Brian Todd Mullis1; L. Andrew Lee1; David Quilici2; Rebekah Woolsey2; Qian Wang3
1Integrated Micro-Chromatography Systems, Irmo, SC; 2Mick Hitchcock, Ph.D. Nevada Proteomics Center, Reno, NV; 3University of South Carolina, Columbia, SC
Introduction
According to American Cancer Society, colorectal cancer (CRC) is anticipated to cause over 50,000 deaths in 2021. Small molecule chemotherapy drugs, most commonly thymidylate synthase (TS) inhibitors, are used as a first line treatment of CRC. However, TS inhibitor resistance is a major problem in CRC treatment. There have been significant efforts to develop predictive markers of TS inhibitor resistance and to find additional drug targets to help combat resistance. The aim of this work is to probe the phosphoproteomic changes in TS-sensitive cells and in TS-resistant cells when treated with 5-fluoro-2’-deoxyuridine monophosphate (5-FdUMP) and folinic acid (leucovorin). We use quantitative MS-based phosphoproteomics in this study to understand and quantify changes in phosphoproteins in response to the TS-inhibitor treatment.
Methods
TS Inhibitor-treated SILAC-labelled-HCT116 or SILAC-labelled-HCT116/200 cells were harvested and lysed with RIPA buffer (Thermo Scientific Pierce, Rockford, IL) containing 1x of Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific Pierce, Rockford, IL). After lysis, the SILAC samples were combined 1:1, based on protein concentration. The proteins were reduced with TCEP and alkylated with IAA followed by overnight tryptic digestion at 37°C. For automated sample processing, we used the Microlab STAR from Hamilton Robotics with peptide desalting and phosphopeptide enrichment IMCStips. Liquid chromatography mass spectrometry (LC-MS) analysis was performed on an UltiMate 3000 RS LCnano system in tandem with an Orbitrap Fusion mass spectrometer. The data analysis was done using Proteome Discoverer, GraphPad, Kinase-Substrate Enrichment Analysis (KSEA), and Cluster Evaluation (CLUE).
Preliminary Data
To carry out quantitative phosphoproteomic studies we used our automated phosphoproteomic method in conjunction with SILAC-based quantitation to generate reproducible phosphoproteomic data from TS inhibitor-treated SILAC-labelled HCT116 (sensitive) and SILAC-labelled HCT116/200 (resistant) cells. From this data, we show the changes in the phospho-profile over 7 time points (0.5, 1, 2, 4, 8, 24, 48 hr(s)). Using CLUE and KSEA to analyze the generated phosphoproteomic data, several phosphorylation signaling pathways were differentially activated at different time points between the sensitive and resistant cells. Interestingly, the data reveals a differential early response (0.5 hr) to the TS-inhibitor treatment between the HCT116 and HCT116/200 cell line. In particular, phosphoproteins associated with the MAPK, CDK1, and CDK2 phosphorylation signaling pathways were activated in the 0.5-hour TS inhibitor-treated HCT116 cell line. This early response has not been reported prior to this work and highlights a potential opportunity to determine TS-inhibitor sensitivity by mapping the phospho-profile of TS-inhibitor treated cells.
Novel Aspect
Phosphoproteome comparisons reveal varied activation of MAPK, CDK1, and CDK2 phosphorylation signaling pathways for TS-inhibitor resistance cells.
