Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Abstract Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐rasG12C mutation) and PC9‐BrM3 (EGFR Δexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐rasG12C mutation) and PC9 (EGFR Δexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.

better response rate and fewer side effects than conventional chemotherapy in treating patients with EGFR mutant metastatic lung adenocarcinoma. 7-10 K-ras mutation is frequent in patients with lung adenocarcinoma (15%-30%), but there is still no approved effective target therapy for K-ras mutant metastatic lung adenocarcinoma. [10][11][12] Yes-associated protein (YAP), a key mediator protein in the Hippo (also known as the Salvador-Warts-Hippo) signalling pathway, has been reported to promote development of various cancers. [13][14][15] YAP has been suggested as a potential therapeutic target for melanoma, mesothelioma and hepatocellular carcinoma. [16][17][18] YAP was also identified in human non-small-cell lung cancer (NSCLC) and is correlated with drug resistance and tumorigenesis. 14,[19][20][21] A previous study reported that because some NSCLC cell lines harbouring K-ras mutations were not K-ras-dependent cells, K-ras may be not an appropriate therapeutic target. 22 Furthermore, YAP appears to take over K-ras as a cancer driver in NSCLC and pancreatic ductal adenocarcinoma, and YAP was identified as a central driver of compensation for K-ras-dependent NSCLC when there is loss of K-ras signalling. [23][24][25] Metastatic lung adenocarcinoma cell line H2030-BrM3 (K-ras G12C mutation) was derived from lung adenocarcinoma cell line H2030 (K-ras G12C mutation), which has a known cancer-driven K-ras mutation and a high potential of metastasis in murine models. 26,27 Although an important regulator, serpin I1 (neuroserpin), was identified in H2030-BrM3 cells to promote brain metastasis, rare study found effective treatments for metastatic K-ras mutant lung adenocarcinoma in murine models. 27 We sought to determine whether there is increased Hippo/YAP signalling pathway reporter activity and YAP expression in metastatic lung adenocarcinoma cells. In addition, we investigated whether direct inhibition of YAP suppresses human lung adenocarcinoma H2030-BrM3 cell metastasis in vivo. authenticated by erlotinib treatment in a viability assay. 28 Human NSCLC cell line H2030 was obtained from American Type Culture Collections (ATCC) (Manassas, VA) and passaged for fewer than 6 months after receipt from ATCC. Cell lines were maintained in RPMI-1640 supplemented with 10% heat-inactivated foetal bovine serum and streptomycin (100 mg/mL), and were cultured at 37°C in a humid incubator with 5% CO 2 .

| Luciferase reporter assay
The control shRNA-transfected H2030-BrM3 and YAP shRNA-  | 3075 next day. Cells were blocked in 5% goat serum for 1 hour, rabbit anti-YAP primary antibody (Cell Signaling, 14074; 1:100) was added, and cells were incubated in 4°C overnight. Goat anti-rabbit IgG (H+L) for secondary antibody with Alexa Fluor â 488 (Thermo Fisher Scientific, A-11008; 1:800) for primary antibody detection were added, and cells were incubated at room temperature for 1 hour. The cells were then washed with PBS 3 times, and nuclei were stained by 2 lg/mL DAPI in PBS. A fluorescence microscope (Zeiss Axioskop 2 microscope; Carl Zeiss, Inc., Germany) was used to view stained cells, and images were taken under a 209 objective lens.

| Tissue samples, histologic analysis and immunohistochemistry
After mice were killed, the tumour tissues from metastatic sites including lung, bone and brain were harvested and immediately fixed in 10% paraformaldehyde for 24 hours. The tissues were then transferred into 70% ethanol for storage and embedded in paraffin.

| ChIP assay
Chromatin ImmunoPrecipitation (ChIP) Assay Kit (Millipore Corporation) was used for the ChIP assay. Polyclonal antibodies for YAP and control rabbit antibody for IgG used for ChIP were purchased from Cell Signaling Technology.

