LTBP4 Inhibits the Proliferation and Metastasis in Melanoma by Activating Hippo-YAP Signaling

Background: Malignant melanoma is the deadliest of skin cancer. The present study aimed to elucidate potential key candidate genes in melanoma and its molecular mechanism. Methods: Three gene expression prole data sets (GSE46517, GSE52882 and GSE54493) were downloaded from the GEO database, which included data from melanoma tissue samples and cell lines. DEGs were subsequently investigated by GO analysis via using DAVID website. PPI network was constructed using the STRING database and visualized by Cytoscape software and MCODE were utilized to PPI network to pick out meaningful DEGs. Cell proliferation, apoptosis, migration and invasion were measured using CCK-8, colony formation, ow cytometry, transwell and wound healing assays. RT-PCR, western blotting and immunohistochemistry assays were used to detect mRNA and protein expressions. TCGAportal and GEPIA databases were used to perform the bioinformatics analysis of LTBP4 in melanoma. Results: LTBP4 both is the DEG and a key gene from the most signicant module of the PPI network. LTBP4 expression was down-regulation in melanoma tissues and cells relative to controls, which showed positive correlation with invasion, TNM stage, distal metastasis and lymph node metastasis, and predicted the poor prognosis for patients with melanoma. Cox analysis identied LTBP4 low-expression as an independent prognostic variable for overall survival (OS) in patients with melanoma. The results revealed that LTBP4 inhibition reduced cell apoptosis, promoted cell proliferation and metastasis. These changes were correlated caspase-3, ki67 and E-cadherin expressions by western blotting assay. Further in vivo tumor formation study in nude mice indicated that LTBP4 inhibition promoted the progress of tumor formation. LTBP4 gene knockout reduced the phosphorylation level of YAP, MST1 and MOB1 and promoted the nuclear translocation of YAP to inhibit the activation of Hippo signaling pathway. The functions of LTBP4 overexpression (OE) inhibiting the expressions of CTGF, Cyr61 Our results showed that OE of LTBP4 downregulated the expressions of YAP, MST1, and MOB1 and promoted the phosphorylation of YAP, MST1/2, and MOB1 in A101D and A375 cells via using western blotting (Fig. 7a and 7b). Next, the mRNA and protein levels of CTGF, Cyr61, and Birc5 in LTBP4 expressing plasmids transfected A101D and A375 cells were signicantly downregulation via using RT-PCR and western blotting assays (Fig. 7c and 7d). Furthermore, the high expression of YAP was found in cytoplasm of LTBP4 plasmids transfected A101D and A375 cells (Fig. 7e), meanwhile, the low expression of YAP was found in nucleus of LTBP4 plasmids transfected A101D and A375 cells 7f).


Background
Melanoma is a malignant tumor that originates from melanocytes in the skin and other tissues, accounting for more than 75% of all skin cancers [1]. Malignant melanoma is the most invasive skin cancer in the world, which is produced by melanocytes. As we all know, surgical resection is an effective treatment for early melanoma, but many advanced patients with melanoma can't accept surgical treatment, usually with surgery combined with adjuvant chemotherapy [2]. Chemotherapy resistance and high metastasis lead to the poor prognosis in patients with advanced melanoma [3], therefore, it is very important to study the pathogenesis of melanoma and nd effective potential targets for the diagnosis and treatment of melanoma.
Hippo signal pathway is involved in the regulation of many kinds of cell and organ homeostasis, and mainly composed of a series of kinase complexes and downstream transcriptional coactivators, including MST1/2, SAV1, LATS1/2, MOB1 and YAP/TAZ [4][5][6]. Activated Hippo signal pathway induces the binding of MST1 kinase and its junction protein SAV1, thereby activating LATS1/2 and MOB1 kinase. Activated LATS1/2 and MOB1 kinases further promote the phosphorylation of YAP protein [7].
Phosphorylated YAP cannot translocate from the cytoplasm to the nucleus, thus leaving it trapped in the cytoplasm. However, most of the phosphorylated YAP in the cytoplasm were degraded by ubiquitin. Inactivated Hippo pathway rarely could make phosphorylation to YAP protein. The unphosphorylated YAP protein enriches in the nucleus via translocating into the nucleus to promote the expression of related genes that contribute to cell proliferation and survival [8]. Therefore, the activation of Hippo pathway determines the phosphorylation level and subcellular localization of YAP protein, which also determines the role of this pathway in tumorigenesis and development.
Hippo pathway is in a dynamic balance to maintain cell proliferation, apoptosis and the stability of the internal environment of the body. It was reported that inactivated Hippo pathway decreases YAP phosphorylation and promotes YAP nuclear translocation in many kinds of tumors [9][10][11]. For example, inactivated Hippo signal pathway or YAP overexpression (OE) caused abnormal liver development and eventually leaded to the occurrence of liver cancer in mice [12]; SOX2 OE promoted the proliferation and migration of esophageal squamous cell carcinoma cells by inhibiting the activation of Hippo signal pathway, and improve the sensitivity of cells to cisplatin [13]. In addition, YAP OE promoted the proliferation and metastasis and inhibit the apoptosis in A375 cells [14]. It is suggested that Hippo signaling pathway played an important role in the occurrence and development of melanoma.

