Identification of ANXA2 (annexin A2) as a specific bleomycin target to induce pulmonary fibrosis by impeding TFEB-mediated autophagic flux.

Abstract

Bleomycin is a clinically potent anticancer drug used for the treatment of germ-cell tumors, lymphomas and squamous-cell carcinomas. Unfortunately, the therapeutic efficacy of bleomycin is severely hampered by the development of pulmonary fibrosis. However, the mechanisms underlying bleomycin-induced pulmonary fibrosis, particularly the molecular target of bleomycin, remains unknown. Here, using a chemical proteomics approach, we identify ANXA2 (annexin A2) as a direct binding target of bleomycin. The interaction of bleomycin with ANXA2 was corroborated both in vitro and in vivo. Genetic depletion of anxa2 in mice mitigates bleomycin-induced pulmonary fibrosis. We further demonstrate that Glu139 (E139) of ANXA2 is required for bleomycin binding in lung epithelial cells. A CRISPR-Cas9-engineered ANXA2E139A mutation in lung epithelial cells ablates bleomycin binding and activates TFEB (transcription factor EB), a master regulator of macroautophagy/autophagy, resulting in substantial acceleration of autophagic flux. Pharmacological activation of TFEB elevates bleomycin-initiated autophagic flux, inhibits apoptosis and proliferation of epithelial cells, and ameliorates pulmonary fibrosis in bleomycin-treated mice. Notably, we observe lowered TFEB and LC3B levels in human pulmonary fibrosis tissues compared to normal controls, suggesting a critical role of TFEB-mediated autophagy in pulmonary fibrosis. Collectively, our data demonstrate that ANXA2 is a specific bleomycin target, and bleomycin binding with ANXA2 impedes TFEB-induced autophagic flux, leading to induction of pulmonary fibrosis. Our findings provide insight into the mechanisms of bleomycin-induced fibrosis and may facilitate development of optimized bleomycin therapeutics devoid of lung toxicity.

• Authors: Kui Wang, Tao Zhang, Yunlong Lei, Xuefeng Li, Jingwen Jiang, Jiang Lan, Yuan Liu, Haining Chen, Wei Gao, Na Xie, Qiang Chen, Xiaofeng Zhu, Xiang Liu, Ke Xie, Yong Peng, Edouard C Nice, Min Wu, Canhua Huang, Yuquan Wei
• Journal: Autophagy (Autophagy) Vol. 14 Issue 2 Pg. 269-282 ( 2018) ISSN: 1554-8635 [Electronic] United States
• PMID: 29172997 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• ANXA2 protein, human
• Annexin A2
• Antibiotics, Antineoplastic
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
• TFEB protein, human
• Bleomycin

Topics

• A549 Cells
• Alveolar Epithelial Cells (drug effects, metabolism, pathology)
• Animals
• Annexin A2 (genetics, metabolism)
• Antibiotics, Antineoplastic (adverse effects, pharmacology)
• Autophagy (drug effects)
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors (antagonists & inhibitors)
• Bleomycin (adverse effects, pharmacology)
• HEK293 Cells
• Humans
• Mice
• Mice, Inbred C57BL
• Proteomics
• Pulmonary Fibrosis (chemically induced)