Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC

Abstract YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1‐1, which contains a single WW domain, and YAP1‐2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF‐induced epithelial‐mesenchymal transition (EMT) in non‐small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1‐1 and YAP1‐2 isoforms—although when compared to YAP1‐1, YAP1‐2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF‐induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1‐1 stable expression cells exhibited a stronger migration ability than YAP1‐2 expressing cells. However, upon EGF treatment, YAP1‐2 stable cells showed more robust migration than YAP1‐1 expressing cells. The protein stability and nuclear localization of YAP1‐2 were preferentially enhanced with EGF treatment. Moreover, EGF‐induced EMT and YAP1‐2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1‐2 is the main isoform that is functionally relevant in promoting EGF‐induced EMT and ultimately NSCLC progression.

Tumour metastasis and recurrence are main contributors to the high mortality rate of NSCLC, 3 whereas epithelial-mesenchymal transition (EMT) is a key initial step of cancer metastasis. EMT refers to a biological process in which epithelial cells lose their polarity and acquire a mesenchymal phenotype and can be induced by intrinsic cell properties as well as extracellular cues, including growth factor stimulation. 4 During EMT, epithelial cells shift their marker expression profile towards that characteristic of mesenchymal cells, notably decreased E-cadherin, and increased N-cadherin as well as vimentin. 3 In EMT-associated tumour invasion and metastasis, epithelial-derived cancer cells lose their epithelial polarity and the ability to adhere to the basolateral membrane and acquire mesenchymal characteristics, such as migration and invasive abilities, allowing them to detach from the primary tumour, enter circulation, and metastasize to distant sites where they may form new lesions. 5 Therefore, a better understanding of the regulatory mechanisms underlying EMT process will facilitate novel strategies targeting EMT for cancer treatment.
The Hippo pathway is a highly conserved and critical pathway that regulates cell proliferation, apoptosis and self-renewal. 6 Numerous studies have established YAP1, the key downstream effector of Hippo pathway, as an important oncogene for tumorigenesis, and a promoter of cancer stemness and metastasis. 7,8 However, some controversies remain regarding the roles of YAP1 in different cancer cell types and models. 9 These discrepancies may partly be due to the existence of YAP1 in different isoforms, for which we have recently shown to exhibit distinct functional properties and regulatory mechanisms. 10 In mammals, at least eight YAP1 protein isoforms derived from alternative mRNA splicing have been identified. YAP1 isoforms can be divided into two subgroups based on the presence of either a single WW motif (YAP1-1) or two tandem WW domains (YAP1-2). 11 The WW domain of YAP1 is responsible for interactions with a number of PPxY motif-containing proteins in the Hippo pathway (where P is proline, x is any amino acid and Y is tyrosine), 12 such as LATS1/2, 13 AMOT, 14 WBP2 15 and PTPN14. 16 The presence of either a single WW or double WW domains may influence the affinity as well as the specificity of YAP1 during its interactions with these PPxY motifs. 17 We recently demonstrated that, under high cell density, the YAP1-2 protein exhibits stronger interactions with several negative regulators making it less stable compared to the YAP1-1. YAP1-2 protein is preferentially degraded under high cell density, and likely in the setting of dense solid tumour. 10 Our data suggest that YAP1-1 and YAP1-2 isoforms are differentially regulated by the upstream Hippo pathway and may be subject to regulation by diverse stimuli such as cell-cell contact, mechanical cues, as well as EMT. Although several studies have documented direct and indirect roles of YAP1 in the EMT process, 18,19 the detailed functional properties and regulatory mechanisms underlying each specific YAP1 isoform during EMT remain unclear.
In our current work, we examined the role of YAP1 in NSCLC with a focus on the YAP1-1 and YAP1-2 isoforms in the EGF-induced EMT process. Our results indicated that YAP1 plays an essential role in maintaining the EMT phenotype such as cell migration and EMT marker expression in NSCLC. However, YAP1-2 exhibited a stronger effect than YAP1-1 in promoting EGF-induced EMT in NSCLC cells.
Mechanistically, we showed that activation of AKT pathway by EGF treatment preferentially leads to YAP1-2 stabilization and its nuclear localization.