Functional enrichment analysis
The GO enrichment was used to analyze DEGs at the functional level with DAVID (http://david.ncifcrf.gov/,version 6.8). DAVID is a comprehensive database of functional annotation tools for connecting functional terms with gene lists using a clustering algorithm. In order to elucidate the functional pro les of the DEGs, we used DAVID to obtain the enriched biological process (BP), cellular component (CC), molecular function (MF) and KEGG pathway. P < 0.05 was considered statistically signi cant.

PPI network analysis
The STRING database (http://string-db.org/) was used to identify the interactions between proteins encoded by DEGs based on experimental data, databases, text mining, and predictive bioinformatics data. A combined score of > 0.4 was set as threshold value. PPI networks were constructed with Cytoscape software. The plug-in MCODE was used to screen the modules of PPI network in Cytoscape software. MCODE score ≥ 3 and number of nodes ≥ 3 were considered as threshold value.
Expression and survival analysis of IFITM1 and LTBP4 TCGAportal (http://www.tcgaportal.org) and GEPIA (http://gepia.cancer-pku.cn/) were used to investigate survival probability and the expression of IFITM1 and LTBP4 in tumor tissues and corresponding paracarcinoma tissues.

Cells and surgical specimens
Seven melanoma cell lines used in this study. A101D, 451Lu, SK-MEL-1, VMM5A, G-361, A375, and MeWo cells and control cell (HaCaT cell) were purchased from the Cell Culture Collection of the Chinese Academy of Sciences (Beijing, China) and cultured in DMEM medium containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 mg/mL streptomycin, and 100 U/mL penicillin in a humidi ed incubator containing 5% CO 2 at 37°C for 2-3 days. Specimens of melanoma tissues and adjacent noncancerous tissues were collected from 76 patients with melanoma between October 2018 and October 2019, following surgical resection at Tianjin Medical University Eye Hospital. All experiments were approved by the Medical Ethics Committee of Tianjin Medical University Eye Hospital and written informed consent documents were signed by all of the patients. Table 1

Rescue experiment
This experiment was divided into the following four groups:1) SK-MEL-1 or A375 cells without any treatment was viewed as CTRL group; 2) SK-MEL-1 or A375 cells transfected with LTBP4 OE was viewed as LTBP4-OE group; 3) SK-MEL-1 or A375 cells co-transfected with LTBP4 OE and YAP OE was viewed as LTBP4-OE+YAP-OE group; 4) SK-MEL-1 or A375 cells co-transfected with LTBP4 OE and MST1 OE was viewed as LTBP4-OE+MST1-OE group. Cell proliferation, apoptosis, migration and invasion in above groups were measured using CCK-8, colony formation, ow cytometry, transwell and wound healing assays.
Cell viability assay Cells were transfected with shRNAs or pcDNA3.1 for 48 h to detect cell viability according to CCK-8 kit instructions (Beyotime, Shanghai, China). Transfected cells (5,000 cells per well) were incubated in a cell incubator for 0.5-4 h and tested at 0, 6, 12, 24, 48 and 72 h, respectively, using a microplate reader. Absorbance was measured at 450 nm.

