Understanding the proteome changes arising throughout 5-Fluorouracil chemoresistance development in colorectal cancer cells, with special focus on the methyl proteome

Introduction: Comprehensive proteomics strategies for analysing post-translational modifications (PTMs) have showcased the potential use of protein modifications for defining novel cancer-driving mechanisms and drug-resistance markers. Recent studies highlight the aberrant methyl proteome changes arising in the development of various cancers, but the changes arising throughout the development of chemoresistance in most cancers has not been explored in depth. This study is currently making use of colorectal cancer (CRC) cell lines as a cancer model to uncover the changes in the global proteome and methyl proteome arising following development of 5-fluorouracil (5FU) chemoresistance.

Method: Three CRC cell lines (HCT116, Caco2, DLD1) were cultured by regular exposure to 5FU over a long period (3 months to 1 year), and stable 5FU-resistant cells were successfully established and isolated for downstream analysis. Whole cell lysates from sensitive and resistant cells (periodically exposed for 6 months) were used, and the peptide digests extracted were measured in label-free quantification (LFQ) DDA-mode using an Easy-nanoLC-Orbitrap-Fusion-Tribrid system, equipped with Nikkyo-C18-nano-Column. The data was processed using Proteome Discoverer (v.2.4), statistical analysis and cluster analysis was performed with Perseus, while Gene Ontology and network analyses were assisted by DAVID, KEGG, and KeyMolnet.

Results: Through global proteomics, approximately 7500 proteins (FDR < 1%) were quantitatively identified, with each cell line exhibiting more than 800 dysregulated proteins (-1.5 ≤ Log2 FC ≥1.5,p < 0.05). Network analysis for the common dysregulated proteins revealed that signalling pathways related to transcriptional and translational regulatory factors and amino acid metabolism, were significantly altered. Furthermore, more than 70 types of methyltransferases were identified, with over 1000 methylated peptides being detected (without enrichment). Manual filtering resulted in a list of just over 600 methylated peptides in each cell line, with di-methylated residues being among the most identified (>300 sites) in each cell line pair. The common proteins with variable methylation sites revealed dysregulated signalling pathways related to alternative splicing.

Conclusions: From the combined data of global proteomics and methyl proteomics, the preliminary data collected has revealed significantly altered methylated proteins and methyltransferases in 5FUR CRC cell lines. It is anticipated that following further validation, the data generated will provide a better understanding of how to improve the effectiveness of anti-cancer drugs from a methyl proteomic standpoint.