YAP‐TEAD up‐regulates IRS2 expression to induce and deteriorate oesophageal cancer

Abstract Oesophageal cancer (EC) represents a significant cause of cancer worldwide. Yes‐associated protein (YAP) is reported to correlate with the initiation of multiple cancers including EC, but the underlying mechanism remains elusive. The current study aimed to investigate the molecular mechanism of YAP‐TEAD in the occurrence and progression of EC. EC tissues and cells were obtained, followed by determination of the expression of YAP, c‐Jun, pc‐Jun and IRS2. The effect of YAP‐TEAD on the biological EC cell processes was explored through gain‐ and loss‐of‐function approaches. The interaction between YAP and TEAD was detected by co‐immunoprecipitation. The binding of TEAD to the c‐Jun promoter was determined using chromatin immunoprecipitation. Tumour formation in the nude mice was detected in order to ascertain the effect of YAP and IRS2 in vivo. We found elevated YAP in the EC tissues and cells. YAP silencing led to a decrease in EC cell proliferation, invasion and sphere formation. YAP‐TEAD complex bound to the promotor of c‐Jun, and c‐Jun led to an increase in the expression of IRS2 through the JNK/c‐Jun pathway. Additionally, pc‐Jun and phosphorylated JNK were localized in the nuclear in addition to displaying enhanced expression in the EC tissues. IRS2 overexpression negated the inhibition of cell proliferation, invasion and sphere formation triggering YAP silencing. YAP up‐regulated IRS2 and aggravated EC in vivo. Taken together, YAP‐TEAD activates the JNK/c‐Jun pathway to up‐regulate IRS2, ultimately promoting EC progression. Therefore, YAP‐TEAD inhibition could be a promising therapeutic approach for EC treatment.

Yes-associated protein (YAP) was initially regarded as a protein associated with Yes, a src family kinase (SFK). 4 Previous literature has emphasized the dysregulation of YAP function as a crucial driver of tumorigenesis, chemoresistance and metastasis. 5 A previous study provided evidence of high levels of YAP expression in ESCC tissues. 6 Additionally, the oncogenic activity of YAP has been shown to be mediated by the TEA Domain (TEAD) family transcription factors, [7][8][9] which led us to further investigate its role in ESCC in vitro and in vivo. Aberrant expression of TEAD has been documented to influence well-known cancer genes such as KRAS and BRAF, with its transcriptional output implicated in various processes including cancer metabolism, tumour progression and cancer metastasis. 10 Existing literature has highlighted that the YAP/PDZ-binding motif (TAZ)/TEAD interacts with AP-1 to facilitate tumour growth, 11 while inhibition of YAP/TAZ-TEAD has recently emerged as a promising therapeutic target for various types of cancers. 4 Interestingly, TEAD is involved in the promoting effects of YAP on EC. 12 The c-Jun N-terminal kinase (JNK), a member of the mitogenactivated protein kinase (MAPK) family, regulates both cancer cell apoptosis and survival. 13 The aberrant activation of JNK has been shown to consequently result in the deterioration in different cancers, including oral, 14 prostate 15 and pancreatic cancer. 16 Among the vast substrates of JNK, the oncogene c-Jun stands out due to its strong association with cancer invasiveness. 17 Considering the c-Jun promoter contains TEAD binding site, 18 we asserted the hypothesis that the JNK/c-Jun pathway is regulated by YAP-TEAD.
Yes-associated protein has been previously reported to positively regulate insulin receptor substrate 2 (IRS2) to affect the activity of non-small cell lung cancer cells, 19 highlighting the relationship between YAP and IRS2. IRS2 represents a signalling molecule capable of mediating the effects of insulin/insulin-like growth factor 1 (IGF1).
IRS2 is expressed in various types of cancer and has been reported to contribute to tumour cell metabolism. 20 Suppression of IRS2 has been shown to confer an inhibitory effect on the progression of liver cancer 21 neuroblastoma 22 and ESCC. 23 Moreover, IRS2 is the target gene of the JNK/c-Jun pathway in breast cancer cells. 24 Here, we set out to determine whether YAP-TEAD could induce and deteriorate ESCC by means of regulating IRS2 via the JNK/c-Jun axis by conducting in vitro and in vivo assays.

