GSTZ1低表达激活Keap1/Nrf2通路重编程细胞代谢并促进肝癌发生的机制研究

Glutathione transferase ζ 1(GSTZ1), also known as malelacetoacetate isomerase (MAAI), is a member of the cytosolic GST superfamily. It catalyzes the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), the penultimate step in the tyrosine degradation pathway. Previous studies have reported that GSTZ1-/- mice induce oxidative stress and develop liver failure with metabolic stress like tyrosine loading, indicating the GSTZ1 deficiency may alter the metabolism of some xenobiotics and increase hepatotoxicity. Nevertheless, the effects of GSTZ1/MAAI deficiency have never been fully investigated in humans...Our preliminary data indicated that the expression of GSTZ1 were significantly downregulated in tumor tissue compared with nontumorous tissues. Deletion of GSTZ1 promoted hepatoma cell proliferation, increased ROS levels and induced activation of Keap1/Nrf2 pathway. Moreover, compared with the wild-type mouse, the concentration of succinylacetone (SA) increased in liver tissue of GSTZ1 knockout mouse. Phenylalanine or SA loading induced nuclear translocation of Nrf2 and activated oxidative stress pathway. However, restoration of GSTZ1 inhibited hepatoma cells proliferation and migration. ..Based on our preliminary data, we hypothesized that deficiency of GSTZ1 could activate Nrf2 signaling pathway through abnormal accumulation of “oncogenic metabolites”, thus involving in the pathogenesis of HCC. In this study, HCC tissue samples will be used to examine the GSTZ1 expression and explore the relationship between GSTZ1 and clinicopathological parameters, such as tumor-related death, metastasis and recurrence. The effect of GSTZ1 on cell proliferation and migration capacity will be evaluated in GSTZ1 overexpression (OE) or depleted (GSTZ1-/-) hepatoma cells. In vitro cell model and GSTZ1-/- mice will be used to identify the abnormal accumulated metabolites in the tyrosine degradation pathway, owing to GSTZ1 deficiency. And we will further investigate the molecular mechanism that metabolites induced modification of cysteine residues with the Kelch-like ECH-associated protein 1(Keap1), abrogating its ability to repress the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway. In GSTZ1-/- mice model, we will explored the interaction between “oncogenic metabolites” and Nrf2 activation in the development of HCC. Our study will provide new insights into the molecular mechanism underlying alternative oncogenic action of metabolites in the pathogenesis of HCC, and could potentially lead to novel therapeutic approaches for the treatment of HCC in patients.

谷胱甘肽S-转移酶ζ 1（GSTZ1）是参与苯丙氨酸/酪氨酸代谢的关键酶，GSTZ1-/-小鼠在苯丙氨酸负荷下出现慢性进行性肝损伤。课题组前期发现临床肝癌组织中GSTZ1低表达，GSTZ1敲除细胞增殖加快、Nrf2通路活化，ROS水平增加，而回复其表达抑制HCC增殖；GSTZ1敲除小鼠肝内琥珀酰丙酮酸（SA）异常堆积；苯丙氨酸或SA处理激活Nrf2通路。由此我们提出“GSTZ1缺失激活Nrf2信号通路重编程细胞代谢促进肝癌发生”的科学假说。本研究拟明确因GSTZ1缺失异常集聚的代谢物，解析代谢物对Keap1转录后修饰，激活Nrf2通路参与肝癌发生的机制。同时在GSTZ1-/-小鼠予化学诱变剂DEN和/或苯丙氨酸处理，观察代谢物诱导Nrf2通路激活在肝癌发生中的作用。本研究将阐明GSTZ1缺失诱导Nrf2重编程细胞代谢促进肝癌发生的机制，为设计靶向苯丙/酪氨酸代谢异常的抗肿瘤药物提供新思路。

细胞的代谢重编程是肿瘤的重要特征之一。因此，研究肿瘤代谢重编程的特点，并以此为靶点恢复正常的代谢途径有望成为肿瘤治疗的新手段。课题组前期研究发现：苯丙氨酸/酪氨酸分解代谢过程中的关键代谢酶谷胱甘肽S-转移酶Zeta 1（GSTZ1）在HCC中显著低表达，且其表达下调与HCC患者预后不良密切相关。此外，GSTZ1低表达的HCC细胞中代谢物SA显著累积，NRF2信号通路明显激活，其增殖侵袭能力也明显增强，由此我们提出“GSTZ1低表达可通过NRF2信号通路促进HCC发生发展”的科学假说。我们通过LC-QQQ-MS 质谱分析发现GSTZ1缺失可导致酪氨酸代谢产物SA显著堆积；Western blot实验发现Phe和SA处理增加NRF2核转位及NRF2下游靶基因的蛋白表达水平。进一步的机制研究表明：SA通过烷基化修饰KEAP1的关键半胱氨酸位点使其失活，进而导致NRF2核转位增加并招募SP1富集于IGF1R启动子区，促进IGF1R表达，从而发挥抗凋亡作用。另一方面，我们发现GSTZ1在索拉非尼耐受的肝癌细胞中明显下调。机制分析提示GSTZ1缺失通过激活NRF2信号通路，进而增强GPX4的表达水平，从而抑制索拉非尼诱导的铁死亡。联合使用索拉非尼与GPX4抑制剂RSL3可显著抑制GSTZ1缺失细胞的生存活力，促进肝癌细胞发生伴有脂质过氧化物异常积累的铁依赖性死亡。上述研究从肿瘤代谢重编程的角度阐释氨基酸代谢酶GSTZ1缺失参与HCC恶性进展的发生机制，并为临床优化肝癌的诊断、治疗与预后监测提供了新的思路。