Identifying SREBP-1 activation mechanism in glioblastoma and its new role in regulating glutamine metabolism

摘要 胶质母细胞瘤(GBM)是最具致命性的原发脑肿瘤，其预后未见明显改善。 在过去的二十年里。迫切需要找到有效的策略来治疗GBM，但我们对此只有部分认识 基底膜生物学的研究显著阻碍了这一进展。我们的实验室长期以来一直致力于了解GBM 生物学和最近证明，GBM重新编程脂代谢，以支持其快速增长。我们 发现固醇调节元件结合蛋白-1(SREBP-1)是一种主要的转录因子，调节 脂质合成在基底膜中高度激活，是肿瘤生长所必需的。然而，分子 激活癌细胞中SREBP-1和脂肪生成的机制尚不清楚。而且，无论是 高活性的SREBP-1在未知的恶性肿瘤中发挥着新的作用。在正常细胞中，SREBP的激活是 被胰岛素诱导基因(Insig)强烈抑制，Insig是一种内质网(ER)锚定蛋白。它绑定到 SREBP转运体，SREBP裂解蛋白(SCAP)，防止SREBP内质网到高尔基体的易位和 随后的激活。因此，SREBP-1激活的第一个关键步骤是将SCAP从Insig解离。多么 癌细胞能够释放Insig在SCAP上的紧密抑制以促进高水平的SREBP- %1激活仍未应答。我们最近发现，葡萄糖介导的N-糖基化可以稳定SCAP。 有趣的是，我们新的初步数据显示，在没有谷氨酰胺的情况下，SREBP-1未能被切割， 尽管SCAP仍然是糖基化的。抑制谷氨酰胺转化为谷氨酸和氨 抑制谷氨酰胺酶(GLS)可显著减少SREBP-1的裂解。这些数据有力地表明， 谷氨酰胺在SREBP-1的激活中起关键作用。有趣的是，表达活性的N-末端形式 GBM细胞中的SREBP-1显著促进谷氨酰胺摄取。对GBM患者样本的分析显示 谷氨酰胺转运蛋白SLC1A5在肿瘤组织中高表达，与患者的病情呈负相关 生死存亡。启动子分析显示，在SLC1A5启动子区域有多个可能的SREBP-1结合位点。 基于这些新的初步数据和我们发表的研究，我们假设SCAP从 Insig被谷氨酰胺衍生的代谢物激活，导致SREBP-1激活；反过来，激活 SREBP-1转录上调SLC1A5的表达，导致前馈环 增加谷氨酰胺摄取和脂肪合成，促进GBM生长。在这项研究中，我们将确定 AIM 1基因中谷氨酰胺激活SREBP-1和脂肪生成的分子机制及新发现 SREBP-1在调节谷氨酰胺代谢中的作用目的2。我们将进一步研究联合 抑制SREBP-1与抑制SLC1A5或GLS联合抑制GBM生长具有很强的协同作用 (目标2)。这项研究的完成将极大地促进我们对基底膜生物学和代谢的理解 重新连接，为GBM的治疗提供了有前景的方法。

ABSTRACT Glioblastoma (GBM) is the most lethal primary brain tumor, and its prognosis has no significantly improved in the past two decades. It is urgent to identify effective strategies to treat GBM, but our only partial understanding of GBM biology significantly hinders this progress. Our laboratory has long focused on understanding GBM biology and recently demonstrated that GBM reprograms lipid metabolism to support its rapid growth. We identified that sterol regulatory element-binding protein-1 (SREBP-1), a master transcription factor regulating lipid synthesis, is highly activated in GBM and is essential for tumor growth. However, the molecular mechanism activating SREBP-1 and lipogenesis in cancer cells remains unclear. Moreover, whether the highly activated SREBP-1 plays new roles in malignancy is unknown. In normal cells, SREBP activation is strongly repressed by Insulin-induced gene (Insig), an endoplasmic reticulum (ER)-anchored protein. It binds to the SREBP transporter, SREBP-cleavage protein (SCAP), preventing SREBP ER-to-Golgi translocation and subsequent activation. Thus, the first key step for SREBP-1 activation is SCAP dissociation from Insig. How cancer cells are able to release the tight repression of Insig on SCAP to promote high levels of SREBP- 1 activation remains unanswered. We recently found that glucose-mediated N-glycosylation stabilizes SCAP. Interestingly, our new preliminary data showed that in the absence of glutamine, SREBP-1 failed to be cleaved, although SCAP remained glycosylated. Inhibiting glutamine conversion to glutamate and ammonia by suppressing glutaminase (GLS) markedly reduced SREBP-1 cleavage. These data strongly suggest that glutamine plays a critical role in SREBP-1 activation. Interestingly, expressing the active N-terminal form of SREBP-1 in GBM cells significantly enhanced glutamine uptake. Analysis of GBM patient samples showed that the glutamine transporter, SLC1A5, is highly expressed in tumor tissues and is inversely correlated with patient survival. Promoter analysis showed multiple putative SREBP-1 binding sites in the SLC1A5 promoter region. Based on these new preliminary data and our published studies, we hypothesize that SCAP dissociation from Insig is activated by glutamine-derived metabolites, leading to SREBP-1 activation; in turn, activated SREBP-1 transcriptionally upregulate the expression of SLC1A5, resulting in a feedforward loop to increase glutamine uptake and lipid synthesis to promote GBM growth. In this study, we will identify the molecular mechanism activating SREBP-1 and lipogenesis by glutamine in GBM in Aim 1, and reveal the new role of SREBP-1 in regulating glutamine metabolism in Aim 2. We will further examine whether combining inhibition of SREBP-1 together with SLC1A5 or GLS suppression provides strong synergy to inhibit GBM growth (Aim 2). Completion of this study will significantly advance our understanding of GBM biology and metabolism rewiring, and provide promising approaches for GBM therapy.