Colony Formation
For the colony formation assay, 600 cells per group were plated in triplicates in a six-well plate. Cells were allowed to grow till visible colonies appeared, stained with crystal violet (Beyotime), washed with PBS, and counted.

Flow cytometry analysis
For apoptosis measurements, the percentages of apoptotic cells were determined by ow cytometry using the Annexin V-FITC/PI cell apoptosis detection kit (Promega) according to the manufacturer's instructions.

Wound-healing assay
After transfection with the different plasmids for 24 h, cells were seeded in a 24-well plate and grown to con uence. The con uent monolayer of cells was wounded using a standard 200 µL pipette tip and then washed three times to remove the non-adherent cells. Wounds were monitored and photographed at the time the scrape was created and 24 h later. Cell migration capacity was calculated according to the width of the wounds at 0 h and 24 h. The migration rate is described as a percentage of the migration observed in the control group under an inverted microscope (Olympus).

Transwell invasion assays
Transwell assays were conducted to detect the invasion capabilities of cells. Cells (4×10 5 /mL) were plated within the top chamber coated with Matrigel membrane. FBS 10% was used as a chemoattractant in the lower chamber. Cells were incubated for 48 hours, and the cells that did not invade through the membrane were removed by a cotton swab. Cells on the lower chamber were then stained with crystal violet and counted under an inverted microscope (Olympus).

In vivo tumorigenicity
Animal experiments were done according to Institutional Animal Care and Use Committee (IACUC) protocol and approved by Tianjin Medical University Animal Center for Use and Care of Animals. The establishment of the subcutaneous melanoma tumor model was treated by subcutaneous injection of SK-MEL-1 cells. A total of 12 4-week-old female BALB/C nude mice (Tianjin Medical University Animal Center, Tianjin, China) were required to establish melanoma model mice for 4 weeks. Mice were sacri ced 30 days after the injection and size of the tumor was measured by vernier caliper and weight of the tumor was measured by electronic balance. Immunohistochemistry (IHC) staining LTBP4 expression between melanoma tissues and adjacent noncancerous tissues in patients with melanoma was determined by IHC staining. For IHC, sections were incubated with anti-LTBP4 (1:300 dilution) antibody. LTBP4 staining was scored by two independent pathologists. LTBP4 or cleaved caspase-3 expression between NC group and LTBP4-KD group in tumor tissues of BALB/C nude mice was determined by IHC staining. For IHC, sections were incubated with anti-LTBP4 (1:500 dilution) antibody or anti-cleaved caspase-3 (1:200 dilution) antibody. LTBP4 or cleaved caspase-3 staining was scored by researcher. The scoring system was based on the staining intensity and extent. Staining intensity was classi ed as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Staining extent depended on the percentage of positive cells and was divided into 0 (<5%), 1 (5-25%), 2 (26-50%), 3 (51-75%), and 4 (>75%). According to the staining intensity and the staining extent scores, the IHC result was classi ed as 0-1, negative (−); 2-4, weakly positive (+); 5-8, moderately positive (++); and 9-12, strongly positive (+++).

