Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression
Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression
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DOI：
10.1080/15548627.2022.2044651
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发表时间：
2022-03
期刊：
影响因子：
13.3
通讯作者：
W. Xiao;Jian-qun Wang;Xiaojing Wang;Shuang Cai;Yanhua Guo;Lin Ye;Dan Li;A. Hu;Shikai Jin-Shikai-Ji
中科院分区：
文献类型：
--
作者：
W. Xiao;Jian-qun Wang;Xiaojing Wang;Shuang Cai;Yanhua Guo;Lin Ye;Dan Li;A. Hu;Shikai Jin-Shikai-Ji
摘要自噬是一种与肿瘤发生和侵袭性相关的保守的细胞过程，而在癌症中调节自噬机制基因表达的机制仍然不清楚。在此，我们确定E2 F4（E2 F转录因子4）作为一种新的转录激活剂的细胞保护性自噬的锌稳态在癌细胞中至关重要。功能获得和功能丧失研究表明，E2 F4以细胞周期依赖性方式促进自噬，导致MT（金属硫蛋白）蛋白的易化降解，自噬体内Zn 2+的分布升高，细胞内不稳定的锌离子减少，以及胃癌细胞的生长、侵袭和转移增加。在机制上，E2 F4直接调节ATG 2A（自噬相关2A）和ULK 2（unc-51 like autophagy activating kinase 2）的转录，导致MT 1 E、MT 1 M和MT 1X的自噬降解，而USP 2（泛素特异性肽酶2）稳定E2 F4蛋白，通过物理相互作用和癌细胞中的去泛素化诱导其反式激活。拯救实验表明，USP 2通过E2 F4促进的自噬和锌稳态具有致癌特性。Emetine是一种小的自噬化学抑制剂，能够阻断UPS 2和E2 F4之间的相互作用，增加不稳定的细胞内锌离子，并抑制肿瘤发生和侵袭性。在临床胃癌标本中，USP 2和E2 F4均上调，并与患者预后不良相关。这些发现表明，USP 2-E2 F4轴的治疗靶向抑制癌症进展中锌稳态所必需的自噬机制。缩略语：3-MA：3-甲基腺嘌呤;方差分析：方差分析; ATG2A：自噬相关2A; ATG5：自噬相关5例; ATP：三磷酸腺苷; BECN 1：1; BiFC：双分子荧光互补; CCND 1：细胞周期蛋白D1; CDK：细胞周期蛋白依赖性激酶; ChIP：染色质免疫沉淀; CHX：放线菌酮; Co-IP：免疫共沉淀; DAPI：4 '，6-二脒基-2-苯基吲哚; E2 F4：E2 F转录因子4; eATP：细胞外腺苷三磷酸; EBSS：Earle平衡盐溶液; FP：第一进展; FRET：荧光共振能量转移; FUCCI：基于荧光泛素化的细胞周期指示剂; GFP：绿色荧光蛋白; GST：谷胱甘肽S-转移酶; HA：血凝素; MAP 1 LC 3 B/LC 3 B：微管相关蛋白1轻链3 β; MDM 2：MDM 2原癌基因; MKI 67/Ki-67：增殖标志物Ki-67; MT：金属硫蛋白; MT 1 E：金属硫蛋白1 E; MT 1 M：金属硫蛋白1 M; MT 1X：金属硫蛋白1X; MTT：3-（4，5-二甲基三唑-2-基）-2，5-二苯基溴化四唑; OS：总生存期; PECAM 1/CD 31：血小板和内皮细胞粘附分子1; PIK 3C 3：磷脂酰肌醇3-激酶催化亚基3型; qPCR：定量PCR; RFP：红色荧光蛋白; SQSTM 1/p62：多价螯合体1; UBXN 1：UBX结构域蛋白1; Ub：泛素; ULK2：unc-51样自噬激活激酶2; USP 14：遍在蛋白特异性肽酶14; USP 2：遍在蛋白特异性肽酶2; USP 5：遍在蛋白特异性肽酶5; USP 7：遍在蛋白特异性肽酶7; ZnCl 2：氯化锌。
ABSTRACT Macroautophagy/autophagy is a conserved cellular process associated with tumorigenesis and aggressiveness, while mechanisms regulating expression of autophagic machinery genes in cancers still remain elusive. Herein, we identified E2F4 (E2F transcription factor 4) as a novel transcriptional activator of cytoprotective autophagy crucial for zinc homeostasis in cancer cells. Gain- and loss-of-function studies showed that E2F4 promoted autophagy in a cell cycle-dependent manner, resulting in facilitated degradation of MT (metallothionein) proteins, elevated distribution of Zn2+ within autophagosomes, decreased labile intracellular zinc ions, and increased growth, invasion, and metastasis of gastric cancer cells. Mechanistically, E2F4 directly regulated the transcription of ATG2A (autophagy related 2A) and ULK2 (unc-51 like autophagy activating kinase 2), leading to autophagic degradation of MT1E, MT1M, and MT1X, while USP2 (ubiquitin specific peptidase 2) stabilized E2F4 protein to induce its transactivation via physical interaction and deubiquitination in cancer cells. Rescue experiments revealed that USP2 harbored oncogenic properties via E2F4-facilitated autophagy and zinc homeostasis. Emetine, a small chemical inhibitor of autophagy, was able to block interaction between UPS2 and E2F4, increase labile intracellular zinc ions, and suppress tumorigenesis and aggressiveness. In clinical gastric cancer specimens, both USP2 and E2F4 were upregulated and associated with poor outcome of patients. These findings indicate that therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. Abbreviations: 3-MA: 3-methyladenine; ANOVA: analysis of variance; ATG2A: autophagy related 2A; ATG5: autophagy related 5; ATP: adenosine triphosphate; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CCND1: cyclin D1; CDK: cyclin dependent kinase; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; E2F4: E2F transcription factor 4; eATP: extracellular adenosine triphosphate; EBSS: Earle’s balanced salt solution; FP: first progression; FRET: fluorescence resonance energy transfer; FUCCI: fluorescent ubiquitination-based cell cycle indicator; GFP: green fluorescent protein; GST: glutathione S-transferase; HA: hemagglutinin; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDM2: MDM2 proto-oncogene; MKI67/Ki-67: marker of proliferation Ki-67; MT: metallothionein; MT1E: metallothionein 1E; MT1M: metallothionein 1M; MT1X: metallothionein 1X; MTT: 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide; OS: overall survival; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; qPCR: quantitative PCR; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; UBXN1: UBX domain protein 1; Ub: ubiquitin; ULK2: unc-51 like autophagy activating kinase 2; USP14: ubiquitin specific peptidase 14; USP2: ubiquitin specific peptidase 2; USP5: ubiquitin specific peptidase 5; USP7: ubiquitin specific peptidase 7; ZnCl2: zinc chloride.