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 ?-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.
The membrane proteome is the primary hub cells use to interact with others. We hope to determine how membrane protein teams change in cancer using new proteomics and antibody detection technologies. Discovering how cancer cells remodel their cell surface should lead to new biomarkers and drug targets to treat cancer.
|Pollock, Samuel B; Hu, Amy; Mou, Yun et al. (2018) Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies. Proc Natl Acad Sci U S A 115:2836-2841|
|Martinko, Alexander J; Truillet, Charles; Julien, Olivier et al. (2018) Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins. Elife 7:|