Western blotting
Protein lysates were prepared from cells using 500 µL of RIPA buffer with 1 mM phenylmethane sulfonyl uoride. Samples were subsequently sonicated for 2 minutes and centrifuged. The supernatants were collected and used for protein analysis. Lysates were separated on 8% polyacrylamide gels and transferred onto polyvinylidene uoride membrane. The membranes were blocked with PBS containing 0.1% Tween-20 (PBST) and 5% nonfat milk (w/v) for 1 hour at room temperature. After they were washed with PBST, the membranes were probed with antibodies overnight at 4°C. Antibody against LTBP4 was obtained from Shanghai Yu Bo Biotech Co,Ltd (Shanghai, China); antibody against cleaved caspase-3/Ki67/E-cadherin/YAP/MST1/CTGF/Cyr61 and phosph-YAP/MST1 antibody was obtained from Abcam (Cambridge, UK); antibody against MOB1 and phospho-MOB1 were obtained from Cell Signaling Technology, Inc (USA); antibody against Birc5 and phosphor-Birc5 were obtained from CUSABIO engineering co. Ltd (Wuhan, Hubei, China); antibody against β-actin or LaminB were obtained from Beyotime (China). The membranes were washed again with PBST, then horseradish peroxidase-labeled IgG at 1:5,000 dilution was added at room temperature for 1 h, and the blots were developed using enhanced chemiluminescence western blotting reagents. β-actin or LaminB was used as an internal control.

Statistical analysis
Statistical analyses were performed using SPSS 19.0 (IBM, Armonk, NY, USA). Correlations between LTBP4 expression and the clinicopathological variables were analyzed using the Pearson χ 2 analysis. The average value of LTBP4 > 1.925 as high expression. Survival was analyzed using the Kaplan-Meier method, and differences were evaluated using the log-rank test. The Cox proportional hazards model was used for univariate analysis to examine the potential prognostic value of different variables on OS. Data were evaluated using ANOVA with LSD test for multiple comparisons and Student's t test between two groups. P < 0.05 was considered to indicate a statistically signi cant difference.  (Fig. 1a). A total of 54 overlapping DEGs from GSE46517, GSE52882 and GSE54493 datasets were identi ed by Venn analysis (Fig. 1b). In addition, the expression of 54 overlapping genes in GSE46517, GSE52882 and GSE54493 datasets was shown in Fig. 1c. IFTM1 and LTBP3 were key genes from signi cant modules of the PPI network of 54 overlapping genes was identi ed using STRING database and Cytoscape software (Fig. 1d). There was no signi cant difference for in IFTM1 or LTBP3 at survival probability between high expression group and low expression group, however, LTBP4 was signi cantly downregulation in melanoma tissues based on GEPIA database analysis (Fig. 1e). GO enrichment analysis showed that 54 overlapping DEGs mainly enriched in BP and CC terms via using DAVID database and LTBP4 mainly enriched in "extracellular matrix" (Fig. 1f and 1 g).

Bioinformatics analysis of DEGs
LTBP4 expression was down-regulation in melanoma tissues and cell lines, which was closely related to the poor survival for patients with melanoma The results of RT-PCR and western blotting assays showed that LTBP4 were signi cantly down-regulated in cancer tissues of 76 patients with melanoma, which were associated with invasion, TNM stage, distal metastasis and lymph node metastasis ( Fig. 2a-b (Fig. 2c). Univariate Cox regression analysis revealed that low LTBP4 expression [hazard ratio (HR), 0.062; 95% con dence interval (CI), 0.006-0.694; P = 0.024] was associated with survival (Table 2). Invasion, TNM stage, distal metastasis, lymph node metastasis, age and sex were not associated with survival (all P > 0.05). Likewise, LTBP4 expression at protein level was signi cantly down-regulation in melanoma tissues via using IHC staining ( Fig. 2d and 2e). Next, we found that LTBP4 expression was signi cantly downregulation in melanoma cell lines via using RT-PCR and western blotting assay ( Fig. 2f and 2 g). LTBP4 expression in SK-MEL-1 and VMM5A cells was signi cantly higher than that in A101D and A375 cells. After shRNA-LTBP4 #1 or #2 was transfected into SK-MEL-1 and VMM5A cells to inhibit LTBP4 expression and LTBP4 expressing plasmids were transfected into A101D and A375 cells to overexpress LTBP4 expression ( Fig. 2h and 2i).