| Tissue and cell culture
All the primary ESCC tissues as well as the adjacent tissues were collected via resection of specimens from 47 ESCC patients from Linyi People's Hospital between January 2012 and January 2014.
Normal mucosa and anterior lesions were obtained and regarded as the controls. All patients were yet to undergo endoscopic mucosal resection, palliative resection, preoperative chemotherapy, or radiotherapy. All patients were confirmed to be free of simultaneous or multiple heterogeneous tumours on other organs. Follow-up visits were performed until January 2019 and patients were monitored regularly. The average follow-up time for surviving patients was 44 months (8 to 60 months). Besides, Kaplan-Meier survive analysis was also performed (Table 1). Some patients were found to have little or no residual tumour, while other patients with small resections were excluded. Thus, certain types of the samples in some instances were missing from the patient.

| Immunohistochemistry
The paraffin-embedded tissues were sliced into 4μm-thick sections, dewaxed in xylene, rehydrated in graded ethanol and subjected to antigen retrieval. The sections were then blocked in 10% normal serum and 1% bovine serum albumin (BSA) in TBS for 2 hours at room temperature. After washing with TBS buffer, the sections were incubated

| RNA extraction and quantification assay
The total RNA in the cells and tissues was isolated using a TRIzol

| Nuclear and cytoplasmic extraction experiment
Nuclear and cytoplasmic extraction were conducted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher).
Quantitative analysis of nuclear or cytoplasmic protein was performed by Western blot.

| Western blot assay
Tissue and cell total protein were extracted using PMSF or RIPA lysis buffer at 4℃ for 30 minutes, followed by centrifugation at 8000 g for 10 minutes according to the user manual. After the supernatant had been collected, protein concentration was deter-

| Colony formation assay
The

| Assessment of viability by cell counting kit (CCK)-8 assay
The cells were pre-cultured in 96-well plate for 24 hours at

| Co-immunoprecipitation assay
The cells were lysed in buffer containing Tris-HCl (50 mM, pH 7.5), NaCl (150 mM), Nonidet P-40 (1%), sodium deoxycholate (0.5%), and proteinase (1%; Sigma-Aldrich) and subjected to sonication. After the cell lysis had been centrifuged, the supernatant was incubated overnight at 4℃ with anti-YAP, anti-TEAD and Protein G Plus/Protein A Agarose beads (CalBiochem) or IgG beads of the same type (Sigma-Aldrich). The beads were subsequently washed six times using a lysis buffer and analysed by Western blot.

| Chromatin immunoprecipitation assay
The cells were fixed using 1% formaldehyde and sheared by sonication. The antibody was added and mixed with the promoter. The antibody-promoter complex was precipitated through the addition of Protein A Agarose/Salmon Sperm DNA. Nonspecific binding was washed away prior to eluting and de-crosslinking the promoter complex. The promoter fragments were purified and employed as the RT-qPCR template.

| Assessment of cell sphere formation
The cells were transfected with si-YAP or control si-RNA. After 48 hours, a total of 2500 cells were cultured in serum-free DMEM/

| YAP is overexpressed in ESCC tissues and cells
The differential analysis of GSE29001 in the GEO database by R language revealed 1964 differentially expressed genes, among which genes ( Figure 1A). GEPIA was employed to analyse the genes in relation to EC survival in the TCGA database, with the top 500 genes subsequently identified. To further elucidate the mechanisms of transcription factors in EC, the human transcriptional factor names were subsequently obtained from Cistrome. The analysis results obtained in connection with GSE29001, GEPIA and Cistrome were intersected, with FOS and YAP (YAP1) identified as essential transcriptional factors associated with EC survival ( Figure 1B). The TCGA database was analysed by UALCAN, and we identified the overexpression of YAP in EC ( Figure 1C).
Primary ESCC tissues and adjacent tissues from 47 patients were evaluated for YAP expression detection. Immunohistochemistry staining ( Figure 1D and Figure S1) indicated high levels of YAP in ESCC tissues, and that YAP was mainly localized in nuclear. RT-qPCR confirmed an increased level of YAP in ESCC tissues ( Figure 1E).
Next, to further investigate the difference of YAP expression inside the cells, the nuclear and cytoplasmic extraction experiment were performed, followed by Western blot assay. As depicted in Figure 1F, the level of YAP was significantly elevated in cell nuclear,

| Silencing YAP inhibits the proliferation, invasion and sphere formation of EC cells
Two si-RNAs were specifically designed for YAP gene silencing and transfected into EC9706 cells. Figure 2A illustrates the YAP expression RT-qPCR findings. No.2 si-RNA was selected for subsequent experiments due to its superior silencing efficiency. After loss-of function, evidence was obtained indicating that when YAP was silenced, cell viability, the number of formed clones, invasiveness and sphere-forming capacity reduced significantly ( Figure 2B-E and Figure S2). We repeated the experiments in Eca-109 cells, with similar results to those in EC9706 cells obtained ( Figure S3). The aforementioned observations led to the conclusion that YAP silencing alleviated EC activity.