| Antibodies and reagents
The primary antibodies and their commercial suppliers are YAP1

| Cell culture, transfection and treatment
The NSCLC cell lines A549, H460 and H1975 were purchased from American Type Culture Collection (ATCC). The cells were maintained under the recommended culture conditions and transfected as previously described. 20 For EGF treatment, the cancer cells were treated with 25 ng/ml EGF for 3 days to induce the EMT process. All experiments were performed three times.

| Lentiviral packaging, transduction and stable cell selection
Lentiviral packaging, host cell transfection and puromycin selection of stably cells containing pLKO-ShRNA were performed as previously described. 20 For the stable reconstituted expression of specific YAP1 isoforms, lentiviral particles carrying pLenti6.3-Flag-YAP1 cDNA encoding either YAP1-1γ or YAP1-2γ were used to transduce A549-shYAP1 cells. The cells were then selected in culture medium supplemented with blasticidin (5 μg/ml), and the surviving blasticidin-resistant cells were used as stable overexpression cells.

| RNA isolation, real-time PCR and YAP1 isoform detection
Total RNA was extracted with RNAiso Plus (TaKaRa, JPN). The

| Trans-well assay
The cell migration assay was performed using a Boyden chamber in

| Statistical analysis
The real-time PCR, MTT assays and tumour growth curve data are presented as the mean ± SD. p values showing differences were calculated by an unpaired two-tailed t test, and those showing no differences were calculated by a one-tailed t test.

| YAP1 expression positively correlates with EGFR activity and NSCLC malignancy
We collected clinical samples from 4 NSCLC patients to analyse the expression of YAP1 by immunohistochemistry. The results showed that the YAP1 expression in TNM stage 3 patients (15877 and 16285) was higher than that of TNM stage 2 patients (22144 and 21687; ( Figure 1A,B). Kaplan-Meier analysis was used to analyse the relationship between YAP1 expression and patient survival in NSCLC (data from TCGA). We found that the overall survival (OS) in the patients with high YAP1 expression was significantly lower than in those with lower YAP1 expression ( Figure 1C) suggesting that high YAP1 expression correlates to poor survival (data from TCGA). In lung adenocarcinoma, oncogenic EGFR expression and mutations co-occur with many other oncogene alterations. Here, we found that the expression of YAP1 and EGFR is positively correlated ( Figure 1C) and patients harbour EGFR mutations are more likely to have higher YAP1 expression, although the TCGA data do not discern YAP1-1 from YAP1-2 isoforms ( Figure 1D).

| EGF upregulates YAP1 expression and promotes EMT in NSCLC
To determine the effect of EGF on the EMT process in NSCLC, showed that the wound healing efficiency as well as the migrated cell number of A549, H460 and H1975 were dramatically increased upon EGF treatment. These results indicated that EGF stimulation induces EMT in NSCLC cells and upregulates YAP1 expression.

| YAP1 knockdown impairs EGF-induced EMT
To assess the potential role of YAP1 in the EMT process, endogenous YAP1 was suppressed in A549 cells using pLentiviral shRNA vector ( Figure 3A,B). EMT markers were examined in A549-shYAP1 cells and H460-shYAP1 cells. Western blot analysis showed that the epithelial marker E-cadherin was increased in the YAP1 knockdown cells, while the mesenchymal markers Vimentin and Snail were decreased, and these changes became more significant after EGF stimulation ( Figure 3C and Figure S1A). The qPCR results show that the changes of mRNA and protein were basically consistent ( Figure 3D and Figure S1B). Together, these results suggest that sup-  or YAP1-2 were designed to detect the mRNA level of each isoform in NSCLC cells. The mRNA level of YAP1-2 was much higher than that of YAP1-1 in NSCLC cells ( Figure 4B). To further investigate the difference between these two isoforms, A549 stable cell lines expressing a single YAP1 isoform were constructed based on A549-shYAP1 (shYAP1 #1, the target is YAP1 3' UTR sequence); these cells were named A549-YAP1-1 cells and A549-YAP1-2 cells, respectively.

