Phosphoproteomic analysis of cell-based resistance to BRAF inhibitor therapy in melanoma

The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, Protein Kinase C, IGF signaling and melanosome maturation were highly divergent after transition to a drug resistant phenotype.

采用小分子化合物对突变BRAF激酶进行靶向抑制的黑色素瘤治疗方案，仅能暂时抑制转移性疾病的进展。在化学治疗压力下，肿瘤的固有与适应性可塑性可通过重塑可激活增殖与迁移系统的细胞通路的生化与遗传特征，促进肿瘤存活。为探究这一过程，本研究采用高分辨率质谱技术对体外模型中该转变过程的磷酸化蛋白质组（phosphoproteome）进行表征。研究采用一种简便精准的无标记定量（label-free quantitative）方法，对数千个磷酸化位点完成定位与定量分析。此外，我们还将磷酸化蛋白质组的变化与总蛋白质组（proteome）进行关联分析，以通过激酶全景图谱（kinase landscape profiling）更精准地确定调控激酶的活性变化。在转变为耐药表型后，参与细胞骨架调控、GTP/GDP交换、蛋白激酶C（Protein Kinase C）、胰岛素样生长因子（IGF）信号通路以及黑素体成熟过程的磷酸肽丰度均存在显著差异。