| YAP activates JNK/c-Jun pathway by binding to TEAD
In order to elucidate the downstream mechanism of YAP, we predicted ten YAP (YAP1)-related genes using STRING and twenty genes using GeneMANIA. In Figure 3A,B, we obtained the related genes of YAP1 through STRING and GeneMANIA databases and constructed the PPI network of the YAP-related genes, respectively.
Next, by taking the intersection of the obtained related genes with the downstream genes of YAP predicted by Cistrome, we finally obtained 2 candidate genes, SMAD7 and TEAD1 ( Figure 3C). Existing literature has suggested that YAP influences EC occurrence by binding to TEAD (TEAD1), 6 which encouraged us to analyse the expression of TEAD in EC. As indicated by GEPIA analysis in Figure 3D, YAP and TEAD (TEAD1) revealed the existence of a distinct link. Besides, the targeting relationship between YAP and TEAD was solidified by analysing the transcriptional gene database hTFtarget ( Figure 3E).
Thus, we chose TEAD for subsequent research instead of SMAD7.
Considering that the JNK/c-Jun pathway is over-activated in more than 80% oesophageal adenocarcinoma specimens, 25 and YAP-TEAD can monitor the progression of basal cell carcinoma via JNK/c-Jun pathway, 18 we set out to investigate the same pathway in EC. The expression of pc-Jun (S63), pc-Jun (S73), and phosphorylated JNK1/2 in primary ESCC tissues and adjacent tissues were identified using immunohistochemistry methods. As depicted in Figure 3F and Figure S4A, both the expression and the nuclear localization of the three above proteins exhibited increases in ESCC tissues.
Quantification from Western blot assay in ESCC tissues provided evidence validating this observation ( Figure 3G). In the event of YAP silencing with si-RNA, expression of pc-Jun (S63), pc-Jun (S73), c-Jun, phosphorylated JNK1/2 and JNK1/2 was reduced in EC9706 cells, demonstrated by Western blot assay ( Figure 3H).
Previous reports have suggested that in basal cell carcinoma, the c-Jun promoter and enhancer contain not only a TEAD binding site but also multiple AP1 recognition sites which can be bound by c-Jun and regulate c-Jun expression. 18 Besides, genome-wide correlation of YAP/TAZ/TEAD with AP-1 promotes tumour growth. 11 Therefore, the relationship between YAP and JNK/c-Jun pathway in EC was examined. Initially, evidence was obtained highlighting interactions between YAP and TEAD based on the co-immunoprecipitation assay results ( Figure 3I). Next, data were obtained showcasing that TEAD bound to c-Jun promoters using Chromatin immunoprecipitation (ChIP)-PCR. The enrichment got lessened when YAP was silenced ( Figure 3J). Finally, Western blot assay displayed that the overexpression of YAP triggered an increase in the expression of pc-Jun (S63), pc-Jun (S73), c-Jun, phosphorylated JNK1/2 and JNK1/2 in EC9706 cells, which was rescued following TEAD silencing in the YAP-overexpressed EC9706 cells ( Figure 3K). Taken together, the aforementioned findings suggested that YAP interacted with TEAD, and that YAP activated the JNK/c-Jun pathway by binding to TEAD.

