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 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.

Anlotinib attenuated bleomycin-induced pulmonary fibrosis via the TGF-β1 signalling pathway

“…Currently, BLM‐induced pulmonary fibrosis is the most classic model for exploring the pathogenesis of IPF and testing the effectiveness of new antifibrotic drugs . Recent studies have shown the mechanism of bleomycin‐induced pulmonary fibrosis: ANXA2(membrane‐associated protein A2) is a specific bleomycin target, and bleomycin binding with ANXA2 blocks TFEB (transcription factor EB)induced autophagic flux and further promotes apoptosis of epithelial cells, leading to pulmonary fibrosis . In short, BLM‐induced fibrosis animal model mimics the clinicopathological features of IPF, which is an effective model to test the effects of novel pharmacological agents.…”

“…[18] Recent studies have shown the mechanism of bleomycin-induced pulmonary fibrosis: ANXA2(membrane-associated protein A2) is a specific bleomycin target, and bleomycin binding with ANXA2 blocks TFEB (transcription factor EB)induced autophagic flux and further promotes apoptosis of epithelial cells, leading to pulmonary fibrosis. [19] In short, BLM-induced fibrosis animal model mimics the clinicopathological features of IPF, which is an effective model to test the effects of novel pharmacological agents. In this study, we established two models: bleomycin-induced inflammatory model and pulmonary fibrosis model to evaluate the antifibrotic effects of anlotinib.…”

Ellagic Acid Attenuates BLM-Induced Pulmonary Fibrosis via Inhibiting Wnt Signaling Pathway

“…Insu cient autophagy might result in the senescence of pulmonary epithelial cells and the activation of myo broblasts in pulmonary brosis [15], and autophagy was inhibited in IPF patients [16]. In lung epithelial cells, bleomycin could induce pulmonary brosis in mice by impeding TFEB-mediated autophagic ux [17]. The AMPK-dependent activation of autophagy enhances collagen turnover to deactivate myo broblasts, which attenuates BLM-induced pulmonary brosis [18].…”

Cell-surface translocation of annexin A2 contributes to bleomycin-induced pulmonary fibrosis by mediating inflammatory response in mice

“…However, the precise molecular mechanisms of IPF remain to be fully revealed, and thus the optimal therapeutic treatment has not yet been developed [6][7][8][9]. In previous study, we found that ANXA2 was a special target of bleomycin and contributed to the development of pulmonary fibrosis by blocking TFEB-induced autophagy [30]. ANXA2 is required for multiple biological functions such as signal transduction, inflammation, EMT, oxidative balance, angiogenesis, cell proliferation and apoptosis, airway epithelial wound repair etc., all of which are also believed to be important pathologic mechanisms of pulmonary fibrosis [20,21,37,[48][49][50][51][52][53][54][55].…”

“…In previous study, we found that ANXA2-bleomycin binding inhibited TFEB-induced autophagy flux, which contributed to bleomycin-induced pulmonary fibrosis [30]. The pathologic mechanisms of pulmonary fibrosis is complex, in addition to autophagy, chronic inflammation and cytokines production were widely accepted to play a role in pulmonary fibrosis [5,[36][37][38][39].…”