PO.TB07.02 · 肿瘤生物学
通过PDK3依赖性的SOX2乳酸化对胰腺癌干性的代谢控制
Metabolic control of pancreatic cancer stemness through PDK3 dependent SOX2 lactylation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
癌干细胞(CSCs)因其独特的耐受治疗并驱动肿瘤复发的能力，在胰腺癌(PC)的管理中构成关键挑战。CSCs表现出代谢灵活性，通过在糖酵解和氧化磷酸化之间切换以适应应激。丙酮酸脱氢酶激酶3(PDK3)通过磷酸化并使丙酮酸脱氢酶复合物(PDHA)失活来主导这一转变，从而促进糖酵解表型。理解维持癌干细胞的代谢重编程可能揭示破坏其存活和增殖的策略。在本研究中，对基于球体的PCSC模型进行RNA测序，随后进行无偏倚的计算机分析，鉴定出14个上调的代谢调控基因，其中PDK3成为首要候选者。PDK3表达在CSC亚型群体、患者的胰腺肿瘤组织和Kras;PdxCre(KC)小鼠模型中升高，但在正常胰腺组织中极低。双重IHC染色证明PDK3与干性标志物CD44在人胰腺肿瘤组织中共表达，突显其与CSC表型的关联。稳定慢病毒介导的shRNA敲低(KD)或用青蒿琥酯(artesunate，本研究中鉴定的一种靶向PDK3的化合物)进行药理学抑制，显著降低了干性标志物(CD44、SOX2和CD133)、糖酵解基因(GLUT1和LDHA)及磷酸化PDHA水平。功能上，PDK3耗竭减少了侧群比例，损害了成球和集落形成能力，提示自我维持和自我更新特性受到抑制。通过LC-MS进行的代谢组学分析揭示，与PDK3 KD细胞相比，乳酸是对照PC细胞中最富集的代谢物之一。与之一致，代谢组学分析揭示PDK3 KD后乳酸释放和葡萄糖摄取显著减少。Seahorse分析进一步表明PDK3耗竭降低了ECAR并升高了OCR，指示代谢从糖酵解向氧化磷酸化转变。有趣的是，在对照和经乳酸处理的PC细胞中观察到赖氨酸乳酸化和SOX2表达升高，而在糖酵解抑制和PDK3 KD后两者均显著降低，指示对PDK3介导的代谢活性有强烈依赖。机制上，免疫沉淀和免疫荧光揭示PDK3促进SOX2的乳酸依赖性赖氨酸乳酸化，增强其稳定性和干性活性。原位植入PDK3 KD人PC细胞导致体内肿瘤生长显著减少，突显了PDK3在驱动CSC介导的肿瘤进展中的关键作用。总之，我们的发现确立PDK3作为PCSC维持和肿瘤进展的核心代谢调控因子，为开发破坏胰腺癌中乳酸驱动干性的疗法提供了新机遇。
查看英文原文 English abstract
Cancer stem cells (CSCs) pose a critical challenge in Pancreatic cancer (PC) management because of their unique ability to survive treatment and drive tumor relapses. CSCs exhibit metabolic flexibility to adapt to stress by switching between glycolysis and oxidative phosphorylation. Pyruvate dehydrogenase kinase 3 (PDK3) governs this transition by phosphorylating and inactivating the pyruvate dehydrogenase complex (PDHA), thereby promoting a glycolytic phenotype. Understanding the metabolic reprogramming that sustains cancer stem cells may reveal strategies to disrupt their survival and propagation . In this study, RNA sequencing of spheroid-based PCSC model followed by unbiased in-silico analysis identified 14 upregulated metabolic regulatory genes, with PDK3 emerging as the top candidate. PDK3 expression was elevated in CSC subtype population, pancreatic tumor tissues from patients and Kras; PdxCre (KC) mouse models, but minimal in normal pancreatic tissues. Dual-IHC staining demonstrated co-expression of PDK3 with the stemness marker CD44 in human pancreatic tumor tissues, highlighting its association with CSC phenotypes. Stable lentiviral-mediated shRNA knockdown (KD) or pharmacologic inhibition with artesunate (a PDK3 targeting compound identified in this study) significantly reduced stemness markers (CD44, SOX2, and CD133), glycolytic genes (GLUT1 and LDHA), and phosphorylated PDHA levels. Functionally, PDK3 depletion decreased the side-population fraction, impaired sphere-formation, and colony-forming capacities, suggesting suppressed sustenance and self-renewal properties. Metabolomic profiling by LC-MS revealed lactic acid as one of the most enriched metabolites in scramble PC cells compared to PDK3 KD cells. Consistently, metabolomic analysis revealed a significant reduction in lactic acid release and glucose uptake upon PDK3 KD. Seahorse analysis further demonstrated that PDK3 depletion lowered ECAR and elevated OCR, indicating a metabolic shift from glycolysis toward oxidative phosphorylation. Interestingly, elevated lysine lactylation and SOX2 expression were observed in control and lactate-treated PC cells, whereas both were markedly reduced upon glycolysis inhibition and PDK3 KD, indicating a strong dependence on PDK3-mediated metabolic activity. Mechanistically, immunoprecipitation and immunofluorescence revealed that PDK3 promotes lactate-dependent lysine lactylation of SOX2 , enhancing its stability and stemness activity . Orthotopic implantation of PDK3 KD human PC cells resulted in significantly reduced tumor growth in vivo , underscoring PDK3's pivotal role in driving CSC-mediated tumor progression. In conclusion, our findings establish PDK3 as a central metabolic regulator of PCSC maintenance and tumor progression, offering new opportunities to develop therapies that disrupt lactate-fueled stemness in pancreatic cancer.
利益披露 Disclosure
N. Gayen, None..
S. Marimuthu, None..
M. P. Ponnusamy, None.