Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

Abstract

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signalregulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival. [Cancer Res 2007;67(17):8240-7]

Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab

“…170 These findings were confirmed using immunofluorescence (IF) microscopy and flow cytometry. In contrast to the study by Lu et al 168 this study demonstrated that EGFR does not seem to be associated with c-Cbl in cetuximabresistant lines, while the parental cetuximab-sensitive line maintains this association. These data support a previously published mechanism for EGFR degradation via c-Cbl recruitment to tyrosine 1045.…”

Genetic modifiers of EGFR dependence in non-small cell lung cancer

“…The finding that Src family kinases are sufficient to modify EGFR dependence is in line with considerable evidence linking EGFR activity with this family: c-Src itself has been characterized extensively with respect to its cooperative relationship with EGFR (27), and the introduction of dominant-active c-Src can reduce the inhibitory effects of erlotinib in head and neck squamous cell carcinoma models (28). Src family kinase activation has been observed in cetuximab-resistant colorectal adenocarcinoma and NSCLC squamous cell carcinoma in vitro models (29,30), and it has been reported that CRIPTO1-mediated EGFR TKI resistance in NSCLC is Src-dependent (31). Furthermore, a recent report described increased expression and activation of Src, mediated by integrin activation, in EGFR TKI-resistant lung adenocarcinoma models (32).…”

Cetuximab Resistance in Squamous Carcinomas of the Upper Aerodigestive Tract Is Driven by Receptor Tyrosine Kinase Plasticity: Potential for mAb Mixtures

“…To investigate mechanisms driving cetuximab resistance in SCCUAT, we established cetuximab-resistant clones of two of the most sensitive cell lines, HN5 and FaDu, by continuous cetuximab pressure in vitro and in vivo, respectively. It has previously been demonstrated that acquired resistance to cetuximab can occur as a result of altered functional status of EGFR but that resistant cells remain fully or partially EGFR dependent (39,40). We therefore investigated the status of EGFR in the resistant cells.…”