National Institute of Cancer Research, National Health Research Institutes, No. 367, Sheng-Li Rd., North District, Tainan, 70456, Taiwan.
Ph.D. Program in Tissue Engineering and Regenerative Medicine, Biotechnology Center, National Chung Hsing University, Taichung, 402, Taiwan.
J Biomed Sci. 2021 Jul 23;28(1):55. doi: 10.1186/s12929-021-00751-5.
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.
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.
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.
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.
在接受 HSP90 抑制剂（如 AUY922）治疗的癌症患者中，眼部不良反应是常见的剂量限制毒性；然而，介导 AUY922 诱导的视网膜毒性的病理学和分子机制仍未被描述。
采用基于等重同位素标记相对和绝对定量（iTRAQ）的蛋白质组学谱分析和途径富集分析、免疫组织化学和免疫荧光染色、末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）检测、MTT 检测、集落形成检测和 Western blot 分析等方法，全面研究了 AUY922 对小鼠视网膜和细胞系的影响。通过免疫共沉淀技术，对 AUY922 对瞬时受体电位阳离子通道亚家族 M 成员 1（TRPM1）-HSP90 伴侣复合物的影响进行了表征。通过 RNAseq 分析和基因集富集分析（GSEA）分析了 TRPM1 调节的基因表达。通过功能丧失和功能获得两种方法评估了 TRPM1 的作用。
本研究表明，AUY922 处理诱导视网膜损伤和细胞凋亡，使光感受器和视网膜色素上皮（RPE）层失调，并降低 TRPM1 表达。差异表达蛋白的蛋白质组学谱分析和功能注释表明，那些与应激反应、蛋白质折叠过程、细胞凋亡、细胞周期和生长、活性氧（ROS）反应、细胞连接组装和粘附调节、质子跨膜转运相关的蛋白显著富集于 AUY922 处理的细胞中。我们发现 AUY922 触发了 caspase-3 依赖性细胞凋亡，增加了 ROS 的产生并抑制了细胞生长。我们确定 TRPM1 是 HSP90 的真正客户，并发现 AUY922 可能通过破坏 CDC37-HSP90 伴侣复合物来降低 TRPM1 的表达。此外，GSEA 显示，TRPM1 调节的基因与相机型眼睛的视网膜形态发生和 JAK-STAT 级联反应相关。最后，功能获得和功能丧失分析验证了 TRPM1 介导 AUY922 诱导的细胞凋亡、ROS 产生和生长抑制的发现。
本研究在体内证明了 AUY922 诱导的视网膜毒性的病理学。TRPM1 是 HSP90 的客户，调节光感受器的形态和功能，并介导 AUY922 诱导的细胞毒性。
