Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer

Abstract

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.

Cav1/EREG/YAP Axis in the Treatment Resistance of Cav1-Expressing Head and Neck Squamous Cell Carcinoma

“…This can primarily be attributed to a deregulation of EGFR itself (overexpression and strong activity in more than 80% of HNSCC) [ 42 , 43 ], the expression of a mutant type III variant of EGFR [ 44 , 45 ], aberrant downstream signaling pathways [ 46 , 47 , 48 ], an oncogenic switch [ 49 , 50 , 51 , 52 ] or compensatory mechanisms (such as the activity of other tyrosine-kinase receptor ALK [ 53 ], MET [ 54 , 55 ] or AXL [ 56 , 57 ]). CTX even seems to promote its own resistance by inducing mutations in KRAS/NRAS/HRAS [ 58 ] or missense mutations in the ectodomain of EGFR, mimicking the activation of the receptor [ 59 ]. In this study, we reported that Cav1 may also play a role in this process.…”

Covalent N-arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor

“…These mutations may lead to conformational changes in the extracellular regions of EGFR, resulting in the induction of ligand-independent EGFR phosphorylation ( 48 , 49 ). Furthermore, the G33K and N56K mutations, which are also located in domain I, cause ligand-independent EGFR phosphorylation in head and neck cancer ( 50 ). These mutations may also cause abnormal phosphorylation by altering EGFR structural dynamics.…”

Evaluating the Targeting Efficiency of Anti-EGFR Functionalised Nanoparticles to Head and Neck Cancer Cells for Use in NIR-II Optical Window

“…However, the amount of uptake was dependant on the cell line, as it was significantly higher in the KYSE-30 compared to CAL-27 cancer cells. We hypothesise that the increased number of AuNRs in EGFR positive cancer cells is likely due to ligand-receptor binding activity [37] as opposed to simple passive diffusion mechanism or other forms of endocytosis. The work of Van Lehn et al showed increased internalisation of AuNPs into HeLa cells when incubated at 37 ℃ compared to ℃, depending on their size and functionalisation [38], indicating the importance of active transport for the cell internalisation of nanoparticles.…”