| Statistical analysis
Data are expressed as mean AE standard deviation (SD) from three independent experiments. All statistical analyses were performed with GraphPad Prism (version 5.0; GraphPad Software, San Diego, CA, USA). t Tests were used to compare the differences between two groups, and one-way ANOVA followed by Tukey's multiple comparisons was used to compare differences among >2 groups. A Kaplan-Meier survival curve was calculated to determine survival in the animal experiments and patients in tissue microarray. All P values were 2-sided and considered to be statistically significant if P was less than .05 (*P < .05, **P < .01, ***P < .001).
When the cell viability of PC9-BrM3 and parental PC9 cells treated by erlotinib was assayed, we found that the IC50 of erlotinib was 0.600 lmol/L for PC9-BrM3 cells and 0.222 lmol/L for parental PC9 cells ( Figure 1C). In PC9-BrM3 cells, YAP protein expression decreased after dose-dependent erlotinib treatment ( Figure 1D).
Western blotting showed that YAP protein expression did not change in K-ras siRNA-transfected H2030-BrM3 cells and that YAP protein expression was decreased in EGFR siRNA-transfected PC9-BrM3 cells ( Figure 1E).
The finding that p-YAP(S127)/YAP ratio decreased in metastatic NSCLC cell lines indicates that YAP stability increased. In the EGFR mutant cell line PC9-BrM3 (EGFR Dexon19 mutation), erlotinib treatment decreased YAP protein expression. In K-ras mutant H2030-BrM3 cells (K-ras G12C mutation), K-ras knockdown by K-ras siRNA did not decrease YAP protein expression.

| YAP activation originates at the transcription level in the metastatic NSCLC cell line H2030-BrM3
Quantitative PCR analysis of DNA copy number showed that parental H2030 and H2030-BrM3 cells had two copies of YAP (Figure 2A). YAP mRNA expression and that of the downstream genes CTGF and CYR61 significantly increased in H2030-BrM3 compared to parental H2030 cells (P < .01) ( Figure 2B,C). Finally, immunofluorescence staining showed that H2030-BrM3 cells had increased YAP staining compared to parental H2030 cells ( Figure 2D). Collectively, these findings indicate that YAP activation originates at the transcription level in the H2030-BrM3 cell line. Collectively, these results show that inhibition of YAP decreased expression of Hippo pathway downstream genes. We further found that YAP binds to the metastatic regulator serpin l1 promoter, and inhibition of YAP decreased serpin l1 expression at the protein and mRNA level. 3.6 | Inhibition of YAP by shRNA decreased nucleus YAP immunohistochemistry stain of mouse brain tumour tissues Brain tissues were collected from control and YAP shRNA groups for histological analysis after mice were killed. Haematoxylin and eosin (H&E) stain showed tumour metastasis within brain tissues (Figure 6A). YAP IHC staining was performed to see the late effect of shRNA transfection. The results showed that YAP IHC staining decreased in YAP shRNA brain tissues compared to control brain tissues ( Figure 6A). Our result shows that inhibition of YAP by shRNA decreased nucleus YAP IHC stain of mouse brain metastatic tumour tissues.

