Background
Head and neck squamous cell carcinoma (HNSCC) affects over 600,000 patients worldwide, where 80% are non-responsive or resistant to an immunotherapy, cetuximab. This treatment is the only selective FDA-approved agent for HNSCC, targeting epidermal growth factor receptor (EGFR) which is involved in dysregulated cellular signalling. Studies to identify differences between the endocytic machineries of cetuximab-sensitive and -resistant cancer cells in vitro may pinpoint trafficking mechanisms and novel biomarkers responsible for cetuximab-resistance that may help advance HNSCC treatments. Thus, 1 SCC cell line that is cetuximab-resistant (KJD) and 4 SCC cell lines that are cetuximab-sensitive (A431, SCC9, COLO16 and SCC25) were used as cell line models in these studies.
Methods and results
Clathrin-mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME) are the 2 main endocytic pathways which traffic EGFR from the cell membrane. Whilst CME is dependent on clathrin and dynamin, FEME is clathrin-independent but involves dynamin and endophilin. Whilst CME is a well-established route, the downstream endocytic mechanisms of FEME are mostly unknown. Immunoblotting revealed KJD cells have relatively higher amounts of dynamin and endophilin relative to the other 4 cell lines. EGFR endocytosis after 5 min with EGF-Alexa488 revealed that EGFR internalisation was significantly faster in only the KJD cells. Although this is characteristic of FEME, the uptake of transferrin (a classic CME marker) was also faster in KJD cells relative to the 4 cetuximab-sensitive lines. FEME begins upon receptor activation by high concentrations of ligand. Immunofluorescence showed that a small population of internalised EGFRs colocalise with endophilin in KJD cells stimulated with a high EGF ligand concentration and a larger population of EGFR colocalises with early endosomes. These are characteristic properties of FEME whereby endophilin remains on FEME carriers after they are fully internalised until fusion with early endosomes. There was also a reduction in EGF ligand uptake upon dynamin inhibition, but not clathrin inhibition, a feature that distinguishes FEME from CME in this cetuximab-resistant cell line.
Conclusion
The combined characteristics observed in cetuximab-resistant KJD cells suggest that EGFR may traffic through a clathrin-independent, dynamin-dependent route involving endophilin in these cells. However, the unexpected faster rate of transferrin uptake raises uncertainty regarding whether FEME is the major pathway for EGFR trafficking in these cells. In turn, this reduces prospects of a potential link between cetuximab resistance and the FEME pathway in cell line models.