Proteomic and phosphoproteomic analysis of cisplatin resistance in patient derived serous ovarian cancer

Understanding the mechanism of resistance in platinum-based regimens for the treatment of high-grade serous ovarian cancer (HGSOC) is important for identifying new therapeutic targets to improve the clinical outcome of ovarian cancer patients. Mass spectrometry-based proteomic strategy was applied to spheroidal cisplatin sensitive and resistant HGSOC generated cell lines in the absence and presence of cisplatin drug. A complete expressed HGSOC proteome and phosphoproteome was characterized in cisplatin sensitive and resistant HGSOC cell lines providing insight into the mechanism of resistance development. PCA analysis showed that phosphorylation of a few proteins provides better classification than the whole proteome of the cellular subtypes. Specifically, a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines in the absence of drug was observed. This same phosphoproteomic signature was observed in our cisplatin sensitive cell line in the absence and presence of drug, indicating a vital role for phosphorylation of proteins in resistance development to cisplatin. The most phosphorylated protein was sequestosome (p62/SQSTM1). Differential expressions of apoptosis by the prognostic factor ratio of Bcl-2/Bax and autophagy, known to be regulated by p62/SQSTM1, was validated in the proteome data and by western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while autophagy revealed increased expression in the resistant relative to sensitive cell line. Furthermore, site specific phosphorylation on 20 modified residues of sequestosome was characterized. Elevated expression of phosphorylation of sequestosome in resistant HGSOC cell lines was validated with western blot analysis. Here, we propose phosphorylation of sequestosome to be a marker and key in cisplatin resistance development in HGOSC ovarian cancers by shuttling ubiquitinated proteins to the autophagy pathway and influencing down-regulation of apoptosis.

解析铂类化疗方案治疗高级别浆液性卵巢癌（high-grade serous ovarian cancer, HGSOC）的耐药机制，对于发掘全新治疗靶点以改善卵巢癌患者的临床结局具有重要意义。本研究采用基于质谱的蛋白质组学策略，对顺铂存在及缺失条件下构建的顺铂敏感型与耐药型高级别浆液性卵巢癌球体细胞系展开分析。本研究对顺铂敏感型与耐药型高级别浆液性卵巢癌细胞系的完整表达蛋白质组及磷酸化蛋白质组进行了系统表征，为阐明耐药发生的分子机制提供了全新视角。主成分分析（Principal Component Analysis, PCA）结果显示，相较于全蛋白质组，少量蛋白质的磷酸化修饰即可实现细胞亚型的更精准分类。具体而言，在未施加顺铂的培养条件下，顺铂敏感型与耐药型细胞系之间存在显著差异的磷酸化蛋白质组特征谱。在未施加及施加顺铂的顺铂敏感型细胞系中，同样可观测到该磷酸化蛋白质组特征谱，这表明蛋白质磷酸化修饰在顺铂耐药发生过程中发挥关键调控作用。磷酸化修饰水平最高的蛋白质为隔离体蛋白（sequestosome, p62/SQSTM1）。以Bcl-2/Bax比值作为预后因子所反映的细胞凋亡差异表达，以及已知受p62/SQSTM1调控的自噬活性，均在蛋白质组数据及蛋白质印迹（western blot）分析中得到了验证。仅在顺铂敏感型细胞系中，施加顺铂后可观测到细胞凋亡水平显著升高；而相较于敏感型细胞系，耐药型细胞系的自噬活性则呈现上调趋势。此外，本研究还对隔离体蛋白上20个特定位点的磷酸化修饰残基进行了系统表征。耐药型高级别浆液性卵巢癌细胞系中隔离体蛋白磷酸化修饰水平升高的现象，经蛋白质印迹分析得到了验证。本研究提出，隔离体蛋白的磷酸化修饰可作为顺铂耐药发生的标志物及关键调控因子，其通过将泛素化蛋白转运至自噬通路并抑制细胞凋亡，从而介导高级别浆液性卵巢癌（HGSOC）的顺铂耐药进程。