Defining the molecular mechanisms regulating the hexosamine-N-glycosylation pathway in glioblastoma

定义胶质母细胞瘤中调节己糖胺-N-糖基化途径的分子机制

• 批准号: 10204955
• 负责人: ARNAB CHAKRAVARTI
• 金额: $ 30.15万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204955
• 关键词: Adult; Anabolism; Automobile Driving; Binding; Binding Sites; Biology; Cancer Biology; Cells; Clinical; Data; Development; Enzymes; Epidermal Growth Factor Receptor; Foundations; Genes; Genetic; Genetic Transcription; Glioblastoma; Glucose; Glycolipids; Glycoproteins; Growth; Hexosamines; In Vitro; Knowledge; Label; Laboratories; Lentivirus; Lipid Synthesis Pathway; Lipids; Malignant Neoplasms; Measurement; Measures; Mediating; Messenger RNA; Metabolism; Modification; Molecular; Molecular Target; Mutate; Mutation; N-Glycosylation Site; Oncogenic; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmacology; Play; Polysaccharides; Primary Brain Neoplasms; Promoter Regions; Proteins; Public Health; Regulation; Reporting; Research; Role; SRE-1 binding protein; Sampling; Signal Transduction; Testing; Therapeutic; Transcriptional Regulation; Tumor Tissue; Up-Regulation; Xenograft Model; base; brain tissue; cancer cell; cohort; combat; epidermal growth factor receptor VIII; experimental study; fructose-6-phosphate; glycosylation; improved; in vivo; insight; lipid biosynthesis; lipid metabolism; mutant; novel; small hairpin RNA; transcription factor; tumor growth

ABSTRACT Glioblastoma (GBM) is the most common primary brain tumor with a median survival of only 12-15 months despite advanced therapies. These disappointing clinical outcomes indicate that more efforts are required to better understand GBM pathogenesis in order to provide the foundation for identifying new effective approaches to target GBM. Research in our laboratories has focused on understanding the underlying mechanisms driving metabolism alterations in GBM. We demonstrated that SREBP-1, a master transcription factor regulating lipogenesis, is highly upregulated in GBM by oncogenic EGFR/PI3K/Akt signaling. More recently, we found a novel molecular connection between the hexosamine biosynthesis pathway (HBP) and SREBPs. We demonstrated that glucose, through the HBP pathway, increases the N-glycosylation of SCAP, the key transporter for SREBPs, promoting SREBP activation and lipid synthesis. However, the mechanisms regulating the HBP-N-glycosylation pathway in GBM and other malignancies are poorly understood. Moreover, the importance of this pathway in GBM growth is also unknown. HBP is regulated by the GFAT1, GNPNAT1, PGM3 and UAP1 enzymes that sequentially convert fructose-6-phosphate to UDP-GlcNAc, the key metabolite for initial synthesis of N-glycan, which is regulated by the DPAGT1 enzyme. To explore how the HBP- N-glycosylation pathway is regulated in GBM, we performed multiple preliminary experiments showing that: 1) all the enzymes above are highly expressed in tumor tissues from GBM patients; 2) EGFRvIII, a constitutively active EGFR mutant, significantly upregulates the expression of these enzymes in GBM cells; 3) pharmacological inhibition of SREBP-1 markedly reduces the protein levels of all enzymes, while only downregulating the mRNA levels of GFAT1, PGM3 and UAP1; 4) removing N-glycan binding using PNGase significantly increased the mobility of GNPNAT1 and DPAGT1 on SDS-PAGE, indicating that both enzymes are modulated by N- glycosylation. Based on these preliminary data, we hypothesize that SREBP-1, activated by oncogenic EGFR signaling, transcriptionally upregulates GFAT1, PGM3 and UAP1 expression to enhance hexosamine synthesis, which in turn promotes the N-glycosylation of GNPNAT1, DPAGT1 and SCAP to increase their stability and activity, leading to a feedforward loop enhancing HBP-N-glycosylation and lipogenesis to promote GBM growth. In this study, we will determine the transcriptional regulation of GFAT1, PGM3 and UAP1 by EGFR-SREBP-1 signaling, and delineate the regulation and role of the N-glycosylation on GNPNAT1 and DPAGT1 proteins in GBM (Aim1). We will further elucidate the importance of HBP-N-glycosylation on GBM growth by examining the effects of genetic inhibition of GFAT1 or mutation of GNPNAT1 or DPAGT1 N-glycan binding sites on tumor growth (Aim 2). Completion of this study will significantly improve our understanding of GBM biology and provide insights for the development of potential new strategy to combat this deadly cancer.

摘要 胶质母细胞瘤（GBM）是最常见的原发性脑肿瘤，中位生存期仅为12-15个月 尽管有先进的治疗方法这些令人失望的临床结果表明，需要更多的努力， 更好地了解GBM的发病机制，以便为确定新的有效方法提供基础 以GBM为目标我们实验室的研究集中在理解驱动 GBM的代谢改变。我们证明了SREBP-1，一个调节转录的主转录因子， 脂肪生成在GBM中通过致癌EGFR/PI 3 K/Akt信号传导高度上调。最近，我们发现了一个 氨基己糖生物合成途径（HBP）和SREBP之间的新分子连接。我们 证明葡萄糖通过HBP途径增加SCAP的N-糖基化，这是SCAP的关键。 SREBP转运蛋白，促进SREBP活化和脂质合成。然而，机制 对GBM和其它恶性肿瘤中HBP-N-糖基化途径的调节知之甚少。 此外，该途径在GBM生长中的重要性也是未知的。HBP受GFAT 1调节， GNPNAT 1、PGM 3和UAP 1酶依次将果糖-6-磷酸转化为UDP-GlcNAc， 在DPAGT 1酶的调节下，N-glycan的初始合成代谢产物。为了探索HBP- N-糖基化途径在GBM中受到调节，我们进行了多个初步实验，结果表明：1） 所有上述酶在GBM患者的肿瘤组织中高度表达; 2）EGFRvIII，组成型 活性EGFR突变体，显著上调GBM细胞中这些酶的表达; 3）药理学 SREBP-1的抑制显著降低了所有酶的蛋白水平，而仅下调了mRNA水平。 GFAT 1、PGM 3和UAP 1水平; 4）使用PNGase去除N-聚糖结合显著增加了 GNPNAT 1和DPAGT 1在SDS-PAGE上的迁移率，表明这两种酶都受N- 糖基化基于这些初步数据，我们假设SREBP-1，由致癌EGFR激活， 信号，转录上调GFAT 1，PGM 3和UAP 1表达，以增强己糖胺 合成，这反过来又促进GNPNAT 1，DPAGT 1和SCAP的N-糖基化，以增加它们的 稳定性和活性，导致前馈环增强HBP-N-糖基化和脂肪生成， 促进GBM生长。在本研究中，我们将确定GFAT 1，PGM 3和UAP 1的转录调控 通过EGFR-SREBP-1信号转导，并描述了GNPNAT 1和 GBM中的DPAGT 1蛋白（Aim 1）。我们将进一步阐明HBP-N-糖基化对GBM的重要性 通过检查GFAT 1的遗传抑制或GNPNAT 1或DPAGT 1 N-聚糖突变的影响， 肿瘤生长的结合位点（Aim 2）。这项研究的完成将大大提高我们对 GBM生物学，并为开发对抗这种致命癌症的潜在新策略提供见解。