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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Tumor Cell Biology Study Section (TCB)
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Salnikow, Konstantin
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University of Pittsburgh
Schools of Medicine
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Nagashima, Takeshi; Inoue, Norihiko; Yumoto, Noriko et al. (2015) Feedforward regulation of mRNA stability by prolonged extracellular signal-regulated kinase activity. FEBS J 282:613-29
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Duex, Jason E; Mullins, Michael R; Sorkin, Alexander (2010) Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking. Exp Cell Res 316:2136-51
Sorkin, Alexander; Duex, Jason E (2010) Quantitative analysis of endocytosis and turnover of epidermal growth factor (EGF) and EGF receptor. Curr Protoc Cell Biol Chapter 15:Unit 15.14
Duex, Jason E; Sorkin, Alexander (2009) RNA interference screen identifies Usp18 as a regulator of epidermal growth factor receptor synthesis. Mol Biol Cell 20:1833-44

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