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ENO1 promotes liver carcinogenesis through YAP1-dependent arachidonic acid metabolism

Nature Chemical Biology volume 19, pages 1492–1503 (2023)Cite this article

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Abstract

Enolase 1 (ENO1) is a glycolytic enzyme that plays essential roles in various pathological activities including cancer development. However, the mechanisms underlying ENO1-contributed tumorigenesis are not well explained. Here, we uncover that ENO1, as an RNA-binding protein, binds to the cytosine-uracil-guanine-rich elements of YAP1 messenger RNA to promote its translation. ENO1 and YAP1 positively regulate alternative arachidonic acid (AA) metabolism by inverse regulation of PLCB1 and HPGD (15-hydroxyprostaglandin dehydrogenase). The YAP1/PLCB1/HPGD axis-mediated activation of AA metabolism and subsequent accumulation of prostaglandin E2 (PGE2) are responsible for ENO1-mediated cancer progression, which can be retarded by aspirin. Finally, aberrant activation of ENO1/YAP1/PLCB1 and decreased HPGD expression in clinical hepatocellular carcinoma samples indicate a potential correlation between ENO1-regulated AA metabolism and cancer development. These findings underline a new function of ENO1 in regulating AA metabolism and tumorigenesis, suggesting a therapeutic potential for aspirin in patients with liver cancer with aberrant expression of ENO1 or YAP1.

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Fig. 1: ENO1 promotes liver cancer progression by activating YAP1 translation as an RBP.
Fig. 2: ENO1 inversely regulates PLCB1 and HPGD expression in a YAP1-dependent manner.
Fig. 3: ENO1 activates the alterative AA biosynthetic pathway via YAP1-regulated PLCB1 and HPGD expression.
Fig. 4: YAP1-regulated AA metabolism via PLCB1 and HPGD expression is important for ENO1-mediated cancer progression.
Fig. 5: Aspirin inhibits ENO1- and YAP1-induced liver cancer progression in vivo.
Fig. 6: Activation of the ENO1-YAP1-AA axis predicts poor prognosis of patients with liver cancer.

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Data availability

RNA-seq data and RIP-seq data have been deposited in the Gene Expression Omnibus under accession code GSE183703. The KEGG used for gene pathway analysis of RNA-seq data can be accessed at https://www.genome.jp/kegg/. All other data are available in the Source data and Supplementary Information that are provided with this paper.

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Acknowledgements

This work is supported in part by the National Natural Science Foundation of China (grant nos. 91957203 to P.G., 82192893, 81930083 and 81821001 to H.Z., 82130087 to P.G., 82273221 to L.S. and 82203556 to C.S.), the Chinese Academy of Sciences (grant no. XDB39020100 to H.Z.) and the National Key R&D Program of China (grant nos. 2022YFA1304504 to H.Z. and 2018YFA0800300 to P.G.).

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Author notes

  1. These authors contributed equally: Linchong Sun, Caixia Suo, Tong Zhang, Shengqi Shen.

Authors and Affiliations

  1. Medical Research Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, China

    Linchong Sun, Tong Zhang, Shengqi Shen, Haoran Wei & Ping Gao

  2. Department of Colorectal Surgery, Guangzhou First People’s Hospital, South China University of Technology, Guangzhou, China

    Caixia Suo & Jie Cao

  3. Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, China

    Shengqi Shen, Haoran Wei & Ping Gao

  4. School of Medicine, South China University of Technology, Guangzhou, China

    Xuemei Gu, Shiqiao Qiu, Rui Chen & Ping Gao

  5. Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Pinggen Zhang, Wenhao Ma, Ronghui Yan & Huafeng Zhang

  6. The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China

    Weidong Jia & Huafeng Zhang

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Contributions

L.S., H.Z. and P.G. conceived the study and supervised the experiments. L.S., C.S., T.Z., S.S., H.Z. and P.G. designed the experiments. L.S. and T.Z. performed RIP and ChIP assays to identify the targets of ENO1 and YAP1, respectively. L.S. and C.S. performed animal studies and analysis of clinical HCC specimens. L.S., C.S. and T.Z. performed immunoblotting, cloning, cell biology and biochemistry experiments. S.S. analyzed RNA-seq and RIP-seq data. X.G., S.Q., P.Z., H.W., W.M., R.Y. and R.C. analyzed the data. W.J. provided clinical specimens. J.C. provided constructive guidance. L.S., C.S., T.Z., H.Z. and P.G. wrote the paper. All the authors read and approved the manuscript.

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Correspondence to Linchong Sun, Huafeng Zhang or Ping Gao.

