Zila Nina, Bileck Andrea, Muqaku Besnik, Janker Lukas, Eichhoff Ossia M, Cheng Phil F, Dummer Reinhard, Levesque Mitchell P, Gerner Christopher, Paulitschke Verena
1Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
2Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria.
Clin Proteomics. 2018 Mar 9;15:13. doi: 10.1186/s12014-018-9189-x. eCollection 2018.
MAP kinase inhibitor (MAPKi) therapy for BRAF mutated melanoma is characterized by high response rates but development of drug resistance within a median progression-free survival (PFS) of 9-12 months. Understanding mechanisms of resistance and identifying effective therapeutic alternatives is one of the most important scientific challenges in melanoma. Using proteomics, we want to specifically gain insight into the pathophysiological process of cerebral metastases.
Cerebral metastases from melanoma patients were initially analyzed by a LC-MS shotgun approach performed on a QExactive HF hybrid quadrupole-orbitrap mass spectrometer. For further validation steps after bioinformatics analysis, a targeted LC-QQQ-MS approach, as well as Western blot, immunohistochemistry and immunocytochemistry was performed.
In this pilot study, we were able to identify 5977 proteins by LC-MS analysis (data are available via ProteomeXchange with identifier PXD007592). Based on PFS, samples were classified into good responders (PFS ≥ 6 months) and poor responders (PFS [Formula: see text] 3 months). By evaluating these proteomic profiles according to gene ontology (GO) terms, KEGG pathways and gene set enrichment analysis (GSEA), we could characterize differences between the two distinct groups. We detected an EMT feature (up-regulation of N-cadherin) as classifier between the two groups, V-type proton ATPases, cell adhesion proteins and several transporter and exchanger proteins to be significantly up-regulated in poor responding patients, whereas good responders showed an immune activation, among other features. We identified class-discriminating proteins based on nearest shrunken centroids, validated and quantified this signature by a targeted approach and could correlate parts of this signature with resistance using the CPL/MUW proteome database and survival of patients by TCGA analysis. We further validated an EMT-like signature as a major discriminator between good and poor responders on primary melanoma cells derived from cerebral metastases. Higher immune activity is demonstrated in patients with good response to MAPKi by immunohistochemical staining of biopsy samples of cerebral melanoma metastases.
Employing proteomic analysis, we confirmed known extra-cerebral resistance mechanisms in the cerebral metastases and further discovered possible brain specific mechanisms of drug efflux, which might serve as treatment targets or as predictive markers for these kinds of metastasis.
BRAF 突变型黑色素瘤的丝裂原活化蛋白激酶抑制剂（MAPKi）治疗具有高缓解率的特点，但在中位无进展生存期（PFS）为 9 - 12 个月内会出现耐药性。了解耐药机制并确定有效的治疗替代方案是黑色素瘤最重要的科学挑战之一。我们希望通过蛋白质组学，具体深入了解脑转移的病理生理过程。
首先对黑色素瘤患者的脑转移灶采用 QExactive HF 混合型四极杆 - 轨道阱质谱仪进行 LC - MS 鸟枪法分析。在生物信息学分析后的进一步验证步骤中，采用靶向 LC - QQQ - MS 方法以及蛋白质免疫印迹、免疫组织化学和免疫细胞化学方法。
在这项初步研究中，我们通过 LC - MS 分析能够鉴定出 5977 种蛋白质（数据可通过 ProteomeXchange 获取，标识符为 PXD007592）。根据 PFS，样本被分为良好缓解者（PFS≥6 个月）和不良缓解者（PFS＜3 个月）。通过根据基因本体（GO）术语、KEGG 通路和基因集富集分析（GSEA）评估这些蛋白质组学图谱，我们能够描述这两个不同组之间的差异。我们检测到 EMT 特征（N - 钙黏蛋白上调）作为两组之间的分类指标，V 型质子 ATP 酶、细胞黏附蛋白以及几种转运蛋白和交换蛋白在不良缓解患者中显著上调，而良好缓解者则表现出免疫激活等特征。我们基于最近收缩质心确定了区分组别的蛋白质，通过靶向方法对该特征进行验证和定量，并使用 CPL/MUW 蛋白质组数据库将该特征的部分与耐药性相关联，通过 TCGA 分析将其与患者生存相关联。我们进一步在源自脑转移的原发性黑色素瘤细胞上验证了一种类似 EMT 的特征作为良好和不良缓解者之间的主要区分指标。通过对脑黑色素瘤转移活检样本进行免疫组织化学染色，在对 MAPKi 反应良好的患者中显示出更高的免疫活性。
通过蛋白质组学分析，我们在脑转移中证实了已知的脑外耐药机制，并进一步发现了可能的脑特异性药物外排机制，这可能作为这类转移的治疗靶点或预测标志物。
