Laboratory of Neuro-oncology, Tianjin Neurological Institute, Department of Neurosurgery, Tianjin Medical University General Hospital, Key Laboratory of Post-Neuro Injury Neuro-Repair and Regeneration in Central Nervous System, Ministry of Education and Tianjin City, Tianjin, China.
Department of Gynecology and Obstetrics, Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China.
Neuro Oncol. 2024 Aug 5;26(8):1405-1420. doi: 10.1093/neuonc/noae040.
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.
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.
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.
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.
缺氧是大多数癌症（包括胶质母细胞瘤（GBM））的病理标志。缺氧信号激活和致癌蛋白的翻译后修饰（PTM）在癌症中得到了广泛研究。越来越多的研究表明，糖酵解酶 PGK1 在肿瘤发生中起着至关重要的作用，但潜在的机制尚不清楚。
我们首先使用 ChIP 测定法揭示了 HIF1α 和 ATF3 之间的串扰及其在 P4HA1 调节中的作用。进行蛋白降解分析、LC-MS/MS 和体外琥珀酸产生测定，以检查蛋白琥珀酰化对 GBM 病理学的影响。 Seahorse 测定法测量了 PGK1 在 K191/K192 或其突变体上的琥珀酰化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。
我们证明 HIF1α 和 ATF3 分别正向和负向调节 P4HA1 的转录，导致琥珀酸产量增加和 HIF1α 信号的激活增强。P4HA1 表达增加了琥珀酸的浓度，导致 PGK1 在 K191 和 K192 位点的琥珀酰化增强。PGK1 的琥珀酰化抑制蛋白酶体降解显著增加有氧糖酵解以产生乳酸。此外，ATF3 过表达和 P4HA1 敲低降低了 GBM 细胞中的琥珀酸和乳酸水平，抑制了免疫反应和肿瘤生长。
总之，我们的研究表明，HIF1α/ATF3 参与了 P4HA1/琥珀酸信号，这是 GBM 中琥珀酸生物合成和 PGK1 在 K191 和 K192 位点琥珀酰化的主要调节因子。 P4HA1/琥珀酸途径可能是 GBM 有氧糖酵解的一个新的有前途的靶点。