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Extended data

Extended Data Fig. 1 ENO1 regulates the expression of YAP1 target genes.

a, Protein copy numbers of glucose metabolism enzymes in NIH3T3 mouse fibroblast cells (Schwanhausser et al., 2011), and HeLa cells (Hein et al., 2015; Itzhak et al., 2016). b, Venn diagram showing the 21 overlapped target genes enriched by ENO1 IP in our RIP-seq data from HEK293T-Flag-ENO1 cells and the previously published HITS-GBP-CLIP data from HeLa-ENO1-YFP cells (Castello et al., 2012). c, qPCR-based expression analysis of known YAP1 target genes including NPPB, CYR61, CTGF, S1PR1, NEXN, NTF3, KDR, CPA3, PSG1, CCDC80, EGR3, EGR4, DDIT4, and TRAIL in PLC cells expression shRNAs targeting ENO1 or YAP1 (n = 3 independent assays). Error bars denote the mean ± s.e.m. (c).

Source data

Extended Data Fig. 2 ENO1 upregulates YAP1 expression by recruiting YTHDF3.

a, Co-IP assay in HEK293T cells transfected with Flag-ENO1 or Hep3B-Flag-ENO1 cells. b, Protein levels of eIF5A, YTHDF3, and YAP1 were measured by immunoblotting analysis in Hep3B cells with eIF5A or YTHDF3 knockdown. Actin served as a loading control. Par: parental; NC: negative control. c, Binding of endogenous YAP1 mRNA transcripts by Flag-ENO1 in Hep3B-Flag-ENO1 cells with or without YTHDF3 knockdown (n = 3 independent assays). Protein levels of ENO1, YTHDF3, and YAP1 were measured. Actin served as a loading control. d, HEK293T cells expressing shRNA targeting endogenous ENO1 were further transfected with plasmids to overexpress Flag-tagged wild-type ENO1, ENO1S40A, or ENO1D245R mutants (n = 1 sample detected at different time points). Then the ENO1 enzyme activity of these cells was measured by the consumption of NADH in an assay buffer that includes pyruvate kinase (PK), lactate dehydrogenase (LDHA), and the required substrates including 2-phospho-D-glycerate (2PG), phosphoenolpyruvate (PEP), and pyruvate. The consumption of NADH is monitored as a decrease of absorbance at 340 nm. e, Flag and YAP1 protein levels were measured in HEK293T cells expressing Flag-tagged wild-type ENO1 and indicated ENO1 mutant variants. Actin served as a negative control. f, g, Photographs showed xenografts described in Fig. 1l (f). ENO1 and YAP1 protein levels were measured (g). Actin served as a negative control. Immunoblots are representative of two independent experiments (a-c,e). Error bars denote the mean ± s.e.m. (c,d). Statistical analyses were performed by two-tailed Student’s t-test (c,d).

Source data

Extended Data Fig. 3 ENO1 and YAP1 transcriptionally activate arachidonic acid synthetic enzymes.

a, Venn diagram of the RNA-seq data showing the overlapped genes regulated by ENO1 and YAP1 in PLC cells with ENO1 or YAP1 knockdown. The statistical significance between ENO1-regulated genes and YAP1-regulated genes was determined using Fisher’s exact probability test, P < 0.001. b, KEGG mapper analysis of the pathways in which the co-regulated genes of ENO1 and YAP1 are enriched. 66 genes of metabolic pathways are shown in Supplementary Table 1. c, Gene Ontology (GO) enrichment analysis. The top ten GO terms are listed. Supplementary Table 2 provides a complete list of terms and corresponding gene lists. P-value was computed using the one-sided Fisher’s exact test and corrected for multiple hypothesis testing using false discovery rate (FDR). d, qPCR analysis of ENO1, YAP1, PLCB1, PTGS2 and HPGD expression in PLC cells expressing shRNAs targeting ENO1 or YAP1 (n = 3 independent assays). e, Immunoblotting analysis of ENO1, YAP1, and HPGD expression in PLC cells expressing shRNAs targeting ENO1 or YAP1. Actin served as a negative control. f, Protein levels of ENO1, YAP1, PLCB1, and HPGD were measured in Hepa 1–6 cells expressing shRNAs targeting ENO1 or YAP1. Actin served as a negative control. g, h, Protein levels of ENO1, YAP1, PLCB1, COX2, and HPGD were measured in PLC cells (g) or Hep3B cells (h) expressing ENO1 or YAP1. Actin served as a negative control. i, Protein levels of YAP1, PLCB1, COX2, and HPGD were measured in HEK293T cells expressing wild-type YAP1 or YAP-5SA mutant. Actin served as a negative control. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. (d). Statistical analyses were performed by two-tailed Student’s t-test (d).

