AIDS Prevention and Research Center, National Yang-Ming University, Shih-Pai, Taipei, Taiwan.
Mol Med. 2012 Mar 30;18(1):286-96. doi: 10.2119/molmed.2011.00331.
Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05-11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60-12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.
甘氨酸 N-甲基转移酶（GNMT）是肝细胞癌（HCC）的肿瘤抑制因子。高比例的 Gnmt 基因敲除小鼠发展为 HCC。表观遗传改变和几条通路的失调，包括无翅型 MMV 整合位点（Wnt）、丝裂原激活的蛋白激酶（MAPK）和 Janus 激酶和信号转导及转录激活因子（JAK-STAT），与 Gnmt 基因敲除小鼠的 HCC 发展有关。我们假设 GNMT 可能通过与其他蛋白质直接相互作用来调节信号转导。在本报告中，我们通过酵母双杂交筛选，鉴定出雷帕霉素的哺乳动物靶标（mTOR）抑制剂（DEP 结构域包含 MTOR 相互作用蛋白 [DEPDC6/DEPTOR]）作为 GNMT 结合蛋白。荧光共振能量转移（FRET）测定表明，GNMT 的 C 端与 DEPDC6/DEPTOR 的 PSD-95/Dlg1/ZO-1（PDZ）结构域相互作用。免疫组织化学染色显示，在肿瘤组织中，27.5%（14/51）的 HCC 患者的 DEPDC6/DEPTOR 表达水平高于肿瘤旁组织，尤其是乙型肝炎病毒感染（优势比 10.3，95%置信区间 [CI] 1.05-11.3）或预后不良（死亡风险比 4.51，95% CI 1.60-12.7）的 HCC 患者。在分子机制方面，在 HuH-7 细胞中敲低 DEPDC6/DEPTOR 的表达会导致 S6K 和 4E-BP 的激活，但抑制 Akt。DEPDC6/DEPTOR 的过表达会激活 Akt 并增加 HCC 细胞的存活率。过表达 GNMT 会导致 mTOR/raptor 下游信号的激活，并延迟 G2/M 细胞周期进程，从而导致细胞衰老。此外，GNMT 减少 HuH-7 细胞的增殖，并在体外和体内增强它们对雷帕霉素治疗的敏感性。总之，GNMT 通过与 DEPDC6/DEPTOR 相互作用并调节 mTOR/raptor 信号通路来调节 HCC 的生长。GNMT 和 DEPDC6/DEPTOR 都是开发 HCC 治疗方法的潜在靶点。
