Proteomics of Melanoma Progression

黑色素瘤进展的蛋白质组学

• 批准号: 6633801
• 负责人: KATHERYN A RESING
• 金额: $ 24.4万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633801
• 关键词: apoptosis; athymic mouse; cell line; enzyme activity; gel electrophoresis; guanine nucleotide binding protein; immunocytochemistry; immunofluorescence technique; melanocyte; melanoma; metastasis; phosphatidylinositol 3 kinase; polymerase chain reaction; posttranslational modifications; proteomics; transfection; tumor antigens

Our current understanding of carcinogenesis indicates that cancer cells incur increasing mutational burden, with each mutation defeating new protective mechanisms encountered during tumor etiology, so that there is a progression in morphology and behavior. In melanoma, this progression is reflected in morphological "stages": atypical naevus, radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Although these changes are driven by signaling pathways, the complexity of these pathways makes it impossible to directly or quantitatively connect them to specific signaling events. A more precise method of delineating the phenotype of the cell in a way that is quantifiable and directly related to the underlying signaling status would be valuable both for diagnostics and for basic research. The goal of this proposal is to show that the new proteomics methodologies can be used in this way. Using two-dimensional gel electrophoresis (2D), we have surveyed approximately1500 proteins in three cell lines derived from metastatic melanoma. We find 43, 47, and 58 proteins in the pI range 4-7 with >10 fold change in expression, compared to an immortalized, non-- tumorigenic melanocyte cell line. When the metastatic cell lines are compared to each other, we find that each line shows a progressively altered proteome, exactly as predicted by the progression model for carcinogenesis. We hypothesize that (a) other stages can be identified in a similar manner, using cell lines representing different stages in melanoma progression, (b) the proteins in common between the three lines are general markers for metastasis, and (c) successive mutations in protooncogenes, survival factors, or tumor suppressors which regulate tumor progression direct the expression of each marker subset. In this proposal, we will test these hypotheses by (i) comparing additional cell lines derived from different stages of melanoma and identifying the marker proteins by in-gel digestion and mass spectrometric sequencing of the peptides, (ii) confirming marker expression in melanoma biopsies, and (iii) determining the involvement of specific signaling pathways in regulating the expression of these markers, focusing particularly on the role of Ras in RGP to VGP transition, and PI3K in metastasis. The complexity of signaling pathways require that cancer research shift from an emphasis on linear signaling pathways to paradigms that consider the entire signaling pattern, and we believe that the methodology used in this proposal will be an important driving force for that change.

我们目前对癌变的理解表明，癌细胞会导致突变负担增加，每个突变都击败肿瘤病因学遇到的新保护机制，因此形态学和行为的进展。在黑色素瘤中，这种进展反映在形态的“阶段”中：非典型Naevus，径向生长阶段（RGP），垂直生长阶段（VGP）和转移。尽管这些变化是由信号通路驱动的，但这些途径的复杂性使得不可能直接或定量地将它们连接到特定的信号事件。一种更精确的方法，是以可量化和直接相关的基本信号状态直接相关的方式来描述细胞的表型，这对于诊断和基础研究都很有价值。该提案的目的是表明可以以这种方式使用新的蛋白质组学方法。使用二维凝胶电泳（2D），我们调查了来自转移性黑色素瘤的三种细胞系中的大约1500蛋白。与永生，非肿瘤性黑色素细胞细胞系相比，我们在PI范围4-7中发现43、47和58个蛋白质，表达变化> 10倍。当将转移细胞系彼此比较时，我们发现每条线都显示出逐渐改变的蛋白质组，这完全如癌变进程模型所预测的。我们假设（a）使用代表黑色素瘤进展的不同阶段的细胞系可以以相似的方式识别（a）（b）三条线之间的共同蛋白质是转移的一般标记，（c）（c）（c）Protooncentes，Protooncogen，生存因子或TUMOR抑制肿瘤进展的肿瘤进展指导每个标记的表达的蛋白质。在该提案中，我们将通过（i）比较来自黑色素瘤不同阶段的其他细胞系，并通过凝胶凝胶的消化和质谱测序来识别标记蛋白的其他细胞系，（ii）确认在黑色素瘤活检中的表达，以及（iii）确定这些指导层的表达，（iii）确定这些标记的表达，（iii），（iii）的表达构成了这些在范围内的表达，并确定了这些标志物的参与，并确定了这些标记的表达。 RGP到VGP过渡，并在转移中进行PI3K。信号通路的复杂性要求癌症研究从对线性信号通路的强调转变为考虑整个信号模式的范例，我们相信该提案中使用的方法将是该变化的重要驱动力。