Abstract LB-2: Cullin-2 facilitates EGFR ubiquitination and degradation after trafficking of the receptor to the nucleus

Abstract

Abstract Objectives of the study: The epidermal growth factor receptor (EGFR) can be overexpressed in bladder cancer and other malignancies and is a molecular risk factor associated with poor prognosis. Surface EGFR is able to traffic to the nucleus, where it can regulate gene expression as a component of transcription complexes. This non-canonical function for the EGFR correlates with tumor aggressiveness and unfavorable clinical outcome. We have identified cullin-2, a ubiquitin E3 ligase cofactor that associates with the Von Hippel-Lindau tumor suppressor protein (pVHL) and elongin B/C, by mass spectrometry as a putative component of an EGFR nuclear trafficking complex in human bladder cancer cells. The goal of the present study was to determine the potential role of cullin-2 in EGFR nuclear trafficking and function. Methodology: Various biochemical approaches, including co-IP/Western blotting and fluorescence imaging, were performed to analyze the association of EGFR and ubiquitin E3 ligase components, including cullin-2 and pVHL. Forced expression or silencing of cullin-2 was also performed. Results: Accumulation of EGFR in nuclei was shown within 30min after treatment of TCCSUP bladder cancer cells with the EGFR ligand HB-EGF. Ubiquitination and degradation of nuclear EGFR were observed 1h after stimulation. Cullin-2 binds to EGFR in the nucleus, leading to recruitment of pVHL upon HB-EGF treatment. The association of nuclear EGFR with the Rpt10/S5a and Rpt1 proteasome subunits implicates the proteasome in nuclear EGFR degradation, which is consistent with data indicating that nuclear EGFR degradation was suppressed by proteasomal inhibition. Silencing of endogenous cullin-2 or pVHL suppressed the degradation of nuclear EGFR. Collectively, these results suggest that cullin-2 is a mediating factor that connects nuclear EGFR to the ubiquitin E3 ligase and the proteasome, leading to proper feedback regulation after EGFR activation. Our findings provide new insight into the diverse intracellular mechanisms in which the EGFR participates and, in particular, its signaling roles in the nucleus in bladder cancer and possibly other cell types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-2.