Discovering how oncogenes remodel the surfaceome of cells

发现癌基因如何重塑细胞表面组

• 批准号: 10212408
• 负责人: JAMES A WELLS
• 金额: $ 53.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212408
• 关键词: Amines; Antibodies; Antineoplastic Agents; BRAF gene; Bacteriophages; Bar Codes; Biological Markers; Cell Line; Cell Surface Proteins; Cell surface; Cells; Collaborations; DNA; Data Set; Drug Targeting; Epidermal Growth Factor Receptor; Event; Foundations; Goals; Human; KRAS2 gene; Lead; Logic; MEKs; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Membrane; Membrane Proteins; Methods; Molecular; Mutation; N-terminal; Oncogenes; Oncogenic; Patients; Phage Display; Polysaccharides; Population; Proteins; Proteome; Proteomics; Recombinant Antibody; Regulation; Sampling; Signal Transduction; Surface; Technology; Tissues; antibody detection; base; cancer cell; metaplastic cell transformation; new technology; new therapeutic target; next generation sequencing; particle

Title: Discovering how oncogenes remodel the surfaceome of cells Abstract: The cell surface is the primary hub for cells to communicate with the outside world. Cancer cells have numerous challenges for survival and we hypothesize many of these start at the cell surface made up of some 3000-4000 proteins. Our primary goal is to systematically understand how cancer cells remodel their membrane proteomes (surfaceomes) during oncogenic transformation to survive. We propose to develop enabling technologies for surfaceomics at the population, single cell, and tissue level. We will generate foundational data sets to understand the molecular logic and signaling mechanisms behind the coordinated remodeling events that drive cellular transformation. (Fig 1). We will focus our studies on mutationally activated KRAS and five other highly proliferative oncogenes (EGFR, BRAF, MEK, Akt and PI3K) that together are found in nearly half of all human cancers. We will study how these oncogenes induce coordinate changes in the surfaceome compared to isogenic cells of the same type, in patient derived cancer cells and tissues. We propose new mass spectrometry-based methods to allow for quantification of membrane proteins using two complementary enrichment methods that target surface glycans or surface N-terminal α-amines. We will also develop a new, highly sensitive and multiplexed technology (phage-antibody next generation sequencing, or PhaNGS) to simultaneously detect 100s of surface proteins in very small Figure 1. (A) To understand how oncogenes such as KRAS induce massive cellular changes we will (B) develop new technologies to measure surfaceome changes to understand coordinate regulation, and identify new drug targets. samples or even single cells. The Antibiome Center, which I direct, in collaboration with the Recombinant Antibody Network, has produced recombinant antibodies to 100s of cell surface proteins using Fab-phage display. Each Fab-phage is essentially a DNA barcoded antibody because it has a functional Fab displayed from the phage particle with the DNA encoding it inside. PhaNGS has tremendous potential for highly multiplexed, inexpensive and ultrasensitive means of probing 1000s of cells or cell lines for changes in their surfaceomes. I am excited that these studies will reveal how coordinate remodeling of membrane protein teams contributes to cell state changes. I believe we will also discover new biomarkers and cancer drug targets.

标题：发现癌基因如何重塑细胞的表面 摘要：细胞表面是与外界通信的细胞交流的主要枢纽。癌细胞 生存面临许多挑战，我们假设许多 这些从大约3000-4000蛋白组成的细胞表面开始。 我们的主要目标是系统地了解癌细胞如何 重塑其膜蛋白质组织（表面体） 致癌转化以生存。我们建议发展 在人群，单细胞中启用表面组学技术， 和组织水平。我们将生成基础数据集 了解后面的分子逻辑和信号传导机制 驱动细胞转换的协调重塑事件。 （图1）。我们将把研究重点放在突变激活的KRAS上 还有另外五个高度增殖的癌基因（EGFR，BRAF，MEK， Akt和pi3k）在几乎所有人类的一半 癌症。我们将研究这些癌基因如何诱导坐标 与同一的同源细胞相比，表面体的变化 类型，在患者衍生的癌细胞和组织中。我们提出了新的建议 基于质谱的方法，以量化 膜蛋白使用两种完整的富集方法 该靶向表面聚糖或表面N末端α-胺。我们将 还开发了一种新的，高度敏感和多重技术 （噬菌体抗体下一代测序，或phangs） 类似地检测到很小的表面蛋白的100秒 图1。（a）了解如何 癌基因（例如KRAS） 大规模的细胞变化我们将（b） 开发新技术 测量表面的变化 了解坐标法规， 并确定新药物靶标。 样品，甚至单个单元。我指导的抗抗菌中心与重组合作 抗体网络已使用Fab-Phage产生了100s细胞表面蛋白的重组抗体 展示。每个Fab-phage本质上都是DNA条形码抗体，因为它具有功能性晶圆厂 从噬菌体粒子的DNA中编码内部。 Phangs具有高度的高度潜力 多路复用，廉价且超敏感的方法，用于探测1000秒的细胞或细胞系，以改变其变化 表面体。我很高兴这些研究将揭示如何坐标膜蛋白的重塑 团队为细胞状态变化做出了贡献。我相信我们还会发现新的生物标志物和癌症药物 目标。