背景:缺氧是大多数癌症的病理标志,包括胶质母细胞瘤(GBM)。癌基因蛋白的缺氧信号激活和翻译后修饰(PTM)在癌症中得到了充分研究。积累的研究表明,糖酵解酶PGK1在肿瘤发生中起着至关重要的作用,然而潜在的机制仍然未知。
方法:我们首先使用ChIP测定法来揭示HIF1α和ATF3之间的串扰及其在P4HA1调节中的作用。蛋白质降解分析,LC-MS/MS,进行了体外琥珀酸生产测定以检查蛋白质琥珀酰化对GBM病理的影响。海马测定法测量了PGK1琥珀酰化在K191/192或其突变体对葡萄糖代谢的影响。我们利用体内颅内小鼠模型进行生化研究,以阐明ATF3和P4HA1对有氧糖酵解和肿瘤免疫微环境的影响。
结果:我们证明了HIF1α和ATF3分别正向和负向调节P4HA1的转录,导致琥珀酸产生增加和HIF1α信号激活增加。P4HA1表达升高了琥珀酸浓度,导致PGK1在K191和K192位点的琥珀酰化增强。通过琥珀酰化抑制PGK1的蛋白酶体降解显著增加了产生乳酸的有氧糖酵解。此外,ATF3过表达和P4HA1敲低降低GBM细胞中琥珀酸和乳酸水平,抑制免疫反应和肿瘤生长。
结论:一起,我们的研究表明HIF1α/ATF3参与了P4HA1/琥珀酸盐信号传导,它是GBM中K191和K192位点琥珀酸酯生物合成和PGK1琥珀酰化的主要调节剂。P4HA1/琥珀酸途径可能是GBM有氧糖酵解的新靶标。
BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown.
METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment.
RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth.
CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.