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

摘要 胶质母细胞瘤(GBM)是最常见的原发脑肿瘤，中位生存期仅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，需要做出更多努力来 更好地了解基底膜的发病机制，为寻找新的有效治疗方法提供基础 将目标对准GBM。我们实验室的研究重点是了解驱动的潜在机制 基底膜的代谢改变。我们证明了SREBP-1，一个主要的转录因子，调节 脂肪生成，通过致癌的EGFR/PI3K/Akt信号在GBM中高度上调。最近，我们发现了一个 己糖胺生物合成途径(HBP)与SREBPs之间的新的分子联系。我们 证明葡萄糖通过HBP途径增加SCAP的N-糖基化，这是关键 SREBPs的转运体，促进SREBP的激活和脂质的合成。然而，这些机制 在GBM和其他恶性肿瘤中，对HBP-N-糖基化途径的调控知之甚少。 此外，该途径在GBM生长中的重要性也是未知的。HBP受GFAT1监管， GNPNat1、PGM3和UAP1酶依次将果糖-6-磷酸转化为UDP-GlcNAc，这是关键 N-糖链初始合成的代谢物，由DPAGT1酶调节。为了探索HBP是如何- N-糖基化途径在GBM中受到调控，我们进行了多个初步实验，结果表明：1) 所有这些酶在GBM患者的肿瘤组织中都高度表达；2)EGFRvIII，一个结构性的 活性的EGFR突变体显著上调GBM细胞中这些酶的表达；3)药理学 抑制SREBP-1显著降低所有酶的蛋白水平，而只下调mRNA GFAT1、PGM3和UAP1的水平；4)用PNGase去除N-糖链结合显著增加 GNPNat1和DPAGT1在SDS-PAGE上的迁移率，表明这两种酶都受到N- 糖基化。基于这些初步数据，我们假设SREBP-1，由致癌的EGFR激活 信号，转录上调GFAT1，PGM3和UAP1的表达以增强氨基己糖 合成，进而促进GNPNat1、DPAGT1和SCAP的N-糖基化，从而增加它们的 稳定性和活性，导致前馈环路增强HBP-N-糖基化和脂肪生成 促进GBM增长。在本研究中，我们将确定GFAT1、PGM3和UAP1的转录调控 通过EGFR-SREBP-1信号转导，阐明N-糖基化对GNPNA1和GNPNA1的调节和作用 GBM中的DPAGT1蛋白(Aim1)。我们将进一步阐明HBP-N-糖基化对GBM的重要性 检测GFAT1基因抑制或GNPNat1或DPAGT1 N-葡聚糖突变对生长的影响 肿瘤生长的结合部位(目标2)。这项研究的完成将大大提高我们对 GBM生物学，并为开发潜在的新战略来抗击这种致命的癌症提供了见解。

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.