Yes-associated protein (YAP) promotes progression of glioma and poor prognosis of human

Background: Yes-associated protein(YAP) plays an important role in signal transduction and gene transcription regulation in 1 normal cells, with elevated and over-expressed YAP levels observed in various malignant tumors. The aim of this study was 2 to investigate the expression of YAP in malignant glioma, and to study the possible relationship of YAP expression with the 3 occurrence and development of malignant glioma. 4 Methods: Immunohistochemical staining was used to assess the expression of YAP and phosphor-YAP in malignant glioma 5 tissue and normal brain tissue, and their protein and mRNA levels were evaluated through Western blotting and reverse 6 transcription-polymerase chain reaction (RT-PCR), respectively. Normal brain tissue obtained from the functional lesion of 7 the epilepsy patients. After transfection of YAPsiRNA oligonucleotides or pcDNA3.1-hYAP plasmid, their effects on glioma 8 cells were investigated using western blot, cell proliferation, cycle, apoptosis and invasion, respectively. We conducted the 9 co- Immunoprecipitation to verify the combination of YAP and PPARγ, explore t he mechanism of action. 10 Results: YAP-positive expression was found in 9 cases of normal brain and 60 cases of glioma. A significantly higher 11 expression of YAP in glioma tissue as compared with normal brain tissue at both protein and mRNA levels, and YAP proteins 12 mainly expressed and located in the nucleus and only a small percentage in the cytoplasm of glioma tissue. Phosphor-YAP 13 protein expression showed high staining of the cytoplasm, but no staining of the nuclear. While, with the enhancement of 14 the malignant degree, the cytoplasm YAP(p-YAP) expression is lower gradually than normal brain tissues. Further study in 15 glioma cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted the cell 16 proliferation, cycle, invasion a nd inhibited the cell apoptosis. Moreover, YAP in company with PPARγ regulat es the cell 17 proliferatin and effects the gliomagenesis. 18 Conclusion: These results indicate that YAP plays an important role in glioma and might be a useful therapeutic target of 19 glioma. 20


Introduction
Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain 23 tumors. Despite the advances in surgery, radiation therapy, and chemotherapy, the prognosis of patients with GBM has not 24 been improved significantly over the past 20 years [1] . It is imperative to have a detailed and comprehensive understanding 25 of the molecular pathogenesis of the gliomas for developing novel strategies in treatment. 26 The Hippo pathway is an important regulator of cell growth, proliferation, and apoptosis. It was first discovered by genetic 27 mosaic based screens in Drosophila melanogaster [2,3] ; however, there is an increasing body of evidence demonstrating that 28 the Hippo pathway also limits organ size in mammalian systems [4] by inhibiting cell proliferation and promoting apoptosis. 29 Components of this pathway are highly conserved in mammals, and include Mst1/2; WW45; Lats1/2; Mob1; YAP; TAZ; NF; 30 and 118 glioma cell which had transfented the pcDNA3.1-hYAP plasmid. Then we observed the glioma cell growth by MTT 75 assay. 76 The YAPsiRNA were obtained from Genepharma Co,Ltd(Shanghai,China): (5'-CUG CCA CCA AGC UAG AUA 77 ATT--3'),targeting the YAP gene; and the negative control duplex,(5'--UUA UCU AGC UUG GUG GCA 78 GTT--3').Oligonucleotides (20pmol/µl) were transfected into SNB19 and LN229 cells at 70% confluence using X-tremeGENE 79 siRNA Transfection reagent according to the manufacture's instruction (Roche,Germany).

98
Glioma cells were seeded into 96-well plates at 4000 cells per well. After transfection as described previously, on each day 99 of consecutive 6 days,20µl MTT (5mg/mL) was added into each well and the cells were incubated at 37℃ for additional 4 00 hours, and the supernatant was discarded. The reaction was then terminated by lysing the cells with 200µl of DMSO. 01 Optical density was measured at wavelength of 490nm and expressed as percentage of control. The data are presented as 02 the mean±SEM, which were derived from triplicate samples of at least three independent experiments. 03 and placed in the lower chamber. After 24-hour incubation at 37℃, the filters were gently taken out, the medium was 19 removed from the upper chamber. The non-invaded cells on the upper surface of the inserted filter were scraped off with a 20 cotton swab. The cells that had migrated into the lower surface of the inserted filter were fixed with methanol.The number 21 of cells invading through the matrigel was counted using three randomly selected visual fields under inverted microscope. 22

