Yes-Associated Protein (YAP) Modulates Human Glioma Cell Growth

Gliomas are the common and incurable primary primary brain tumors in the adult population. Glioblastoma multiforme (GBM) is one of the most malignant and highly invasive glioma, representing up to 50% of all primary gliomas. Despite progression in surgical techniques, radiotherapy, chemotherapy and biological therapy, the prognosis remains poor with a median survival less than one year. Glioblastoma is characterized by rapid diffusely infiltrative growth and high level of cellular heterogeneity associated with therapeutic resistance. At present, it has been demonstrated that gliomas associated with genetic disorder, such as epidermal growth factor receptor (EGFR) amplification, PTEN deletion, p53 mutations, Wnt/beta-catenin, PI3K/AKT/mTOR, Notch pathway are involved in the molecular pathogenesis of malignant gliomas. Therefore looking for glioma related genes, has become one of the important hotspots in glioma research area,which will contribute to study of the pathogenesis of glioma and exploring new strategies and potential targets for treatment of glioma.The Hippo pathway is a newly discovered and evolutionally conserved signaling pathway. It regulates organ size by inhibiting cell proliferation and promotes cell apoptosis. Hippo pathway is conserved and plays a role as a tumor suppressor in mammals. YAP/TAZ are the major downstream effectors of the Hippo pathway. Similar to the invertebrate cascade, a series of phosphorylation events via MST and LATS ultimately leads to the phosphorylation of YAP and TAZ. In addition to cytoplasmic detention by 14-3-3,YAP/TAZ phosphorylation by the kinases LATS and CK1 leads to β trcp-dependent proteasomal degradation of YAP/TAZ. In the nucleus, both YAP and TAZ can bind to the TEAD1/4 and activate the transcription of genes required to promote cell growth and inhibit apoptosis. Disorders of the pathway are very frequently detected in human cancers. Indeed, YAP plays a tumor suppressive role in some cancers. However, so far only one study on YAP in gliomas has been reported.This study aimed to examine YAP expression in glioma and explored the effect of YAP on biological characters of glioma cells.In the first part of this study, YAP expression level was detected in 60 glioma samples with dfferent grades and 9 normal brain tissues from tissue array,20 freshly resected glioma samples of different grades and 3 normal brain tissues as well as 8 malignant gliomas cell lines by immunohistochemical staining, real-time PCR and Western blot analysis. The results showed that YAP mRNA was upregulated in 8 malignant gliomas cell lines. YAP expression was increased most significantly in SNB19 and LN229 and a less degree in U87 and 118 cell lines. The result of Western blotting in glioma cell lines was consistent with that of real-time PCR. In addition, the expression level of YAP in gliomas samples of tissue array was significantly increased in comparison to that of the normal brain tissues. Immunostaining of YAP was located in the nucleus of cells, suggesting the activation of YAP. In addition, YAP expression was positively correlated with the tumor grades.The second part focused on the effect of Yes-associated protein on biological behavior of glioma cells. YAPsiRNA mediated by Lipofectamine was transfected to LN229 and SNB19 glioma cells for knocking down the YAP, and pcDNA3.1-hYAP or control pcDNA3.1 plasmid was transfected into U87 and 118 glioma cells for forced overexpression of YAP. YAP expression was identified by Western blot analysis and Real-time PCR. The cell invasion activity was examined by Transwell assay. The cell proliferation ability was determined by MTT. Flow cytometry and Annexin V staining were used to detect the cell cycle and apoptosis respectively. The results showed that:1) the expression of YAP in the cells transfected with YAPsiRNA was significantly reduced. The cell proliferation activity of SNB19 cells and LN229 was inhibited. The cell cycle was arrested in Go/Gi phase, cell invasive ability was attenuated, and apoptotic cells were increased in cells transfected with YAPsiRNA as compared to scramble and control groups.2)The expression of YAP in U87 and 118 cells transfected with pcDNA3.1-hYAP was significantly enhanced. The cell proliferation activity of glioma cells was increased. The proportion of G0/G1 phase was decreased, but S phase was increased, cell invasive ability was strengthened, and apoptotic cells were decreased in cells transfected with pcDNA3.1-hYAP as compared to those of the cells transfected with control pcDNA3.1 plasmid and control cells. After transfected the YAPsiRNA, the expression of proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase 9(MMP-9), CyclinDl and BCL-2 were downregulated. Moreover, these indicators were upregulated, which transfected in pcDNA3.1-hYAP of glioma cells.Conclusions:1. YAP gene expression is upregulated in malignant glioma cell lines and glioma specimens than in the normal counterparts, and is positively correlated to tumor grades, suggesting that YAP may be considered as a gene involved in glioma pathogenesis and the hippo pathway was inhibited in glioma.2. Knocking-down YAP expression in glioma cells by siRNA could inhibit the proliferation activity and invasive ability of glioma cells and induce cells apoptosis. Forced YAP overexpression by transfection with YAP plasmid in glioma cells could increase the proliferation activity and invasive ability of glioma cells and inhibit cells apoptosis.3. The study demonstrates that YAP promotes glioma cell growth. YAP can be considered as a potential target for glioma gene therapy. YAP and Hippo pathway are closely associated with gliomagenesis, and deserved for further study.