Role of Src family kinases in acquired resistance to cetuximab

A187 EGFR is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of proliferation and progression in many human tumors. Following more than 20 years of preclinical development, four EGFR inhibitors, two monoclonal antibodies and two small molecule tyrosine kinase inhibitors (TKIs), have recently gained FDA approval in oncology (panitumumab, cetuximab, erlotinib and gefitinib). Both strategies of EGFR signaling inhibition have demonstrated major clinical tumor regressions in approximately 10-20% of advanced cancer patients. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. The underlying mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain largely unexplored. To explore potential molecular mechanisms of acquired resistance to cetuximab we established a series of cetuximab-resistant clones by chronically exposing the NSCLC (H226) cell line to cetuximab. We have previously shown that cetuximab-resistant clones have a dramatic increase in the steady-state expression of EGFR, due to alterations in trafficking and endocytosis. This increase in steady-state expression of the EGFR led to constitutive activation of HER2 and HER3 and downstream survival signals mediated by AKT. The results of this study indicated that EGFR-dependent activation of HER3 is crucial for acquiring resistance to cetuximab. Further exploration of the EGFR indicated a robust phosphorylation at the Src phosphorylation site on the EGFR, tyrosine 845 (Y845), in all resistant clones. In the present study we hypothesized that increased Src family kinase (SFK) activity augments EGFR function in cetuximab-resistant cells and, in part, leads to acquired-resistance by enhancing signals to HER3. To test this hypothesis we performed a series of experiments to inhibit SFKs and determine the downstream effects on EGFR activity and signals to HER3 in cells with acquired-resistance to cetuximab. Growth proliferation assays using increasing doses of the SFK inhibitors PP1 or PP2 indicated an increased sensitivity of cetuximab-resistant cells to SFK blockade relative to the parental controls. In addition, blockade of SFK activity lead to a decrease in overall tyrosine phosphorylation of the EGFR which correlated with a loss of activity of HER3, AKT and MAPK and proliferation. These results indicate that the EGFR- dependent activation of HER3 is dependent upon SFKs. Furthermore, low doses of SFK inhibitors re-sensitized cells with acquired-resistance to cetuximab therapy. Experiments aimed at knocking down EGFR activity using both the tyrosine kinase inhibitors erlotinib and gefitinib or siRNAs directed against the open reading frame of the EGFR resulted in the loss of active SFKs. Taken together, these results indicate cooperation between SFKs and EGFR in mediating acquired-resistance to cetuximab. Collectively, this data suggests a critical role for SFKs in acquired-resistance to cetuximab and may suggest a rationale for designing clinical trials to examine the value of treating patients that develop cetuximab-resistance with inhibitors of SFKs.