LTBP4 signi cantly regulated the proliferation and apoptosis in melanoma cell lines
Our results showed that LTBP4 KD promoted the viability of SK-MEL-1 and VMM5A cells (Fig. 3a and 3b), whereas LTBP4 OE inhibited the viability of A101D and A375 cells (Fig. 3c and 3d). Not surprisingly, LTBP4 KD promoted the proliferation of SK-MEL-1 and VMM5A cells (Fig. 3e), and LTBP4 OE inhibited the proliferation of A101D and A375 cells (Fig. 3f). Flow cytometry results showed that the apoptosis level in SK-MEL-1 and VMM5A cells was signi cantly suppressed by LTBP4-KD1 or LTBP4-KD2, meanwhile, the apoptosis level in A101D and A375 cells was signi cantly promoted by LTBP4 OE (Fig. 3g and 3 h). However, there was no signi cant differences in cell viability, proliferation and apoptosis between LTBP4-KD1 group and LTBP4-KD2 group.
LTBP4 signi cantly regulated the invasion and migration in melanoma cell lines, and closely related to the expressions of cleaved caspase-3, Ki67 and E-cadherin Transwell assay results showed that silencing of LTBP4 signi cantly promoted the invasion of SK-MEL-1 and VMM5A cells (Fig. 4a) and OE of LTBP4 markedly restrained the invasion of A101D and A375 cells (Fig. 4b). Conversely, wound healing assay results showed that silencing of LTBP4 signi cantly promoted the migration of SK-MEL-1 and VMM5A cells (Fig. 4c) and OE of LTBP4 markedly restrained the migration of A101D and A375 cells (Fig. 4d). Next, the changes of cleaved caspase-3, Ki67 and E-cadherin expression in LTBP4 KD or OE transfected melanoma cell lines were determined via using western blotting. The results showed that silencing of LTBP4 signi cantly inhibited the expressions of cleaved caspase-3 and E-cadherin, and increased Ki67 expression in SK-MEL-1 and VMM5A cells, meanwhile, OE of LTBP4 signi cantly increased the expressions of cleaved caspase-3 and E-cadherin and suppressed Ki67 expression in A101D and A375 cells ( Fig. 4e and 4f).

Silencing of LTBP4 promoted the oncogenicity of melanoma cell in vivo
After shRNA-LTBP4 transfected SK-MEL-1 cells were injected into the nude mice subcutaneously, tumor weight and volume were measured via using electronic balance and vernier caliper (Fig. 5a). LTBP4 KD promoted tumor volume and weight ( Fig. 5b and 5c). IHC staining results showed that the expressions of LTBP4 and cleaved caspase-3 were inhibited by LTBP4 KD and the expressions of Ki67 and E-cadherin were increased by LTBP4 KD (Fig. 5d and 5e).
Silencing of LTBP4 suppressed the activation of Hippo signaling pathway via inhibiting the phosphorylation of YAP to promote the nuclear translocation of YAP, thereby promoting the expressions of CTGF, Cyr61 and Birc5 in SK-