Source data

Extended Data Fig. 4 The NuRD complex is required for ENO1/YAP1-mediated HPGD gene repression.

a, b, qPCR (a) and immunoblotting (b) analysis of HPGD expression levels in PLC cells treated with 1 μM TSA, or expressing shRNAs targeting MTA2, HDAC1, or HDAC2 (mRNA data: n = 3 independent assays). Actin served as a negative control. c, PLC cells expressing Flag-tagged YAP1 or ENO1 were further infected with shRNA viruses targeting HDAC1. Protein levels of YAP1, ENO1, Flag, HDAC1, and HPGD were measured by immunoblotting analysis. Actin served as a negative control. d, A schematic representation of the ENO1-mediated activation of YAP1 transcriptional regulation of the PLCB1 and HPGD loci. Illustration created with Biorender.com. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. (a). Statistical analyses were performed by two-tailed Student’s t-test (a).

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Extended Data Fig. 5 ENO1 and YAP1 activate AA metabolism in a PLCB1- and HPGD-dependent manner.

a, AA and PGE2 contents were measured in PLC cells expressing ENO1 or YAP1 by ELISA (n = 3 independent assays). b, c, Immunoblotting and qPCR analysis of HPGD expression in PLC cells expressing shRNAs targeting HPGD (b), followed by the detection of PGE2 content and cell growth status (c) (n = 3 independent assays). Actin served as a negative control. d, Protein levels of ENO1 or YAP1, and HPGD (lower panel), and PGE2 content (upper panel) were measured in ENO1 or YAP1-overexpressing PLC cells expressing HPGD (n = 3 independent assays). Actin served as a negative control. e, AA content was measured in ENO1- or YAP1-overexpressing Hep3B cells infected with shRNA viruses targeting PLCB1 (n = 3 independent assays). f, Protein levels of HPGD were measured by immunoblotting in endogenous ENO1-knockdown PLC cells expressing wild-type ENO1, ENO1S40A, and ENO1D245R. AA and PGE2 contents in these cell lines were measured by ELISA (n = 3 independent assays). Actin served as a negative control. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. (a-f) or mean ± s.d. (c, lower panel). Statistical analyses were performed by two-tailed Student’s t-test (a-f), or two-way ANOVA (c, lower panel).

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Extended Data Fig. 6 The accelerated tumor progression promoted by ENO1/YAP1 depends on PLCB1 expression.

a, Cell growth was measured in ENO1 or YAP1-overexpressing Hep3B cells infected with shRNA viruses targeting PLCB1 (n = 4 independent assays). b, Photographs show xenografts described in Fig. 4a at the end of the experiment. c, Protein levels of Flag, ENO1, YAP1, and PLCB1 in tumor lysates of Extended Data Fig. 6b were measured by immunoblotting analysis. Actin served as a loading control. d, Protein levels of YAP1 and PLCB1 were measured in YAP1-knockdown Hep3B cells further infected with shRNA viruses targeting PLCB1 (upper panel). Actin served as a negative control. Growth curves were measured by trypan blue counting (lower panel, n = 3 independent assays). e, f, Equal numbers of Hep3B cells expressing shYAP1 or/and shPLCB1 were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (e). Tumor weights (f) were determined at the end of the experiment (day 30). g, Protein levels of YAP1 and PLCB1 in tumor lysates of Extended Data Fig. 6f were measured by immunoblotting analysis. Actin served as a loading control. Immunoblots are representative of two independent experiments (d). Error bars denote the mean ± s.e.m. (a,d-f). Statistical analyses were performed by two-tailed Student’s t-test (f), or two-way ANOVA with tukey’s multiple comparisons test (a,d,e).

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Extended Data Fig. 7 The accelerated tumor progression promoted by ENO1/YAP1 depends on HPGD expression.

