in NSCLC and the potential molecular mechanisms of action for WBP2 have not been reported.
In this study, we found that WBP2 was highly expressed in NSCLC and was associated with poor prognosis of patients. Moreover, WBP2 promoted the proliferation, migration, and metastasis of lung cancer cells in vivo and in vitro. This was consistent with the basic function of WBP2 in breast cancer and other malignant solid tumors. However, the cause of high expression of WBP2 in lung cancer remains unclear. According to the existing literature, the expression of WBP2 could be affected by many underlying factors. In hepatocellular carcinoma, micro non-coding RNA (miRNA) can affect the malignant phenotype of hepatocellular carcinoma cells by downregulating the expression of WBP2 [31]. The transcription factor USF1 can promote the transcription of WBP2 by binding to the E-box region of the WBP2 promoter [32]. ITCH also attenuated CD4 T-cell proliferation in mice by limiting WBP2 protein stability [33]. Additionally, changes in the activity of some classic signaling pathways can play an important role in regulating the expression of WBP2. For example, MST in the Hippo pathway can regulate the expression of WBP2 through the miRNA-Dicer pathway in a kinase-dependent manner [34].
Wnt pathway activation inhibits the degradation of WBP2 mediated by Itch in a post-translational manner, thus affecting the biological phenotype of breast cancer [18]. These experimental data have The molecular mechanism underlying the function of WBP2 in breast cancer is closely related to the steroid hormone signaling pathway (ER-based). Some scholars have determined that WBP2 is also the key node of the PI3K and Wnt signaling transduction pathways in other tumors [14,18,29], and WBP2 also plays a key role in the Hippo pathway [34]. Both Yki/YAP and TAZ in the Hippo signaling pathway are vital binding proteins of WBP2, as the binding of these proteins facilitates the transcription of downstream target genes and affects the development of Drosophila and the proliferation and invasion of tumor cells [8-9, 16, 19-20]. In NSCLC, inactivation of the Hippo pathway is also a common phenomenon that is closely related to the occurrence, development, and drug resistance of lung cancer [35]. Therefore, we also focused on exploring if the potential molecular mechanism of WBP2 in lung cancer is related to Hippo signaling.
First, we used luciferase reporter gene assays and RT-qPCR to verify that WBP2 could promote YAP translocation into the nucleus to upregulate YAP/TEAD transcriptional activity and promote the transcription of the target genes CTGF and CYR61. This dual-directional regulation of WBP2 expression con rmed the negative regulatory effect of WBP2 on the Hippo pathway in lung cancer cells. However, interestingly, we found that WBP2 was localized within the cytoplasm of lung cancer cells according to immunohistochemical staining and cell uorescence, which is in contrast to previous reports that suggested that WBP2 was a co-activator of YAP/TEAD in the nucleus. Thus, WBP2 may modulate Hippo in an indirect YAP/TAZ-dependent manner in the cytoplasm by facilitating upstream molecular processes.
When investigating this hypothesis, we used immunoprecipitation and GST-pulldown assays to show that WBP2 could directly bind via the PPxY motifs to the WW domain of WWC3 upstream of the Hippo pathway. WWC3 promotes the autophosphorylation of LATS1 via the MST-independent pathway, activates the Hippo pathway, and inhibits the proliferation and migration of lung cancer cells [24] and promotes Earle's balanced salt solution (EBSS)-induced apoptosis and inhibits cell growth by inhibiting the autophagy process [36]. Furthermore, WWC3 acts as a tumor suppressor in other tumors such as gastric cancer and glioma [37,38]. We found that WBP2 competitively binds to the WW domain of WWC3 with LATS1 to inhibit the formation of the WWC3-LATS1 complex and reduces the phosphorylation level of LATS1 to promote YAP translocation into the nucleus. This inhibits the Hippo pathway and ultimately promotes the malignant phenotype of lung cancer. Therefore, WBP2 regulates Hippo activity in a manner that is not directly dependent on YAP at the upstream level, and this explains the speci c molecular mechanism of the regulation of the Hippo pathway by WBP2 that is localized to the cytoplasm. In regard to the differences in subcellular localization of WBP2 in breast cancer and lung cancer, future research will determine if these differences are tissue-speci c or regulated by other factors.

Conclusions
WBP2 is highly expressed in NSCLC and is closely related to tumor stage, lymph node metastasis, and poor prognosis in patients.  was used as a loading control. P < 0.05 indicated statistical signi cance, and ** represented P < 0.001.

Figure 3
Knockdown of WBP2 weakens the proliferation, invasion, and metastasis abilities of lung cancer cells.  with YAP was used as a stimulus. TK was used as the internal reference. B. WBP2 down-regulated LATS1 phosphorylation and YAP phosphorylation through an MST-independent pathway. After overexpression and knockdown of WBP2 in H1299 and A549 cells, respectively, the phosphorylation and total amount of key proteins in the Hippo pathway were analyzed by western blotting. GAPDH was used as the loading control. C (a-b). The results of RT-qPCR analysis demonstrated that the mRNA levels of CTGF and CYR61 were upregulated after WBP2 overexpression (C-a), and the mRNA levels of these two target genes were downregulated (C-b) after silencing of WBP2. P < 0.05 indicates statistical signi cance, * P < 0.05, ** P < 0.01. D. Immuno uorescence assay results revealed that the level of YAP nuclear translocation increased in response to overexpression of WBP2 in H1299 cells. WBP2 interacts with the WW domain of WWC3 via PPxY motifs. A. GFP-WWC3 was transfected into H1299, and after 48 h, the cells were collected and lysed. The GFP monoclonal antibody was used for pull down. The presence of WBP2 in the precipitate was detected by western blot analysis using aWBP2 antibody. B. After incubation with puri ed GST or GST coupled-WWC3 protein for 6 h at 4 °C, the binding status of the two proteins was examined using Coomassie brilliant blue staining and western blot WBP2 and LATS1 competitively bind to WWC3 to inhibit Hippo pathway activity. A. Overexpression of WBP2 attenuated the binding between WWC3 and LATS1. In the H1299 cell line, the WBP2 plasmid (1 μg and 2.5 μg) was transfected in a dose gradient manner. After 48 h, the cells were collected and lysed. GFP monoclonal antibody was used for immunoprecipitation. The combination change of WWC3 and LATS1 protein in the sediment was detected according to western blot. B. The binding of WWC3 to LATS1 increased after WBP2 knockdown. siRNA-WBP2 (5 pmol and 10 pmol) was transfected into A549 cells with high expression of WBP2. After cell collection, the binding was detected according to anti-FLAG immunoprecipitation followed by anti-WWC3immunoblotting. C, D: Conversely, after LATS1 was transfected into A549 cells with low LATS1 expression, the binding of WBP2 to WWC3 gradually decreased with an increase of LATS1 (C). In contrast, the binding of WBP2 to WWC3 gradually increased with the downregulation of LATS1 after siRNA-LATS1 was transfected into H1299 cells with high LATS1 expression (D). E, F: After 48 h post transfection with WBP2 plasmid or siRNA-WBP2 into H1299 cells and A549 cells, the cells were collected and lysed. The results of the dual-luciferase reporter assay revealed that WBP2 overexpression reversed the decrease in TEAD transcriptional activity caused by WWC3 (E).
Conversely, WBP2 knockdown further enhanced the decrease in TEAD transcriptional activity caused by  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