| YAP-TEAD activates IRS2 via JNK/c-Jun pathway
IRS2 is reported to be the downstream target gene of the JNK pathway. 26 With immunohistochemistry staining, the data obtained indicated that the expression of IRS2 was markedly elevated in the ESCC tissues ( Figure 4A and Figure S4B). The up-regulation of IRS2 was confirmed by data obtained from RT-qPCR and Western blot assay ( Figure 4B,C and Figure S4C), while a positive correlation between the expression of IRS2 and YAP expression was uncovered ( Figure 4D).
Thereafter, we set out to evaluate whether the expression of IRS2 was regulated by YAP. Following the overexpression of YAP in EC cells, RT-qPCR and Western blot assay results suggested that the expression of IRS2 was elevated ( Figure 4E). Correspondingly, silencing YAP suppressed IRS2 expression ( Figure 4F). We previously found that c-Jun and the phosphorylated c-Jun (pc-Jun) were up-regulated in the YAP-overexpressed cells. In line without predictions, the JNK pathway inhibitor SP600125 deceased JNK-mediated c-Jun phosphorylation and IRS2 expression, thus the overexpression of YAP failed to upregulate IRS2 expression ( Figure 4G). These findings suggested IRS2 expression was regulated by the JNK/c-Jun pathway.
Suppressing the JNK/c-Jun pathway restrained IRS2, which could not be rescued by YAP overexpression. In short, YAP increased IRS2 expression via the JNK/c-Jun pathway.

| YAP elevates IRS2 to induce and deteriorate EC
Next, we set out to ascertain whether YAP deteriorates ESCC by up-regulating IRS2 in EC. In the YAP-silenced EC9706 cell, IRS2 expression was decreased. Compared to treatment with si-YAP alone, silencing YAP and overexpressing IRS2 simultaneously increased the IRS2 level but kept YAP expression unchanged ( Figure 5A). Finally, we investigated tumour formation in nude mice. EC9706 cells transfected with lentivirus pf sh-YAP + oe-NC or sh-YAP + oe-IRS2 were subcutaneously inoculated into the back of the mice. The expression of YAP and IRS2 in xenografted nude mice was quantified by RT-qPCR, as depicted in Figure 5F, with consistent results obtained from the cell experiments. Tumorigenesis after EC9706 inoculation in mice revealed that silencing YAP inhibited the progression of EC and reduced tumour size and weight, which was neutralized following the overexpression of IRS2 ( Figure 5G). Western blot analysis further revealed that silencing YAP markedly decreased the expression of YAP, JNK/p-JNK, c-Jun/p-c-Jun and IRS2, while overexpression of IRS2 significantly increased IRS2 expression but had no effect on other proteins ( Figure 5H). Collectively, the aforementioned results indicated that YAP deteriorated EC via activation of IRS2 both in vivo and in vitro.

| D ISCUSS I ON
Oesophageal cancer remains a significant cause of cancer-related mortality worldwide. The current study set out to elucidate a new  shown to contribute to the development of EC, 12 providing insight into the role of YAP/TEAD signalling in the EC deterioration.
In light of the fact that the c-Jun promoter and enhancer contain TEAD binding sites, we investigated the role of c-Jun in our F I G U R E 3 YAP binds to TEAD to activate the JNK/c-Jun pathway in EC cells. (A) PPI network graph of YAP and related genes constructed by STRING. Colour of nodes from red to blue indicates the transition from hyper-core level to non-core level genes; (B) PPI network graph of YAP and related genes constructed by GeneMANIA. The size of the node indicates the score in the PPI network, the higher the score the larger the node. (C) Venn diagram of gene sets related to YAP predicted from STRING and GeneMANIA, and human transcription factors. Overlapped area indicates SMAD7 and TEAD1; (D) Scatter plot of correlation analysis of YAP and TEAD1 obtained from GEPIA (r = .28, P = 1.2E-04); (E) Targeting relationship of YAP and TEAD predicted by hTFtarget, the last column shows a targeting relationship between the two predicted by website; hence, the current study identified the upstream transcription factors and kinases of JNK1/2.
A positive correlation has been suggested between YAP-TEAD and IRS2 expression in the context of liver cancer 21 as well as nonsmall cell lung cancer. 19 However, direct evidence demonstrating that pc-Jun binds to IRS2 promoter remains elusive. The insulin receptor substrate (IRS) family consists of at least four members, IRS1, IRS2, IRS3 and IRS4. 40 A study with IRS2-knockout mice highlighted the critical role of IRS2 in cell growth and hormone secretion. 41 In ESCC, IRS1 and IRS2 are overexpressed and promote cell proliferation. 23,42 Consistently, IRS2 has been shown to deteriorate EC in our study.
In conclusion, the key observations of our study provide evidence of YAP-TEAD-activated JNK/c-Jun pathway to up-regulate IRS2, thus promoting cell proliferation and invasion of EC cells. The downstream genes of YAP could be diverse; hence, further investigation is required.

ACK N OWLED G EM ENTS
The authors would like to acknowledge the helpful comments on this paper received from the reviewers.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.