The State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, China.
Organ Transplantation Institute of Xiamen University, Xiamen Key Laboratory of Regeneration Medicine, Fujian Provincial Key Laboratory of Organ and Tissue Regeneration, School of Medicine, Xiamen University, Xiamen, China.
Cell Mol Gastroenterol Hepatol. 2023;16(4):541-556. doi: 10.1016/j.jcmgh.2023.06.006. Epub 2023 Jun 17.
BACKGROUND & AIMS: Phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of the de novo serine synthesis pathway (SSP), has been implicated in the carcinogenesis and metastasis of hepatocellular carcinoma (HCC) because of its excessive expression and promotion of SSP. In previous experiments we found that SSP flux was diminished by knockdown of zinc finger E-box binding homeobox 1 (ZEB1), a stimulator of HCC metastasis, but the underlying mechanism remains largely unknown. Here, we aimed to determine how SSP flux is regulated by ZEB1 and the contribution of such regulation to carcinogenesis and progression of HCC.
We used genetic mice with Zeb1 knockout in liver specifically to determine whether Zeb1 deficiency impacts HCC induced by the carcinogen diethylnitrosamine plus CCl. We explored the regulatory mechanism of ZEB1 in SSP flux using uniformly-labeled [C]-glucose tracing analyses, liquid chromatography-mass spectrometry, real-time quantitative polymerase chain reaction, luciferase report assay, and chromatin immunoprecipitation assay. We determined the contribution of the ZEB1-PHGDH regulatory axis to carcinogenesis and metastasis of HCC by cell counting assay, methyl thiazolyl tetrazolium (MTT) assay, scratch wound assay, Transwell assay, and soft agar assay in vitro, orthotopic xenograft, bioluminescence, and H&E assays in vivo. We investigated the clinical relevance of ZEB1 and PHGDH by analyzing publicly available data sets and 48 pairs of HCC clinical specimens.
We identified that ZEB1 activates PHGDH transcription by binding to a nonclassic binding site within its promoter region. Up-regulated PHGDH augments SSP flux to enable HCC cells to be more invasive, proliferative, and resistant to reactive oxygen species and sorafenib. Orthotopic xenograft and bioluminescence assays have shown that ZEB1 deficiency significantly impairs the tumorigenesis and metastasis of HCC, and such impairments can be rescued to a large extent by exogenous expression of PHGDH. These results were confirmed by the observation that conditional knockout of ZEB1 in mouse liver dramatically impedes carcinogenesis and progression of HCC induced by diethylnitrosamine/CCl, as well as PHGDH expression. In addition, analysis of The Cancer Genome Atlas database and clinical HCC samples showed that the ZEB1-PHGDH regulatory axis predicts poor prognosis of HCC.
ZEB1 plays a crucial role in stimulating carcinogenesis and progression of HCC by activating PHGDH transcription and subsequent SSP flux, deepening our knowledge of ZEB1 as a transcriptional factor in fostering the development of HCC via reprogramming the metabolic pathway.
磷酸甘油酸脱氢酶（PHGDH）是从头合成丝氨酸途径（SSP）的限速酶，由于其过度表达和促进 SSP，已被牵连到肝细胞癌（HCC）的癌变和转移中。在之前的实验中，我们发现锌指 E 盒结合同源盒 1（ZEB1）的敲低会减少 SSP 通量，ZEB1 是 HCC 转移的刺激物，但这种调节的潜在机制在很大程度上仍是未知的。在这里，我们旨在确定 ZEB1 如何调节 SSP 通量，以及这种调节对 HCC 癌变和进展的贡献。
我们使用特异性在肝脏中敲除 Zeb1 的遗传型小鼠，以确定 Zeb1 缺陷是否会影响致癌剂二乙基亚硝胺加 CCl 诱导的 HCC。我们使用统一标记的 [C]-葡萄糖示踪分析、液相色谱-质谱联用、实时定量聚合酶链反应、荧光素酶报告测定和染色质免疫沉淀测定来探索 ZEB1 在 SSP 通量中的调节机制。我们通过细胞计数测定、甲基噻唑基四唑（MTT）测定、划痕愈合测定、Transwell 测定和软琼脂测定，以及体外、原位异种移植、生物发光和 H&E 测定，确定了 ZEB1-PHGDH 调节轴对 HCC 癌变和转移的贡献。我们通过分析公共可用数据集和 48 对 HCC 临床标本，研究了 ZEB1 和 PHGDH 的临床相关性。
我们发现 ZEB1 通过结合其启动子区域内的非经典结合位点来激活 PHGDH 转录。上调的 PHGDH 增加 SSP 通量，使 HCC 细胞更具侵袭性、增殖性和对活性氧和索拉非尼的耐药性。原位异种移植和生物发光测定表明，ZEB1 缺陷显著抑制 HCC 的肿瘤发生和转移，而外源性表达 PHGDH 可在很大程度上挽救这种缺陷。这些结果得到了以下观察结果的证实：在小鼠肝脏中条件性敲除 ZEB1 可显著抑制二乙基亚硝胺/CCl 诱导的 HCC 癌变和进展以及 PHGDH 表达。此外，对癌症基因组图谱数据库和临床 HCC 样本的分析表明，ZEB1-PHGDH 调节轴预测 HCC 的预后不良。
ZEB1 通过激活 PHGDH 转录和随后的 SSP 通量，在刺激 HCC 的癌变和进展中起着关键作用，这加深了我们对 ZEB1 作为转录因子通过重编程代谢途径促进 HCC 发展的认识。