| DISCUSSION
Our study provides several lines of evidence that suggest the Hippo/YAP signalling pathway is important in promoting metastasis of lung adenocarcinoma. First, we found that H2030-BrM3 (Kras G12C mutation) and PC9-BrM3 (EGFR Dexon19 mutation) cells have According to recent studies, YAP takes over K-ras as a cancer driver in NSCLC. [23][24][25] A previous study also identified that some NSCLC cell lines harbouring K-ras mutations were not K-ras-dependent cells, and that for those cell lines, K-ras may be not a therapeutic target. 22 Moreover, H2030 cells (K-ras G12C mutation) are not responsive to therapies targeting K-ras, mitogen-activated protein (MAP)-ERK kinase (MEK) and the extracellular signal-regulated kinase (ERK) signalling pathway. [29][30][31][32] In our study, inhibition of K-ras by K-ras siRNA in H2030-BrM3 cells did not decrease YAP protein expression, which suggests these cells may not be K-ras-dependent.
Therefore, we investigated YAP as a therapeutic target for metastasis of H2030-BrM3 cells because they have increased YAP mRNA and YAP protein expression.
We recently reported crosstalk between Hippo/YAP and EGFR/extracellular signal-regulated kinase signalling pathways in human NSCLC cells. 19,21 Collectively, PC9-BrM3 cells are dependent on the EGFR signalling pathway and inhibiting that pathway can inhibit the YAP signalling pathway and control metastasis of PC9-BrM3 cells.
Yes-associated protein transcription can be stimulated by SIRT1, 37 and SIRT1 is highly expressed in NSCLC brain metastasis tissues. 38 Together, these finding may partially explain the increase in YAP mRNA expression level in H2030-BrM3 cells as shown in our study. Much work is needed to verify the source of increased YAP transcription in metastatic NSCLC.
The activation of YAP forms autocrine loops with the ERBB pathway to promote cancer progression, migration and invasion in various cancers. [39][40][41][42] Moreover, Hippo downstream genes CTGF and CYR61 have been reported to activate integrin-mitogen-activated protein kinase (MAPK) and the AKT signalling pathway and promote cancer progression and metastasis in cancer. [43][44][45][46] MAPK and the AKT pathways negatively regulate Hippo kinase and reduce p-YAP (S127). 47,48 The decreased p-YAP(S127)/YAP ratio indicates an increase in YAP stability, and then more YAPs enter nucleus to activate downstream gene expression. These findings may explain our finding that YAP mRNA level increased modestly (about 1.5-fold), but the reduction in p-YAP was more prominent. The mechanism of high metastatic potential in H2030-BrM3 cells is summarized in Fig-ure 5E.
In our study, YAP protein expression decreased more than 70% after YAP knockdown by shRNA and siRNA, but the decrease in GTIIC reporter activity and CTGF mRNA expression was not as great as the decrease in YAP protein in our in vitro experiments. Actually, YAP does not account for all the GTIIC reporter activity and other mediators exist. 49 Like GTIIC reporter activity, CTGF mRNA expression did not decrease as markedly as YAP protein after shRNA and siRNA transfection in H2030-BrM3 cells because YAP is one of several factors that control CTGF expression; for example, TGF-b can control CTGF expression. 50,51 Serpin I1 (neuroserpin) is an important regulator in promoting brain metastasis of lung adenocarcinoma H2030-BrM3 cells in murine models. Serpin I1 helps cancer cells to infiltrate brain by inhibiting plasminogen activator (PA)-plasmin system. 27 In our study, sequence analysis of serpin I1 revealed a putative YAP binding site more than 6 kb upstream of the transcription start site, and it is known that YAP can bind distal enhancers through TEAD to control transcription of downstream genes. 52 Using a ChIP assay, we showed that YAP binds to the serpin l1 enhancer in H2030-BrM3 cells, and inhibiting YAP by YAP shRNA and YAP siRNA decreased serpin I1 protein and mRNA expression in H2030-BrM3 cells. These results suggest that YAP signalling may be involved in regulating serpin I1, and through inhibition of YAP can partly suppress serpin I1 expression and then decrease the potential of brain metastasis in H2030-BrM3 cells. These results also explained that inhibition of YAP by shRNA did not suppress proliferation ability of H2030-BrM3 cells markedly, but brain metastasis occurred late in the in vivo experiment of shRNA group.
To our knowledge, our study is first to provide the key evidence that direct inhibition of YAP by shRNA suppresses lung HSU ET AL.
| 3081 adenocarcinoma H2030-BrM3 cell metastasis in a murine model. This is one the best metastasis models that are currently available using human lung cancer cells. Most unresectable lung cancer patients (e.g., stage IV lung cancer) are at stage of colonization, and the treatment options for these patients are very limited (e.g., TKI and immunotherapy). Targeting this stage can be beneficial to patients with late-stage lung cancer, although we also wish to find a better metastasis models (e.g., orthotopic models) that can help us to validate whether YAP is indeed a driver for the entire metastasis process (as suggested in in vitro transwell experiments). 6,26,27,53 Our study shows that YAP plays a key role in promoting brain metastasis in lung adenocarcinoma H2030-BrM3 cells and that inhibition of YAP can suppress brain metastasis in vivo. These findings may have implications for future potential therapeutic strategies for F I G U R E 5 Inhibition of YAP by shRNA suppressed metastatic ability of H2030-BrM3 cells in a murine model (control shRNA n = 10, YAP shRNA#1 n = 5, YAP shRNA#2 n = 5). A, Bioluminescence images of mice inoculated with control shRNA-transfected H2030-BrM3 cells, YAP shRNA#1-transfected and YAP shRNA#2-transfected H2030-BrM3 cells. B, Tumour metastasis burden in control mice and mice inoculated with YAP shRNA-transfected H2030-BrM3 cells based on photon flux (photons per second) (***P ≤ .001). C, Kaplan-Meier survival curves for control mice and mice inoculated with YAP shRNA-transfected H2030-BrM3 cells. D, Summary of the hypothetical model of our study. In H2030-BrM3 cells, activation of YAP occurs at transcription mRNA level and increases YAP protein expression and mRNA expression of downstream genes CTGF and CYR61. CTGF/CYR61 forms an autocrine loop to activate the MAPK signalling pathway in H2030-BrM3 cells. The enhancement of the MAPK signalling pathway increases the invasion and migration abilities and metastatic potential of H2030-BrM3 cells F I G U R E 6 Histological analysis of mice brain tissue. A, H&E stain of brain tissues from control mice and mice inoculated with YAP shRNA-transfected H2030-BrM3 cells. B, YAP IHC stain of brain tissues from control mice and mice inoculated with YAP shRNA-transfected H2030-BrM3 cells. The scale bar = 100 lm K-ras mutant metastatic lung adenocarcinoma. More studies to validate YAP as a therapeutic target for K-ras mutant human metastatic lung adenocarcinoma are warranted.