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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 ANXA2^E139A 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.

SUBMITTER: Wang K

PROVIDER: S-EPMC5902212 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Wang Kui K Zhang Tao T Lei Yunlong Y Li Xuefeng X Jiang Jingwen J Lan Jiang J Liu Yuan Y Chen Haining H Gao Wei W Xie Na N Chen Qiang Q Zhu Xiaofeng X Liu Xiang X Xie Ke K Peng Yong Y Nice Edouard C EC Wu Min M Huang Canhua C Wei Yuquan Y

Autophagy 20180129 2

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 bleomyci ...[more]

PMID: 29172997

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Project description:BackgroundMigration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood.MethodsMIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration.ResultsClinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca(2+) -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.ConclusionOur data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

Dataset Information

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

Publications

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

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