MilliporeSigma
  • 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.

Autophagy (2022-03-08)
Wenjing Xiao, Jianqun Wang, Xiaojing Wang, Shuang Cai, Yanhua Guo, Lin Ye, Dan Li, Anpei Hu, Shikai Jin, Boling Yuan, Yi Zhou, Qilan Li, Qiangsong Tong, Liduan Zheng
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Acetyl-L-cysteine, Sigma Grade, ≥99% (TLC), powder
Sigma-Aldrich
Cycloheximide, from microbial, ≥94% (TLC)
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)
Sigma-Aldrich
3-Methyladenine, autophagy inhibitor
Sigma-Aldrich
R-SPONDIN-1 human, recombinant, expressed in CHO cells, ≥95% (SDS-PAGE), ≥95% (HPLC)
Sigma-Aldrich
(R)-MG132
Sigma-Aldrich
Fibroblast Growth Factor-Basic, FGF-Basic, from human, recombinant, expressed in E. coli, carrier free
Sigma-Aldrich
DAPI, for nucleic acid staining
Roche
cOmplete, EDTA-free Protease Inhibitor Cocktail, Tablets provided in EASYpacks
Sigma-Aldrich
Wnt3A, human, recombinant, expressed in HEK 293 cells human
Sigma-Aldrich
Noggin human, recombinant, expressed in HEK 293 cells, suitable for cell culture