Elucidation of the physiological regulation of epidermal growth factor receptor (EGFR) is the key to understanding of the mechanisms causing its oncogenic activation. Growth factor binding to the EGFR triggers the signal transduction process. Receptor activation also causes rapid internalization and degradation of EGFR in lysosomes, leading to receptor down-regulation. This endocytic trafficking determines the number of active receptors in the cell and, therefore, the intensity and duration of signaling. Endocytosis also orchestrates EGFR signaling network by determining the localization and temporal dynamics of signaling processes. Importantly, EGFR endocytosis is frequently dysregulated in tumor cells. However, the mechanisms of EGFR endocytosis and the role of endocytosis and signaling remain to be poorly understood. During the previous cycle of this project we have defined the molecular mechanisms of EGFR degradation and ubiquitination, the major modification of the receptor that controls EGFR endocytosis. We have also demonstrated the complexity of the internalization step of EGFR endocytosis that is mediated by multiple redundant mechanisms. One of these mechanisms involves a novel post-translational modification of EGFR by acetylation. We have also developed new reagents, such as degradation- and internalization-impaired EGFR mutants, and used these new tools to demonstrate unexpected inhibitory effects of impaired EGFR internalization on tumorigenic signaling. Stemming from the new data and based on the availability of new technologies, such as high-resolution intravital imaging of tumors, ultra-fast confocal imaging of living cultured cells and quantitative mass-spectrometry, we propose to define the molecular mechanisms of EGFR internalization in tumor cell lines and in vivo models and examine the role of EGFR endocytosis in signaling in tumor models in vivo.
The specific aims of this proposal are: 1) define the mechanisms of EGFR endocytosis, including the mechanisms and role of EGFR acetylation, under physiological conditions in cancer cells in vitro and in vivo in mouse xenograft models of head-and-neck cancer;2) examine the effects of inhibited endocytosis and degradation of EGFR on tumor growth in mouse xenograft models and transgenic mouse inducible model of lung cancer;3) define the regulatory mechanisms of EGFR ubiquitination and deubiquitination, and examine the potential of EGFR-specific deubiquitination enzymes as therapeutic targets in EGFR-dependent tumors.

Public Health Relevance

EGFR is involved in the development and progression of many epithelial cancers where its expression is linked to poor prognosis. Aberrant endocytosis of EGFR is associated with a variety of cancers, and the pharmaceutical interference with the ability of tumor cells to disrupt EGFR endocytosis may constitute a novel target for cancer therapy.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01CA089151-14
Application #
8676443
Study Section
Tumor Cell Biology Study Section (TCB)
Program Officer
Salnikow, Konstantin
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Pinilla-Macua, Itziar; Watkins, Simon C; Sorkin, Alexander (2016) Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes. Proc Natl Acad Sci U S A 113:2122-7
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Pinilla-Macua, Itziar; Sorkin, Alexander (2015) Methods to study endocytic trafficking of the EGF receptor. Methods Cell Biol 130:347-67
Fortian, Arola; Dionne, Lai K; Hong, Sun H et al. (2015) Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation. Traffic 16:1137-54
Tomas, Alejandra; Vaughan, Simon O; Burgoyne, Thomas et al. (2015) WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway. Nat Commun 6:7324
Schmid, Sandra L; Sorkin, Alexander; Zerial, Marino (2014) Endocytosis: Past, present, and future. Cold Spring Harb Perspect Biol 6:a022509
Fortian, Arola; Sorkin, Alexander (2014) Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis. J Cell Sci 127:432-44
Huang, Fangtian; Zeng, Xuemei; Kim, Woong et al. (2013) Lysine 63-linked polyubiquitination is required for EGF receptor degradation. Proc Natl Acad Sci U S A 110:15722-7
Goh, Lai Kuan; Sorkin, Alexander (2013) Endocytosis of receptor tyrosine kinases. Cold Spring Harb Perspect Biol 5:a017459

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