| YAP1-2 is the dominant isoform in the EMT process in A549 cells
Western blots and qPCR confirmed efficient and specific overexpression of YAP1-1 and YAP1-2 isoforms in A549-shYAP1 stable cells, respectively, at both the mRNA and the protein levels ( Figure 4C,D).

| EGF promotes YAP1 protein stability and its nuclear localization
As YAP1 nuclear localization is vital for its regulation and activity, we Significantly, upon treatment with EGF, total YAP1 were increased in both cell lines, in particular, nuclear YAP1 was even higher in A549-YAP1-2 cells, than that in A549-YAP1-1 cells ( Figure 6B). The degradation of YAP1-2 cells was stronger than YAP1-1 under normal culture condition ( Figure 6C,D); however, this trend was reversed after EGF stimulation ( Figure 6E,F). We determined the mRNA expression of CYR61 and CTGF, two downstream target genes of YAP1, to assess the transcriptional activity of YAP1. Without EGF stimulation, the expression of CYR61 and CTGF in the A549-YAP1-1 cells was higher than in the A549-YAP1-2 cells. However, when stimulated with EGF, the expression of CYR61 and CTGF in the A549-YAP1-2 cells became higher than those in the A549-YAP1-1 cells ( Figure 6G,H).

| EGF enhances YAP1 protein stability via the AKT pathway
EGF and its receptors are widely distributed in different cell types and can activate multiple downstream pathways. It has been reported that the AKT signalling pathway regulates the protein stability of YAP1. 21 Here, we applied AKT-specific inhibitor MK2206 to investigate the role of this pathway in YAP1-mediated EMT. As ex- The aberrantly activated EGFR pathway by gene amplification and/or mutation is highly frequent in NSCLC, particularly in patients of East Asian descent. 22 EGFR targeting inhibitors have been widely used in clinical treatment, 23 yet the regulatory machinery underlying its oncogenic activity is still not fully understood. The current work illustrated important roles of YAP1 isoforms in EGF-induced EMT progression in NSCLC. Interestingly, YAP1 isoforms exhibited different responses to EGF stimulation, in that the protein stability of YAP1-2 and its nuclear localization was enhanced more compared to YAP1-1. Moreover, our recent findings suggest that a similar phenomenon exists in TGFβ induced EMT in pancreatic cancer. 24 The identification of factors that influence the stability of YAP1 isoforms may lead to the discovery of possible determinants of NSCLC progression. In this regard, we postulate that the extra WW domain of YAP1-2 may contribute to the higher overall stability of the nuclearlocalized YAP1-2 through binding particular molecules that may also modulate its transcriptional activity, when compared to YAP1-1.
Although YAP1-2 exhibited stronger response to EGF stimulation, the overall amount of YAP1-2 localized to the nuclei was comparable to that of YAP1-1 due to a higher basal level of YAP1-1.
Therefore, mere nuclear localization cannot fully explain the more robust effect of YAP1-2 in promoting invasion and metastasis of cancer cells than YAP1-1 upon EGF treatment. We suggest that the en-  Our data also indicate that the AKT pathway is critical for YAP1-2 stability, which is consistent with previous reports. 30 Although Hippo pathway is the main upstream regulator of YAP1, mainly by cell contacts, the AKT pathway may become important for cancer cells undergoing EMT. During EMT process, the cell contacts become weaker and Hippo signalling is inhibited, leading to YAP1-2 stabilization and enhanced nuclear localization. Therefore, AKT signalling may promote YAP1-2 stabilization and nuclear localization in both direct and indirect ways. Future studies will investigate the possibility that EGF signalling may differentially impact

CO N FLI C T O F I NTE R E S T S
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.