Abstract
PurposeWe hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAFV600 wild-type colorectal cancer (CRC).MethodsA multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAFV600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAFV600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance.ResultsDifferences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone.ConclusionOur findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type CRC.
Highlights
Cetuximab is a monoclonal antibody that targets the epidermal growth factor receptor (EGFR)
A number of phospho-proteins was found to be differentially regulated in the cetuximab sensitive versus resistant cell lines following 1 and 4 h exposure to 306 μg/ml cetuximab, a concentration that was clinically achievable in Cmax concentration trials [11]
After a 4 h exposure another closely related PI3K pathway phospho-protein, phospho-PRAS40, was more likely to be decreased in cetuximab sensitive compared to resistant cell lines (p = 0.04)
Summary
Cetuximab is a monoclonal antibody that targets the epidermal growth factor receptor (EGFR). Even in patients with KRAS WT tumours, with resistance to standard of care chemotherapy, the clinical efficacy of cetuximab is modest, with a monotherapy radiological response rate of 20% [1]. No studies have been reported on the profiling of phospho-proteomic changes upon exposure to cetuximab in KRAS/NRAS/BRAFv600 WT CRC cells. We hypothesised that studying re-wiring of signal transduction in response to cetuximab may provide novel insights into mechanisms of drug resistance in the setting of RAS/BRAFV600 WT CRC.
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