Che-Hung Shen1,2, Chi-Che Hsieh3, Kuan-Ying Jiang3, Chih-Yu Lin3, Nai-Jung Chiang3,4, Ting-Wei Li5, Chun-Ting Yen6, Wan-Ju Chen7, Daw-Yang Hwang3, Li-Tzong Chen8,9,10. 1. National Institute of Cancer Research, National Health Research Institutes, No. 367, Sheng-Li Rd., North District, Tainan, 70456, Taiwan. chshen@nhri.edu.tw. 2. Ph.D. Program in Tissue Engineering and Regenerative Medicine, Biotechnology Center, National Chung Hsing University, Taichung, 402, Taiwan. chshen@nhri.edu.tw. 3. National Institute of Cancer Research, National Health Research Institutes, No. 367, Sheng-Li Rd., North District, Tainan, 70456, Taiwan. 4. Department of Oncology, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. 5. Department of Life Sciences, National Cheng Kung University, Tainan, 704, Taiwan. 6. Institute of Molecular Medicine, National Cheng Kung University, Tainan, 704, Taiwan. 7. Department of Ophthalmology, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. 8. National Institute of Cancer Research, National Health Research Institutes, No. 367, Sheng-Li Rd., North District, Tainan, 70456, Taiwan. leochen@nhri.org.tw. 9. Department of Oncology, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. leochen@nhri.org.tw. 10. Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, 807, Taiwan. leochen@nhri.org.tw.
Abstract
BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.
BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancerpatients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.