Role of O-GlcNAc-ylation on tumor progression

摘要 O-连接的N-乙酰葡糖胺（O-GlcNAc）是连接到丝氨酸或半胱氨酸的侧链羟基的糖。 蛋白质上的苏氨酸残基。O-GlcNAc酰化控制关键的信号传导和生物过程，如信号传导 转导、转录、细胞周期进程和代谢。O-GlcNAc稳态的扰动 与糖尿病癌症和神经退行性疾病有关增加葡萄糖水平通道通量 通过己糖胺生物合成途径（HBP），最终导致O-GlcNAc水平升高。 HBP的活性和因此细胞O-GlcNAc-化在几种癌症类型中升高，包括 乳腺癌我们最近报道，抑制HBP活性可显著降低侵袭性表型， 乳腺肿瘤细胞的我们推测，丰富的葡萄糖，一种容易代谢的碳水化合物， 通过HBP的通量，导致通过O-GlcNAc-化修饰的蛋白质组合的富集。使用 无偏见的蛋白质组学分析，我们确定，高葡萄糖培养条件下富集O-GlcNAc- 修饰的GLI蛋白，刺猬（Hh）途径的转录因子。重要的是，我们发现， 在升高的葡萄糖条件下，GLI的O-GlcNAc修饰加剧Hh/GLI活性; 减轻了这种影响。我们假设HBP指导的O-GlcNAc-ylation从根本上调节了侵袭性和 通过激活Hh/GLI信号传导，在肿瘤细胞中具有化学抗性属性。 在目的1中，我们将确定GLI的HBP-指导的O-GlcNAc-化的分子基础。我们将 确定GLI O-GlcNAc-化的原因和后果。我们将首先确定HBP的参与情况 在GLI蛋白的O-GlcNAc修饰中。下一步，我们将进行调查，以确定 HBP信号传导如何使O-GlcNAc修饰的GLI参与编程侵入性和 肿瘤细胞的化学抗性属性。 在目标2中，我们将评估升高的O-GlcNAc景观对细胞的分子和细胞属性的影响。 乳腺肿瘤和相关的免疫微气候使用两种不同的和互补的同源基因， 小鼠乳腺癌模型。为了丰富相关性，我们还将评估人类TNBC和PDX模型 系统.我们将测试在O-GlcNAc升高的情况下抑制GLI活性是否会消除O-GlcNAc的影响 GlcNAc-化对乳腺肿瘤细胞的侵袭性和化疗耐药性的影响 相关性：我们提出的研究旨在系统地研究O-GlcNAc驱动的代谢 癌细胞中的重编程在分子水平上连接到异常激活Hh/GLI信号传导。的 累积的结果将产生对O-GlcNAc-ylation如何程序化肿瘤侵袭的机制理解， 进展和对抗肿瘤的反应。

Abstract O-linked ß-N-acetylglucosamine (O-GlcNAc) is a sugar attachment to the side chain hydroxyl of a serine or threonine residue on proteins. O-GlcNAcylation controls key signaling and biological processes such as signal transduction, transcription, cell cycle progression, and metabolism. Perturbations in O-GlcNAc homeostasis have been linked with diabetes, cancer, and neurodegenerative diseases. Increased glucose levels channel flux through the Hexoseamine Biosynthetic Pathway (HBP), culminating in increased O-GlcNAc levels. The activity of HBP and consequently cellular O-GlcNAc-ylation are elevated in several cancer types, including breast cancer. We recently reported that inhibiting HBP activity significantly decreased the invasive phenotype of breast tumor cells. We surmised that abundance of glucose, a readily-metabolizable carbohydrate, will drive flux through HBP, resulting in enrichment of a portfolio of proteins that are modified by O-GlcNAc-ylation. Using unbiased proteomics analysis, we identified that elevated glucose culture conditions enrich for O-GlcNAc- modified GLI proteins, transcription factors of the Hedgehog (Hh) pathway. Importantly, we identified that in elevated glucose conditions, O-GlcNAc-modification of GLI exacerbates Hh/GLI activity; and inhibiting HBP mitigated this effect. We hypothesize that HBP-directed O-GlcNAc-ylation fundamentally programs invasive and chemoresistant attributes in tumor cells through activating Hh/GLI signaling. In Aim 1 we will determine the molecular underpinnings of HBP-directed O-GlcNAc-ylation of GLI. We will determine the causes and consequences of GLI O-GlcNAc-ylation. We will first identify engagement of the HBP in O-GlcNAc-modification of GLI proteins. Next, we will undertake investigations to identify establish the mechanistic basis of how HBP signaling engages O-GlcNAc-modified GLI to program invasive and chemoresistant attributes in tumor cells. In Aim 2 we will evaluate the impact of an elevated O-GlcNAc landscape on molecular and cellular attributes of the mammary tumor and the associated immune microclimate using two distinct and complementary syngeneic mouse models of mammary cancer. To enrich the relevance, we will also evaluate human TNBC and PDX model systems. We will test if inhibiting GLI activity, in the context of elevated O-GlcNAc, uncouples the influence of O- GlcNAc-ylation on invasive and chemoresistant attributes of mammary tumor cells. Relevance: Our proposed studies are structured to systematically investigate how O-GlcNAc-driven metabolic reprogramming in cancer cells connects at the molecular level to aberrantly activate Hh/GLI signaling. The cumulative outcomes will create mechanistic understanding of how O-GlcNAc-ylation programs tumor invasion, progression and response to anti-neoplastics.