Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression
Creators: name: Xiao, Wenjing
name: Wang, Jianqun
name: Wang, Xiaojing
name: Cai, Shuang
name: Guo, Yanhua
name: Ye, Lin
name: Li, Dan
name: Hu, Anpei
name: Jin, Shikai
name: Yuan, Boling
name: Zhou, Yi
name: Li, Qilan
name: Tong, Qiangsong
name: Zheng, Liduan
Publisher: name: Taylor & Francis
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Inorganic Chemistry
subject: FOS: Chemical sciences
subject: Plant Biology
subject: Virology

description: 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 <i>E2F4</i> promoted autophagy in a cell cycle-dependent manner, resulting in facilitated degradation of MT (metallothionein) proteins, elevated distribution of Zn<sup>2+</sup> within autophagosomes, decreased labile intracellular zinc ions, and increased growth, invasion, and metastasis of gastric cancer cells. Mechanistically, E2F4 directly regulated the transcription of <i>ATG2A</i> (autophagy related 2A) and <i>ULK2</i> (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 <i>USP2</i> harbored oncogenic properties via <i>E2F4</i>-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 <i>USP2</i> and <i>E2F4</i> were upregulated and associated with poor outcome of patients. These findings indicate that therapeutic targeting of the <i>USP2-E2F4</i> axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. <b>Abbreviations:</b> 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; ZnCl<sub>2</sub>: zinc chloride.