MEL-1 and VMM5A cells
Our results showed that silencing of LTBP4 upregulated the expressions of YAP, MST1, and MOB1 and suppressed the phosphorylation of YAP, MST1, and MOB1 in SK-MEL-1 and VMM5A cells via using western blotting (Fig. 6a and 6b). Next, the mRNA and protein levels of CTGF, Cyr61, and Birc5 in shRNA-LTBP4 transfected SK-MEL-1 and VMM5A cells were signi cantly upregulation via using RT-PCR and western blotting assays ( Fig. 6c and 6d). Furthermore, the low expression of YAP was found in cytoplasm of shRNA-LTBP4 transfected SK-MEL-1 and VMM5A cells (Fig. 6e), meanwhile, the high expression of YAP was found in nucleus of shRNA-LTBP4 transfected SK-MEL-1 and VMM5A cells (Fig. 6f). In addition, there was no signi cant differences for the expressions of YAP, MST1, MOB1, CTGF, Cyr61, and Birc5 in SK-MEL-1 and VMM5A cells between LTBP4-KD1 group and LTBP4-KD2 group.
OE of LTBP4 activated Hippo signaling pathway via promoting the phosphorylation of YAP to inhibit the nuclear translocation of YAP, thereby inhibiting the expressions of CTGF, Cyr61 and Birc5 in A101D and A375 cells Our results showed that OE of LTBP4 downregulated the expressions of YAP, MST1, and MOB1 and promoted the phosphorylation of YAP, MST1/2, and MOB1 in A101D and A375 cells via using western blotting ( Fig. 7a and 7b). Next, the mRNA and protein levels of CTGF, Cyr61, and Birc5 in LTBP4 expressing plasmids transfected A101D and A375 cells were signi cantly downregulation via using RT-PCR and western blotting assays ( Fig. 7c and 7d). Furthermore, the high expression of YAP was found in cytoplasm of LTBP4 plasmids transfected A101D and A375 cells (Fig. 7e), meanwhile, the low expression of YAP was found in nucleus of LTBP4 plasmids transfected A101D and A375 cells (Fig. 7f).
OE of LTBP4 inhibited the proliferation, invasion and migration, and promoted the apoptosis of melanoma cells via Hippo-YAP signaling YAP/or MST1 expressing plasmids were transfected into SK-MEL-1 and A375 cells to induce YAP/or MST1 OE via using RT-PCR and western blotting assays ( Fig. 8a and 8b). Furthermore, the results of CCK-8 and colony formation assays indicated that the functions of LTBP4 OE inhibiting the viability and proliferation of SK-MEL-1 and A375 cells were reversed by YAP OE or MST1 OE ( Fig. 8c and 8d). In addition, YAP OE or MST1 OE signi cantly abolished the effects of LTBP4 OE on promoting the apoptosis of SK-MEL-1 and A375 cells (Fig. 8e). The functions of LTBP4 OE inhibiting the invasion and migration of SK-MEL-1 and A375 cells were reversed by YAP OE or MST1/2 OE ( Fig. 8f and 8 g). Similarly, western blotting results showed that the functions of LTBP4 OE increased the cleaved caspase-3 and E-cadherin expression and reduced Ki67 expression counteracted by YAP OE or MST1 OE in SK-MEL-1 and A375 cells (Fig. 9a-9d). Next, both YAP OE and MST1 OE could inhibit the functions of LTBP4 OE decreasing CTGF, Cyr61, and Birc5 in SK-MEL-1 and A375 cells (Fig. 9e-9 h).

