Proteasome Inhibition Blocks Ligand-Induced Dynamic Processing and Internalization of Epidermal Growth Factor Receptor via Altered Receptor Ubiquitination and Phosphorylation

Abstract

Epidermal growth factor (EGF) receptor (EGFR), a member of the EGF superfamily of receptor tyrosine kinases, is a critical regulator of cell growth and an important target for single agent and combination anticancer therapeutics. To further investigate the dynamics of ligand-induced EGFR processing and regulation noninvasively, we developed a chimeric EGFRfirefly luciferase (FLuc) fusion reporter to directly monitor processing of EGFR in real-time. In a stable HeLa cell line expressing the reporter at physiologically relevant levels, bioluminescence imaging continuously monitored reporter dynamics, correlating with the ligand-induced response of endogenous EGFR as determined by Western blot, subcellular localization of an EGFR-green fluorescent protein (GFP) fusion protein, and validated pharmacologic responses. The signaling competency of the reporter was confirmed by gene rescue experiments in EGFR-null cells. Bioluminescence analysis further showed that proteasome inhibition with bortezomib or MG132 attenuated overall ligand-induced degradation of EGFR. In cells expressing EGFR-GFP, pretreatment with proteasome inhibitors trapped essentially all of the receptor at the cell membrane both before and after ligandinduced activation with EGF. Furthermore, proteasome inhibition enhanced receptor ubiquitination in both the basal and ligand-activated states as well as delayed the processing of ligand-activated phosphorylation of the receptor, kinetically correlating with attenuated receptor degradation. These observations point to a potential mechanism for the synergistic therapeutic effects of combination EGFR-and proteasome-targeted therapies. [Cancer Res 2009;69(3):976-83]

Extracellular Lumican Inhibits Pancreatic Cancer Cell Growth and Is Associated with Prolonged Survival after Surgery

“…We next investigated whether lumican reduces Akt activity via EGFR. As demonstrated in previous studies (39), the proteosome inhibitor MG132 blocks ligand-induced internalization of EGFR through altered receptor ubiquitination and phosphorylation. When cells were pretreated with MG132 and subsequently exposed to lumican, the previously observed EGFR degradation was blocked as was the resultant Akt phosphorylation (Fig.…”

RETRACTED: Antitumor activity of bortezomib in human cancer cells with acquired resistance to anti-epidermal growth factor receptor tyrosine kinase inhibitors

“…Several relevant signalling pathways which are involved in cancer development and progression could be a target for the bortezomib induced inhibition of proteasome activity [10]. The EGFR itself is subject to ubiquitination and subsequent proteolytic breakdown and is thus affected by proteasome activation [11,12]. In this respect, bortezomib treatment caused a marked suppression of the PI3K/AKT controlled cell survival pathway in human breast cancer cell lines [13], suggesting a functional link between activation of the EGFR family of growth factor receptors, PI3K/AKT signalling and proteasome inhibition [14].…”

A role of Rab7 in stabilizing EGFR‐Her2 and in sustaining Akt survival signal

“…EGFR and Her2 normally undergo lysosomal degradation. However, in a recent study, it was reported that EGFR levels are sensitive to proteasome inhibitors and proteosomal inhibition caused accumulation of EGFR (Kesarwala et al, 2009). Consistently, HSP90 inhibitors enhance degradation of EGFR and Her2 via the proteosomal pathway (Sepp-Lorenzino et al, 1995;Smith et al, 2002;Imai et al, 2003;de Candia et al, 2003).…”