Many oncoproteins and tumor suppressive proteins shuttle between the cytosol and nucleus and thus interact with changing sets of protein partners in different sub-cellular compartments. Furthermore, increasing evidence indicates that oncogenic mutations of these proteins can result in alteration of cellular localization and rewirin of oncoprotein interaction networks. Characterization of the dynamic interaction network of oncoproteins and tumor suppressive proteins is therefore crucial to understand their roles in tumorigenesis. Robust technologies for mapping dynamic protein interaction networks under physiological conditions within the cell are not yet available. We propose to develop a novel approach for mapping dynamic oncoprotein interaction networks using endogenously epitope-tagged proteins for affinity- purification and quantitative proteomics for accurate network identification. This application builds upon our successful development of an innovative method to introduce epitope tag- encoding DNA into endogenous loci by homologous recombination-mediated knock-in in human cancer cells and our intensive experience in quantitative proteomics analysis. The endogenously tagged proteins are expressed at physiological levels and provide physiologically-relevant environments and compartments for protein complex identification and are therefore ideal for mapping dynamic protein networks. The goals of this application are twofold. First, we propose a detailed quantitative comparison of our technique in terms of distinguishing mutant and wild-type oncoprotein complexes against two conventional approaches for studying protein complexes. Second we test the potential of our new approach for studying the dynamic interactions that occur in response to cell signaling. Successful development of this technology will provide a platform for understanding the reconfigurations to protein interaction networks that result from oncogenic related translocation or mutation. As large-scale projects such as The Cancer Genome Atlas (TCGA) proceed, many more novel oncogenes and tumor suppressor genes will be discovered;our technology provides an important pipeline for understanding the function of these genes at the interaction network level by mapping their dynamic interaction networks.

Public Health Relevance

Cancer development is driven by mutations in oncogenes and tumor suppressor genes. We aim to develop an innovative approach to facilitate studies of how these gene products function in cancer cells. Such studies will provide the fundamental knowledge required for the development of novel cancer therapeutic approaches.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA160060-02
Application #
8538899
Study Section
Special Emphasis Panel (ZCA1-SRLB-Q (O1))
Program Officer
Knowlton, John R
Project Start
2012-09-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$224,691
Indirect Cost
$81,576
Name
Case Western Reserve University
Department
Genetics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Song, J; Wang, Z; Ewing, R M (2014) Integrated analysis of the Wnt responsive proteome in human cells reveals diverse and cell-type specific networks. Mol Biosyst 10:45-53
Koon, Henry B; Krown, Susan E; Lee, Jeannette Y et al. (2014) Phase II trial of imatinib in AIDS-associated Kaposi's sarcoma: AIDS Malignancy Consortium Protocol 042. J Clin Oncol 32:402-8
Hao, Yujun; Zhao, Shuliang; Wang, Zhenghe (2014) Targeting the protein-protein interaction between IRS1 and mutant p110* for cancer therapy. Toxicol Pathol 42:140-7
Scott, Anthony; Song, Jing; Ewing, Rob et al. (2014) Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications. Acta Biochim Biophys Sin (Shanghai) 46:199-203
Liu, Bo; Cong, Rixin; Peng, Bin et al. (2014) CtIP is required for DNA damage-dependent induction of P21. Cell Cycle 13:90-5
Hao, Yujun; Wang, Chao; Cao, Bo et al. (2013) Gain of interaction with IRS1 by p110*-helical domain mutants is crucial for their oncogenic functions. Cancer Cell 23:583-93