Discussion
Our results showed that LTBP4 is a key gene in melanoma based on bioinformatics analysis (Fig. 1). It is reported that LTBP is a class of extracellular matrix glycoproteins belonging to the brillin superfamily, mainly including four subtypes of LTBP1, LTBP2, LTBP3 and LTBP4 [15][16][17][18]. LTBP family regulated the occurrence and development of cancer via binding to TGF-β [19]. LTBP4 expression might be linked to the carcinogenesis of human mammary carcinomas, ductal carcinoma in situ and mouse mammary tumors and DCIS [20]. In addition, LTBP4 is downregulated in human mammary adenocarcinomas and may be involved in neoplastic transformation of human mammary tumors [20,21]. Disruption of the gene encoding the LTBP4 causes abnormal lung development, cardiomyopathy, and colorectal cancer [22]. However, the study of LTBP4 in malignant melanoma has not been reported. Our results showed that downregulation of LTBP4 was found in melanoma tissues and cell lines and had a poor survival in patients with melanoma (Fig. 2, Table 1 and Table 2). Therefore, we speculated that LTBP4 might be a tumor suppressor gene in melanoma. Furthermore, the results of cell functional experiments indicated that silencing of LTBP4 promoted the viability, proliferation, invasion and migration, and inhibited the apoptosis in melanoma cells, whereas OE of LTBP4 inhibited the viability, proliferation, invasion and migration, and induced the apoptosis in melanoma cells ( Fig. 3 and Fig. 4).
Caspase-3 and Ki67 are important proteins related to proliferation and apoptosis, which are abnormally expressed in many kinds of malignant tumors [23]. E-cadherin is an important tumor suppressor gene related tumor metastasis, and its expression is signi cantly down-regulated in malignant tumor cells with high ability of migration and invasion [24,25]. In this study, we found that LTBP4 KD signi cantly inhibited the expression of cleaved caspase-3 and E-cadherin and up-regulated the expression of Ki67 in melanoma cells. Unsurprisingly, the effect of LTBP4 OE on the expression of cleaved caspase-3, Ecadherin and Ki67 in melanoma cells was opposite to that of LTBP4 KD on the expression of cleaved caspase-3, E-cadherin and Ki67 in melanoma cells. By virtue of nude mouse tumorigenesis test, the tumorigenesis ability in vivo of LTBP4 KD was further con rmed (Fig. 5). To sum up, we can con rm that LTBP4 gene knockout promotes the growth and metastasis of melanoma and affects the survival status of patients. However, the molecular mechanism of the inhibitory effect of LTBP4 on melanoma needed to be further con rmed.
YAP, as a tumor-promoting protein, is one of the core molecules of Hippo signaling pathway, and highly expressed in almost all melanoma tumor cells [26]. YAP gene knockout can delay wound healing and reduce the expression of TGF-β1. In addition, YAP regulates TGF-β signaling pathway [27]. It is reported that there is a molecular interaction between LTBP4 and TGF-βRII, and LTBP4 gene knockout leads to a decrease in the number of TGF-βRII [28]. Studies have shown that the decrease and distribution of TGF-βRII protein results in the inability of TGF-β1 to bind to its receptor, which in turn leads to the inability of TGF-β 1 to inhibit the growth of tumor cells [29]. These reports con rmed laterally that LTBP4 gene knockout promotes the growth of tumor cells. Therefore, we speculated that there was an interaction between LTBP4 and YAP, which was closely related to the activation of Hippo signal pathway. The results showed that LTBP4 KD upregulated the expressions of YAP, MST1, MOB1, and the downstream geneexpression of YAP (CTGF, Cyr61 and Birc5), and inhibited the phosphorylation levels of YAP, MST1, and MOB1 in melanoma cell. Furthermore, LTBP4 KD promoted the nuclear translocation of YAP (Fig. 6). On the contrary, LTBP4 OE downregulated the expressions of YAP, MST1, MOB1, and the downstream geneexpression of YAP (CTGF, Cyr61 and Birc5) [30], and increased the phosphorylation levels of YAP, MST1, and MOB1 in melanoma cell. Furthermore, LTBP4 OE suppressed the nuclear translocation of YAP (Fig. 7). It showed that LTBP4 KD inhibited the activation of Hippo-YAP signaling in melanoma cells.
Rescue experiment results showed that the functions of LTBP4 OE inhibiting the viability, proliferation, invasion, and migration, and promoting the apoptosis in melanoma cells were reversed by YAP OE or MST1 OE (the core molecule of Hippo signaling pathway), meanwhile, the functions of LTBP4 OE inhibiting the expressions of cleaved caspase-3 and E-cadherin, and inhibiting the expressions of Ki67, CTGF, Cyr61 and Birc5 in melanoma cells were abolished by YAP OE or MST1 OE ( Fig. 8 and Fig. 9).
These results showed that LTBP4 affected the activation of Hippo signaling pathway via regulating the nuclear translocation of YAP.

Conclusions
The downregulation of LTBP4 was found in melanoma tissues and cell lines and had a poor survival in patients with melanoma. Silencing of LTBP4 promoted the growth and metastasis of melanoma in vivo and in vitro. LTBP4 knockdown suppressed the activation of Hippo signaling pathway via inhibiting the phosphorylation of YAP to promote the nuclear translocation of YAP, thereby promoting the proliferation, invasion and migration, and inhibiting the apoptosis of melanoma cells. These results also raise the possibility that LTBP4 may function as an important new biomarker for melanoma tumors. Availability of data and materials The datasets generated/analyzed during the current study are available.

Funding
Not applicable.