Flow Cytometric Analysis
Co-Immunoprecipitation 23 For immunoprecipitation using 1.5 million LN229 cells, 30µl of matrix beads (Thermo Scientific; USA) were labeled by 24 resuspension in 200µl of calcium-free PBS containing 10µg of antibody, incubated for 1h at room temperature with 25 end-over-end mixing using a rotator, washed twice with calcium-free PBS, and resuspended in 100 µl of lysis buffer (50 mM 26 Tris HCl, pH7.4, 1% Triton X-100 and protease inhibitors). The cells were resuspended for 15 min in 110µl of lysis buffer. 27 After full-speed centrifugation of the cell lysate (10 min at 4°C), 10µl of supernatant was collected for protein expression 28 analysis and the remaining 100µl was subjected to immunoprecipitation by incubation with the antibody-labeled beads 29 overnight at 4°C using a rotator. Centrifuge the beads for 30 seconds at 5,000- 8,200×g. Remove the supernatants with a 30 narrow-end pipette tip. Wash the beads three times with 500µl of ice-cold TBS. Immunoprecipitated proteins were 31 released by incubation of the beads in SDS-PAGE loading buffer (50 mM Tris HCl, pH7.4, 10% glycerol, 2% SDS, 0.05% 32 bromophenol blue, 100mMβ-mercaptoethanol) for 3 min at 95°C. Non-immunoprecipitated proteins previously collected 33 were used as a control for protein expression level. 34

35
The statistical package SPSS 18.0 (SPSS, Windows) was used for all analyses as described previously. All values are expressed 36 as mean ± SD. The χ2-test was used to determine the correlation between YAP expression and clinicopathologic factors. All 37 P-values were based on the two-sideds tatistical analysis and a P-value of <0.05 was considered to indicate statistical 38 significance. 39

42
Expression of YAP in human glioma and poor prognosis 43 We next evaluated YAP expression and cellular location in 60 tumors of various World Health Organization(WHO) grades. 44 Normal brain tissues (9/9,100%) showed no staining of nucleus. In contrast, very high proportion of WHO grade 45 astrocytoma (7/11,63.6%),WHO grade Ⅱ astrocytoma (8/11,72.7%),WHO grade Ⅲ glioblastoma (21/22,95.5%),and 46 WHO grade Ⅳglioblastoma multiforme (GBM) (16/16,100%) showed at least some nuclear reactivity( Table 2).Within the 47 group of astrocytoma, high-grade tumors, including glioblastoma and glioblastoma multiforme (GBM), showed greater high 48 level staining than normal brain tissues. Moreover, YAP expression contributes to the poor prognosis of glioma. (Figure 1) 49 Cytoplasmic YAP(phosphor-YAP) was detected in 60 tumors of various WHO grades in line with the YAP staining. Total 50 normal brain tissues(9/9,100%)showed high staining of the cytoplasm, but no staining of the nuclear. While, with the 51 enhancement of the malignant degree, the cytoplasm YAP(p-YAP) expression is lower gradually than normal brain tissues. 52 ( Figure 2A) 53 The YAP protein was mainly accumulated in the nucleus with a lesser cytoplasmic presence( Figure 2B).We also verified by 54 qRT-PCR and Western blot which were identical with the results above ( Figure 3). 55

56
YAP activity was analyzed in ten human glioma cell lines by assessing levels of YAP protein relative to phosphor-S127-YAP. 57 Both the level of YAP and ratio of YAP to phosphor-S127-YAP varied greatly between cell lines ( Figure 4A). The results were 58 in line with the qRT-PCR (Table 3) of YAP expression( Figure 4B).For further study, we chose cell lines with low baseline YAP 59 activity (U87 and 118) and the cell line that showed the highest baseline YAP activity(SNB19 and LN229),based on the fact 60 that it had less Hippo pathway-dependent inhibitory phosphorylation at S127(Figure4). 61 To determine whether YAP plays a role in glioma cell proliferation, MTT assays was performed. The mRNA level and protein 63 level were assessed 48 hours later of transfection. Relative expression level of YAP was analyzed by real-time PCR in a panel 64 of glioma cell lines. The SNB19 and LN229 cell lines had higher YAP levels while the U87 and 118 cell lines had lower levels. 65 We knocked down YAP expression in SNB19 and LN229 cells using YAP-specific siRNA. Efficient depletion of YAP expression 66 was confirmed by western blot and qRT-PCR analysis (Fig.5). YAP depletion in SNB19 and LN229 cells reduced the 67 proliferation rate ( Figure 7A-B). 68 We also overexpressed YAP in U87 and 118 cells. We then investigated the difference in apoptosis ratio between the overexpression YAP groups and the control and empty 02 vector group to confirm our retrospective cohort study results. Flow cytometry analysis showed that the apoptosis ratio 03 was decreased in U87 and 118 cells transfected with pcDNA3.1-hYAP plasmid from 9.22%and 10.0% to 2.80%and 2.57% in 04 cells of control groups and 9.31% and 9.08% in empty vector group, respectively. (Figure 10 Right) 05 Collectively, these results indicated that YAP expression played a role in promoting cell proliferation and suppressing cell 06 apoptosis. 07 Moreover,we detected also the expression of Ki-67,MMP-9,CyclinD1,BCL-2 by Western blotting (Figure 11). The results 08 were in line with those above. 09

10
Co-Immunoprecipitation experiments were performed on total protein from the LN229 glioma cells showed that YAP 11 interacted with PPARγ, suggesting a function at the level of protein (Figure 12) . To determine whether PPARγ plays a role in 12 glioma cell proliferation, MTT assays was performed. When we transfected the PPARγ plasmid, the proliferation ability can 13 decrease vividly( Figure 13). 14

