Signaling metabolite succinylacetone activates HIF-1α and promotes angiogenesis in GSTZ1-deficient hepatocellular carcinoma
Huating Luo, Qiujie Wang, Fan Yang, Rui Liu, Qingzhu Gao, Bin Cheng, Xue Lin, Luyi Huang, Chang Chen, Jin Xiang, Kai Wang, Bo Qin, Ni Tang
Huating Luo, Qiujie Wang, Fan Yang, Rui Liu, Qingzhu Gao, Bin Cheng, Xue Lin, Luyi Huang, Chang Chen, Jin Xiang, Kai Wang, Bo Qin, Ni Tang
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Research Article Oncology

Signaling metabolite succinylacetone activates HIF-1α and promotes angiogenesis in GSTZ1-deficient hepatocellular carcinoma

Abstract

Aberrant angiogenesis in hepatocellular carcinoma (HCC) is associated with tumor growth, progression, and local or distant metastasis. Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a major role in regulating angiogenesis during adaptation of tumor cells to nutrient-deprived microenvironments. Genetic defects in Krebs cycle enzymes, such as succinate dehydrogenase and fumarate hydratase, result in elevation of oncometabolites succinate and fumarate, thereby increasing HIF-1α stability and activating the HIF-1α signaling pathway. However, whether other metabolites regulate HIF-1α stability remains unclear. Here, we reported that deficiency of the enzyme in phenylalanine/tyrosine catabolism, glutathione S-transferase zeta 1 (GSTZ1), led to accumulation of succinylacetone, which was structurally similar to α-ketoglutarate. Succinylacetone competed with α-ketoglutarate for prolyl hydroxylase domain 2 (PHD2) binding and inhibited PHD2 activity, preventing hydroxylation of HIF-1α, thus resulting in its stabilization and consequent expression of vascular endothelial growth factor (VEGF). Our findings suggest that GSTZ1 may serve as an important tumor suppressor owing to its ability to inhibit the HIF-1α/VEGFA axis in HCC. Moreover, we explored the therapeutic potential of HIF-1α inhibitor combined with anti–programmed cell death ligand 1 therapy to effectively prevent HCC angiogenesis and tumorigenesis in Gstz1-knockout mice, suggesting a potentially actionable strategy for HCC treatment.

Authors

Huating Luo, Qiujie Wang, Fan Yang, Rui Liu, Qingzhu Gao, Bin Cheng, Xue Lin, Luyi Huang, Chang Chen, Jin Xiang, Kai Wang, Bo Qin, Ni Tang

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Figure 6

Lack of GSTZ1 impairs PHD2-mediated HIF-1α degradation by SA accumulation.

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Lack of GSTZ1 impairs PHD2-mediated HIF-1α degradation by SA accumulatio...
(A) Schematic illustration showing SA is a structural analog of α-KG. (B) Geometry of SA with active PHD2 state and key residues. (C) DARTS assays for identification of direct binding between SA and PHD2 in HepG2 and Huh7 cells. (D) CETSA shows the binding affinity of SA to PHD2 in HepG2 and Huh7 cells. The data are presented as means ± SEM from 3 independent experiments. (E) SPR analysis of the binding between recombinant PHD2 with SA at the indicated concentrations. (F and G) In vitro PHD2 activity assay is performed in GSTZ1-KO HepG2 cells treated with or without NTBC and in GSTZ1-OE Huh7 cells treated with SA. Data are shown as mean ± SEM (n = 3 in each group). (H) In vitro PHD2 activity assay is performed by mixing bacteria-purified recombinant His-PHD2 with increasing amounts of SA. Data are shown as mean ± SEM (n = 3 in each group). (I) Immunoblots of parental or GSTZ1-KO HepG2 cells cultured with NTBC or 1 mM DMOG as indicated. (J and K) Co-IP assays to detect the direct interaction between PHD2 and HIF-1α treated with SA in HepG2 cells and in GSTZ1-KO HepG2 cells treated with or without NTBC. (L) Hydroxylated and total HIF-1α are detected from 0 to 8 hours after proteasomal blockade using 10 μM MG-132 in GSTZ1-KO and parental HepG2 cells. (M) In vitro prolyl hydroxylation of the purified HIF-1α–ODD protein at 0 minute and 15 minutes using lysates from GSTZ1-KO or parental HepG2 cells, incubated for 24 hours with or without NTBC. Hydroxylation of HIF-1α–ODD is determined using the hydroxyprolyl-specific antibody at proline 564 (HIF-1α–OH-ODD), and total HIF-1α is detected (HIF-1α–ODD). DMOG-treated lysates are used as a negative control. Statistical analysis was performed using 1-way ANOVA with Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001. NTBC, 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione.