背景:眼部不良事件是使用HSP90抑制剂治疗的癌症患者中常见的剂量限制性毒性,例如AUY922;然而,介导AUY922诱导的视网膜毒性的病理学和分子机制仍未描述.
方法:使用基于相对和绝对定量(iTRAQ)的蛋白质组学分析和途径富集分析的同量异序标签,全面研究了AUY922对小鼠视网膜和细胞系的影响。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)测定,MTT测定,集落形成试验,和蛋白质印迹分析。AUY922对瞬时受体电位阳离子通道亚家族M成员1(TRPM1)-HSP90伴侣复合物的影响通过共免疫沉淀进行表征。通过RNAseq分析和基因集富集分析(GSEA)分析TRPM1调节的基因表达。使用功能丧失和功能获得方法评估TRPM1的作用。
结果:这里,我们表明,用AUY922治疗诱导视网膜损伤和细胞凋亡,异常调节的光感受器和视网膜色素上皮(RPE)层,和减少TRPM1表达。差异表达蛋白质的蛋白质组学分析和功能注释揭示了那些与应激反应有关的蛋白质,蛋白质折叠过程,细胞凋亡的调节,细胞周期和生长,活性氧(ROS)响应,细胞连接组装和粘附调节,和质子跨膜转运在AUY922处理的细胞中显著富集。我们发现AUY922可触发caspase-3依赖性细胞凋亡,增加ROS产生并抑制细胞生长。我们确定TRPM1是真正的HSP90客户,其特征是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和JAK-STAT级联中的视网膜形态发生有关。最后,功能获得和功能丧失分析证实了TRPM1介导细胞凋亡的发现,AUY922诱导的ROS产生和生长抑制。
结论:我们的研究证明了体内AUY922诱导的视网膜毒性的病理学。TRPM1是HSP90客户端,调节感光体的形态和功能,并介导AUY922诱导的细胞毒性。
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.