Colorectal cancer (CRC) still has a 45% mortality rate, and one of the barriers to therapeutic success is the development of acquired resistance to 5-fluorouracil (5-FU), the most commonly used drug in CRC treatment. Here we establish cell culture models and use state of the art proteomics methods to increase our understanding of how CRC cells develop resistance to 5-FU.
We develop 5-FU resistant CRC sublines with different microsatellite stability (MSS) profiles, since MSS is a key genetic alteration in CRC formation, and aim to use these to identify new biomarkers of 5-FU response. 5-FU resistant sublines for 2 cell lines, DLD-1 (microsatellite instability phenotype (MSI)) and HT-29 (MSS), were developed by continuous 5-FU exposure, and resistance fold changes of 130.2 and 3.5 respectively were achieved. Once 5-FU resistant sublines were developed they were analysed proteomically using a stable isotope labelling with amino acids in cell culture SILAC-approach and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis, to identify new biomarkers of drug resistance.
A total of 3003 proteins were commonly quantified in the parent cell lines (low and high passage numbers), and in the DLD-1 5-FU and HT 29 5-FU resistant sublines. Six proteins were seen to be significantly up-regulated, and eight down-regulated, in both 5-FU resistant sublines when compared to the parent cell lines.
This is the first use of a proteomics approach to study protein expression changes in 5-FU resistant CRC cell lines with varying microsatellite stability status, while accounting for changes which occur in the parent lines over the duration of establishing the resistant sublines. We have identified protein changes that correlate both with acquired resistance and the MSI/MSS status, and validated these finding using immunodetection techniques. We are currently extending the CRC study to look at multiple resistance mechanisms for 5-FU with other commonly used CRC therapeutics, oxaliplatin and irinotecan.
Clinical trial identification
Legal entity responsible for the study
Dr. Steve Shnyder
University of Bradford (Institute of Cancer Therapeutics)
All authors have declared no conflicts of interest.