a, Cell growth was measured in ENO1 or YAP1-knockdown PLC cells infected with shRNA viruses targeting HPGD (n = 3 independent assays). b, Cell growth was measured in ENO1 or YAP1-overxpressing PLC cells expressing HPGD (n = 3 independent assays). c, Photographs show xenografts described in Fig. 4e at the end of the experiment. d, Protein levels of ENO1, YAP1, and HPGD in tumor lysates of Extended Data Fig. 7c were measured by immunoblotting analysis. Actin served as a loading control. e, PLC cells expressing shRNA targeting endogenous ENO1 were further transfected with plasmids to overexpress Flag-tagged wild-type ENO1, ENO1S40A, ENO1D245R, or ENO1M4mutants. Protein levels of ENO1 and Flag were measured by immunoblotting analysis. Actin served as a loading control. f, ENO1 enzyme activity of cells in Extended Data Fig. 7e was measured (n = 3 independent assays). g, Growth curves were measured by trypan blue counting in PLC cells expressing the EV, wild-type ENO1, or indicated ENO1 mutants with endogenous ENO1 knockdown (n = 3 independent assays). h, Photographs show xenografts described in Fig. 4k at the end of the experiment. i, Protein levels of Flag, ENO1, YAP1, and HPGD were detected in the tumor lysates described in Extended Data Fig. 7h. Actin served as a loading control. Immunoblots are representative of two independent experiments (e). Error bars denote the mean ± s.d. (a,b,g) or mean ± s.e.m. (f). Statistical analyses were performed by two-tailed Student’s t-test (f), two-way ANOVA (g), or two-way ANOVA with tukey’s multiple comparisons test (a,b).

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Extended Data Fig. 8 COX2 is responsible for ENO1/YAP1-mediated tumor growth.

a, b, Cell growth was measured in ENO1- or YAP1-overexpressing PLC cells (a) or Hep3B cells (b) infected with shRNA viruses targeting COX2 (n = 3 independent assays). Protein levels of ENO1, YAP1, and COX2 were measured by immunoblotting analysis. Actin served as a loading control. c-f, Equal numbers of Hep3B cells expressing ENO or YAP1 that further knockdown COX2 were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (c, d). Tumor weights (e, f) were determined at the end of the experiment (day 29). g, h, Protein levels of ENO1, YAP1, and COX2 in tumor lysates of Extended Data Fig. 8e, f were measured by immunoblotting analysis. Actin served as a loading control. Immunoblots are representative of three independent experiments (a,b). Error bars denote the mean ± s.d. (a,b) or mean ± s.e.m. (c-f). Statistical analyses were performed by two-tailed Student’s t-test (e,f), or two-way ANOVA with tukey’s multiple comparisons test (a-d).

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Extended Data Fig. 9 Aspirin inhibits ENO1- and YAP1-induced liver cancer in vivo.

a, Protein levels of COX2 were measured in PLC cells treated with different doses of aspirin. Actin served as a negative control. b, COX2 enzyme activity was measured in the tumor lysates of Fig. 5b using ELISA (n = 10). c, Protein levels of ENO1 and YAP1 were measured in the tumor lysates of Fig. 5b. Actin served as a negative control. d, Protein levels of mus-ENO1 or mus-YAP1 were measured in Hepa 1–6 cells stably infected with viruses expressing mus-ENO1 or mus-YAP1. e, COX2 enzyme activity was measured in the tumor lysates of Fig. 5e, g using ELISA (n = 5). f, g, Protein levels of ENO1 (Flag), YAP1, PLCB1, and HPGD were measured in the tumor lysates of Fig. 5e, g. Calnexin served as a loading control. h, COX2 enzyme activity was measured in the tumor lysates of Fig. 5i using ELISA (n = 5). i, j, protein (i) and mRNA (j) levels of YAP1, PTGS2 (COX2), PLCB1, and HPGD were detected in the tumor lysates of Fig. 5i. Calnexin served as a loading control. Immunoblots are representative of three independent experiments (a,d). Error bars denote the mean ± s.e.m. (b,e,h,j). Statistical analyses were performed by two-tailed Student’s t-test (b,e,h,j).

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Extended Data Fig. 10 ENO1 promotes liver cancer progression by regulating PLCB1 and HPGD-mediated arachidonic acid metabolism through YAP1.

a, Summary: ENO1, as an RNA-binding protein, activates YAP1 translation by binding to the (CUG)n triplet rich elements within the 3’UTR of YAP1 mRNA. YAP1 content increases and translocates into the nucleus to promote PLCB1 expression, but directly represses HPGD expression at transcriptional levels, which is responsible for the ENO1-mediated activation of the alterative arachidonic acid pathway, PGE2 accumulation, cell proliferation, and cancer progression. Aspirin, a blocker of the AA pathway by inhibiting COX2 activity and PGE2 production, could inhibit liver cancer progression, especially in liver cancers with elevated expression of ENO1 and YAP1. Illustration created with Biorender.com.

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Sun, L., Suo, C., Zhang, T. et al. ENO1 promotes liver carcinogenesis through YAP1-dependent arachidonic acid metabolism. Nat Chem Biol 19, 1492–1503 (2023). https://doi.org/10.1038/s41589-023-01391-6

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