Secretome Proteomics for VHL Tumor Biomarker Discovery

描述(申请人提供)：由于血清蛋白质组的非凡复杂性，通过直接对血清蛋白质组分析来发现肿瘤生物标记物仍然是一个挑战。作为另一种选择，这一应用程序建议分析培养中的肿瘤细胞分泌的蛋白质，然后使用肿瘤患者和健康对照的血清样本来验证这些候选肿瘤标记物。中心假设是，肿瘤生物标记物可以被识别为在失去肿瘤抑制因子后表现出分泌增强的蛋白质。本申请的目的是以冯·希佩尔-林道(Von Hippel-Lindau，VHL)肿瘤抑制因子作为模型案例来验证这一假说：它旨在识别VHL肿瘤生物标记物，即当VHL肿瘤抑制因子丢失时，显示分泌增加的蛋白质。VHL病是一种以肾透明细胞癌、神经系统血管母细胞瘤和肾上腺嗜铬细胞瘤为特征的遗传性癌症综合征。VHL病患者携带1个野生型VHL等位基因和1个失活VHL等位基因。这些患者中的肿瘤和囊肿是由剩余的野生型等位基因的体细胞突变引起的。双等位基因VHL失活在散发性肾透明细胞癌和血管母细胞瘤中也很常见，目前尚无有效的血清肿瘤生物标志物用于诊断VHL病或肾细胞癌。这种生物标志物将有助于对VHL突变携带者的监测和临床前干预，以及散发性肾癌的早期发现。应用定量蛋白质组学技术，ICAT(同位素编码亲和标签)，对VHL缺失的肾癌细胞株786-O及其VHL再导入的衍生物的蛋白质分泌进行了初步的小规模分析。这项分析发现了一些在VHL丢失时分泌增加的蛋白质，包括血管内皮生长因子(VEGF)，它显示VHL疾病患者的血清水平显著升高。在这些初步数据的指导下，这项应用追求两个具体目标：1)扩大和扩展使用3种不同细胞系统的初步分析，全面识别候选VHL肿瘤生物标记物；2)使用VHL疾病患者和健康对照的血清样本验证这些候选标记物，并确定这些已验证标记物在这些患者和对照中的绝对血清浓度范围。公共卫生相关性：通过分析VHL肿瘤抑制基因缺失时分泌的蛋白质，拟议的项目发现了VHL突变肿瘤的血清生物标志物，这将促进对VHL突变携带者的监测和临床前干预，以及散发性肾细胞癌的早期发现。此外，本项目开发的基于分泌组蛋白质组的癌症生物标记物发现方法将广泛适用于其他抑癌基因和癌蛋白，有助于识别许多有用的癌症生物标记物。

DESCRIPTION (provided by applicant): Because of the extraordinary complexity of the serum proteome, tumor biomarker discovery by direct proteomic analysis of blood serum continues to be a challenge. As an alternative, this application proposes to analyze proteins secreted from tumor cells in culture and later validate these candidate tumor markers using serum samples from tumor patients and healthy controls. The central hypothesis is that tumor biomarkers can be identified as proteins displaying enhanced secretion upon loss of a tumor suppressor. The objective of the present application is to test this hypothesis using the von Hippel-Lindau (VHL) tumor suppressor as a model case: it is directed towards identifying the VHL tumor biomarkers as proteins displaying increased secretion upon loss of the VHL tumor suppressor. VHL disease is a hereditary cancer syndrome characterized by clear cell renal carcinomas, hemangioblastomas of the nervous system, and adrenal pheochromocytomas. Patients with VHL disease harbor one wild-type VHL allele and one inactivated VHL allele. Tumors and cysts in these patients arise by somatic mutation of the remaining wild-type allele. Biallelic VHL inactivation is also common in sporadic clear cell renal carcinomas and hemangioblastomas Currently, there is no effective serum tumor biomarker for the VHL disease or renal cell carcinomas. Such biomarker would facilitate the monitoring and pre-clinical intervention of VHL mutation carriers and early detection of sporadic renal carcinomas. Employing a quantitative proteomics technology, ICAT (isotope-coded affinity tag), a preliminary small- scale analysis of protein secretion from a VHL-null renal carcinoma cell line, 786-O, and its VHL-reintroduced derivative was carried out. This analysis identified a number of proteins that display enhanced secretion upon VHL loss, including Vascular Endothelial Growth Factor (VEGF) which showed significantly elevated serum levels in VHL disease patients. Guided by these preliminary data, this application pursues two specific aims: 1) scale-up and extend the preliminary analysis using 3 different cell systems and comprehensively identify candidate VHL tumor biomarkers and 2) validate these candidate markers using serum samples from VHL disease patients and healthy controls and also determine the absolute serum concentration ranges of the validated markers in these patients and controls. PUBLIC HEALTH RELEVANCE: By analyzing proteins secreted upon loss of the VHL tumor suppressor, the proposed project discovers serum biomarkers for VHL-mutated tumors, which will advance the monitoring and pre- clinical intervention of VHL mutation carriers as well as early detection of sporadic renal cell carcinomas. Furthermore, secretome proteomics-based approach for cancer biomarker discovery developed by this project will be widely applicable to other tumor suppressors and oncoproteins, facilitating the identification of many useful cancer biomarkers.