ZEB1 enhances Warburg effect to facilitate tumorigenesis and metastasis of HCC by transcriptionally activating PFKM.
ZEB1 enhances Warburg effect to facilitate tumorigenesis and metastasis of HCC by transcriptionally activating PFKM.
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ZEB1通过转录激活PFKM增强Warburg效应促进HCC肿瘤发生和转移
作者：
Zhou Y;Lin F;Wan T;Chen A;Wang H;Jiang B;Zhao W;Liao S;Wang S;Li G;Xu Z;Wang J;Zhang J;Ma H;Lin D;Li Q
代谢重编程，尤其是瓦尔堡效应，是肿瘤发生和发展的关键事件。ZEB 1在多种癌症的转移中起着至关重要的作用。我们以前发现ZEB 1在肝细胞癌（HCC）中过度表达，其高表达与HCC的转移和复发密切相关。我们想了解糖酵解酶是否受ZEB 1的调控，并参与HCC的发生和转移。研究方法：为了探索ZEB 1是否可以增强HCC中的糖酵解，我们在MHCC-97 H和HCC-LM 3细胞中通过短发夹RNA（shRNA）敲低ZEB 1，并进行葡萄糖摄取、乳酸产生、ECAR和OCR测定。为了研究ZEB 1如何增强糖酵解，使用Western印迹检测相同细胞系中糖酵解酶的蛋白水平。通过RT-qPCR、荧光素酶报告试验和ChIP试验证实ZEB 1对PFKM mRNA水平的调节作用。为了评估ZEB 1-PFKM轴在细胞增殖中的作用，在ZEB 1敲低并进一步重新表达ZEB 1或PFKM或不表达ZEB 1或PFKM的MHCC-97 H和HCC-LM 3细胞系中进行细胞计数和CCK-8测定。为了探索ZEB 1-PFKM轴是否也在HCC细胞迁移、侵袭和转移中起作用，对相同的MHCC-97 H和HCC-LM 3细胞系进行伤口愈合测定、transwell测定和集落形成测定，同时对MHCC-97 H细胞系进行原位肝移植测定。最后，采用免疫组化和Western blot方法检测ZEB 1和PFKM在人肝癌组织和非肿瘤肝组织中的表达。结果如下：我们发现，ZEB 1转录上调磷酸果糖激酶-1（PFKM）的肌肉亚型的表达，PFKM是糖酵解的限速酶。有趣的是，在PFKM的启动子区域中的非经典的ZEB 1结合序列被鉴定，ZEB 1通过该序列直接激活PFKM的转录。MHCC-97 H和HCC-LM 3细胞中ZEB 1的沉默导致PFKM表达、糖酵解、增殖和侵袭受损，并且这些受损通过PFKM的外源表达而被拯救。重要的是，原位HCC异种移植物测定和来自TCGA数据库的研究表明，ZEB 1-PFKM轴对于HCC的致癌和转移至关重要。结论：我们的研究揭示了ZEB 1通过激活PFKM的转录而促进HCC的新机制，建立了ZEB 1促进糖酵解和瓦尔堡效应的直接联系，提示抑制ZEB 1对PFKM的转录活性可能是HCC的潜在治疗策略。
Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.
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