Epigenetic reprogramming-induced guanidinoacetic acid synthesis promotes pancreatic cancer metastasis and transcription-activating histone modifications

Background Pancreatic ductal adenocarcinoma (PDAC) tends to undergo distant metastasis, especially liver metastasis, leading to a poor prognosis. Metabolic remodelling and epigenetic reprogramming are two important hallmarks of malignant tumours and participate in regulating PDAC tumorigenesis and metastasis. However, the interaction between these two processes during PDAC metastasis has not been fully elucidated. Methods We performed metabolomics analysis to identify the critical metabolites associated with PDAC liver metastasis and focused on guanidinoacetic acid (GAA). Intracellular GAA content was significantly increased in liver metastatic PDAC cells compared to primary cancer cells in mouse xenograft tumour models. The effects of GAA supplementation and glycine amidinotransferase (GATM) knockdown on PDAC metastasis were assessed by analysing cell migration, filopodia formation, epithelial-mesenchymal transition (EMT), and in vivo metastasis in different cell and animal models. Next, ChIP‒qPCR, 3C‒qPCR, and CRISPRi/dCas9-KRAB experiments were used to validate the “epigenome-metabolome" mechanism. Finally, the results of in vitro approaches, including RNA-seq, CUT&RUN, RT‒qPCR, and western blot analyses, as well as luciferase reporter gene assay and transwell assay, revealed the GAA-c-Myc-HMGA axis and transcription-activating histone modifications reprogramming. Results A high level of intracellular GAA was associated with PDAC liver metastasis. GAA could promote the migration, EMT, and liver metastasis of pancreatic cancer cells in vitro and in vivo. Next, we explored the role of GATM-mediated de novo GAA synthesis in pancreatic cancer metastasis. High expression of GATM was positively correlated with advanced N stage in PDAC. Knockdown of GATM significantly reduced the intracellular level of GAA, suppressed EMT, and inhibited PDAC liver metastasis, and these effects were attenuated by GAA supplementation. Mechanistically, we identified the active enhancers looped to the GATM gene locus that promoted GATM expression and PDAC liver metastasis. Furthermore, we found that GAA promoted cell migration and EMT by regulating c-Myc-mediated high mobility group AT-hook protein expression. Moreover, GAA increased the H3K4me3 modification level by upregulating histone methyltransferases, which induced the transcription of metastasis-related genes, including MYC. Conclusions These findings revealed the critical role of the epigenome-metabolome interaction in regulating PDAC liver metastasis and suggested potential therapeutic strategies targeting GAA metabolism and epigenetic regulatory mechanisms. mRNA profiles of pancreatic cancer cell lines with GATM knock-down and the control group.

背景 胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）易于发生远处转移，尤以肝转移最为常见，预后极差。代谢重塑与表观遗传重编程是恶性肿瘤的两大重要特征，二者均参与调控PDAC的发生与转移过程，但目前二者在PDAC转移进程中的相互作用尚未完全阐明。
方法 本研究通过代谢组学分析筛选与PDAC肝转移相关的关键代谢物，并聚焦于胍基乙酸（guanidinoacetic acid, GAA）。在小鼠异种移植瘤模型中，相较于原发肿瘤细胞，肝转移性PDAC细胞内的GAA水平显著升高。我们通过分析不同细胞与动物模型中的细胞迁移、丝状伪足形成、上皮间质转化（epithelial-mesenchymal transition, EMT）以及体内转移情况，评估了GAA补充以及甘氨酸脒基转移酶（glycine amidinotransferase, GATM）敲低对PDAC转移的影响。随后，采用ChIP‒qPCR、3C‒qPCR以及CRISPRi/dCas9-KRAB实验验证“表观组-代谢组”调控机制。最后，通过RNA-seq、CUT&RUN、RT‒qPCR、蛋白质免疫印迹（western blot）、荧光素酶报告基因实验以及Transwell实验等体外手段，揭示了GAA-c-Myc-高迁移率族AT钩蛋白（high mobility group AT-hook protein, HMGA）轴以及转录激活型组蛋白修饰重编程过程。
结果 细胞内高水平GAA与PDAC肝转移密切相关。GAA可在体内外促进胰腺癌细胞的迁移、EMT进程以及肝转移能力。接下来，我们探究了GATM介导的内源性GAA合成在胰腺癌转移中的作用。GATM高表达与PDAC患者较高的N分期呈正相关。敲低GATM可显著降低细胞内GAA水平，抑制EMT进程以及PDAC肝转移，而补充GAA可逆转上述效应。从机制上来说，我们鉴定出了与GATM基因位点形成染色质环的活性增强子，其可促进GATM表达以及PDAC肝转移。此外，我们发现GAA可通过调控c-Myc介导的HMGA蛋白表达，促进细胞迁移与EMT。同时，GAA可通过上调组蛋白甲基转移酶提升H3K4me3修饰水平，进而诱导包括MYC在内的转移相关基因的转录。
结论 本研究揭示了表观组-代谢组相互作用在调控PDAC肝转移中的关键作用，并为靶向GAA代谢与表观遗传调控机制的潜在治疗策略提供了理论依据。本研究附带数据集为GATM敲低胰腺癌细胞系及其对照组的mRNA表达谱数据。