The Mechanism Of HDAC9 In Glioblastoma Cell Proliferation And Tumorigenecity

In this study, to examine the role and mechanism of HDAC9 in glioblastoma, we first analyzed the HDAC9 expression. HDAC9, like most class Ⅱ HDACs, has a conserved histone deacetylase domain, catalyzes the removal of acetyl moieties in the N-terminal tail of histones, and possesses a long regulatory N-terminal domain to interact with tissue-specific transcription factors and co-repressors. The amino-terminal domain contains highly conserved serine residues that are subjected to phosphorylation. Signal-dependent phosphorylation of HDAC9 is a critical event that determines whether it is localized in the cytoplasm or nucleus. High expression of HDAC9 has been reported in many cancers.The cell cycle progression usually regulates cell proliferation. EGFR is a member of ErbB family, which is activated by phosphorylating the tyrosine kinase moiety. Following EGFR activation, three major intracellular signaling pathways are activated, including the PI3K/AKT kinase pathway, the Ras-ERK cascade, and the STAT3-dependent signaling events. EGFR activation drives cell cycle progression. Recently, TAZ has been identified as an oncogene and has an important role in tumorigenicity of many malignancies, including breast cancer, non-small cell lung cancer, gastric cancer, colon cancer and papillary thyroid carcinoma. Furthermore, there’s evidence to show that HDAC inhibitors (HDIs) could decrease the TAZ expression.We performed qRT-PCR and western blot analysis in glioblastoma cell lines and primary cells. To detect the effect of HDAC9 on glioblastoma cell proliferation, we undertook knock-down of HDAC9 in U87 and LN229 cells by infection with lentivirus-expressing shRNA using shGFP as a negative control. And then we performed MTT and BrdU staining to detect the proliferation capabilities of the two groups. To elucidate whether HDAC9 had an effect on cell cycle, the U87 and LN229 cell cycle was analyzed by flow cytometry. We next examined the effect of HDAC9 down-regulation on the phosphorylated status of EGFR and its downstream effectors AKT and ERK1/2 by western blot analysis in U87 and LN229 cells. And then to examine the effect of down-regulation of HDAC9 on glioblastoma cells in vivo, xenograft shHDAC9 tumors and shGFP tumors were established in SCID mice. We detected HDAC9 after immunoprecipitation of endogenous TAZ from lysates of GBM cells. At last, we forcedly expressed TAZ in HDAC9-knockdown cells, which induced TAZ down regulation and found that all the effects induced by HDAC9 silencing were abrogated.In this study, we found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.