15
Glioblastoma is the most common subtype of primary brain tumor in adults. These tumors are very aggressive with a 16 highly invasive capacity and often infiltrate critical neurological areas within the brain [14] . Glioblastoma cells are notoriously 17 resistant to apoptosis, a characteristic that contributes toward the failure of most standard clinical treatments [15] . Current 18 research in this area is driven by the need to discover new agents that will be effective and have few side effects. 19 Overexpression of YAP has been implicated in tumor progression in various human cancers [16] . However, the 20 expression of YAP and its correlation with clinicopathologic factors have not been defined in glioma. In this study, we 21 demonstrated that overexpression of YAP in glioma tissues was significantly correlated with grades malignancy of glioma. 22 Furthermore, YAP overexpression was associated with poor prognosis of glioma patients. 23 In previous studies, YAP expression and nuclear localization was found to be elevated in hepatocellular carcinoma, 24 prostate cancer, colon cancer, and breast cancer [6,10,16] . A study of 28 cases of lung adenocarcinoma subjected to tissue 25 microarray showed that YAP was expressed in 54% of cases [6] . Clinical study in 177 hepatocellular carcinoma patients 26 showed that YAP could serve as an independent predictor for hepatocellular carcinoma-specific disease-free survival and 27 overall survival [16] . Consistent with these findings, the present report showed that the level of YAP was increased in glioma 28 tissues, which was mainly accumulated in the nucleus with a lesser cytoplasmic presence. Importantly, YAP positive 29 expression was associated with reduced overall survival in glioma patients, but did not serve as an independent predictor 30 for patient survival. Taken together, these results indicate that YAP is an oncogenic protein overexpressed in glioma and is 31 associated with glioma progression. 32 YAP is acknowledged as a candidate oncogene, and became the focus of research, after it was identified in human 33 chromosome 11q22 amplicon [17] . To address the impact of YAP on proliferation in glioma, we overexpressed and silenced 34 YAP in glioma cell lines. We found that overexpression of YAP resulted in a marked increase of cell growth rate, while 35 depletion of YAP greatly suppressed cancer cell proliferation. Moreover, consistent with previous studies [10] , we found that 36 YAP was able to overcome cell contact inhibition. The YAP-overexpressed U87 and 118 cells kept proliferating and resulted 37 in a higher saturation density than control cells. 38 In this study, we showed that YAP overexpression significantly promoted glioma cell invasion, accelerated the cycle of 39 glioma cells in the G0/G1 phase and inhibited the cell apoptosis. Nevertheless, YAPsiRNA knockdown blocked cell invasion, 40 reduced cell proliferation, arrested the cycle of glioma cells in the G0/G1 phase and promoted the cell apoptosis. These 41 results implied YAP may play a oncogene role in gliomagenesis. 42 Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that were first 43 discovered 20 years ago [18] . There are three isoforms of PPAR; PPAR-α, PPAR-β/δ (also known as PPAR-β or PPAR-δ), and 44 PPAR-γ [19] . These have been found in all the mammalian species that have been examined to date [20,21] . PPAR-γ is highly 45 expressed in cancer cells, and treatment with PPAR-γ ligands can induce cell differentiation and apoptosis [22,23] . Although it 46 remains unclear whether PPARs are oncogenes or tumor suppressors, research has also been focused on this receptor 47 because of its involvement in various metabolic disorders that are known to be associated with cancer risk [24,25] .To further 48 explore the mechanisms by which YAP promoted glioma cells, we performed the Co-Immunoprecipitation. We observe that 49 YAP protein can deposit PPARγ mutually by western blot. Then, we transduced the PPARγ plasmid into the U87 and 118 50 glioma cells which had transfected the pcDNA3.1-hYAP plasmid, the cell proliferation increased significantly in contrast to 51 control group, suggesting PPARγ maybe acted as tumor suppressor gene in gliomagenesis.However,the concrete 52 machanisms is still unknown. 53 However, YAP was also reported to be a tumor suppressor as its gene locus was deleted in some breast cancers with a 54 correlated loss of YAP protein expression [11] . Yap/Taz transcriptional activity is essential for vascular regression via Ctgf 55 expression and actin polymerization [26] .In addition, some studies have identified YAP as a transcriptional coactivator 56 promoting anti-tumoral effects on the basis of its interaction with p53 family members, p73, and p53-binding 57 protein-2 [27] .These results present a rather intriguing question: is YAP1 an anti-or pro-tumorigenic protein? One possible 58 explanation is that pathways are not fixed but can dynamically change depending on the context [28] . YAP contributes to 59 glioma cell migration and invasion by regulating N-cadherin and Twist, as well as cytoskeletal reorganization [29] .Thus, it is 60 possible that YAP function changes depending on the upstream input, and the variations of binding partners' repertoire 61 between cells. 62 In conclusion, this study identifies YAP as an oncoprotein overexpressed in glioma which is important for the 63 regulation of malignant cell growth, invasion and apoptosis. Moreover, YAP expression contributes to the poor prognosis of 64 glioma, making it a candidate target protein for future cancer therapeutics. Additional work is needed to elucidate the 65 molecular functions of YAP in glioma.

Score Grading
Complete absence of reactivity Negtive