Abstract
Chemotherapeutics cause the detachment and death of adherent cancer cells. When studying the proteome changes to determine the protein target and mechanism of action of anticancer drugs, the still-attached cells are normally used, whereas the detached cells are usually ignored. To test the hypothesis that proteomes of detached cells contain valuable information, we separately analyzed the proteomes of detached and attached HCT-116, A375, and RKO cells treated for 48 h with 5-fluorouracil, methotrexate and paclitaxel. Individually, the proteomic data on attached and detached cells had comparable performance in target and drug mechanism deconvolution, whereas the combined data significantly improved the target ranking for paclitaxel. Comparative analysis of attached versus detached proteomes provided further insight into cell life and death decision making. Six proteins consistently up- or downregulated in the detached versus attached cells regardless of the drug and cell type were discovered; their role in cell death/survival was tested by silencing them with siRNA. Knocking down USP11, CTTN, ACAA2, and EIF4H had anti-proliferative effects, affecting UHRF1 additionally sensitized the cells to the anticancer drugs, while knocking down RNF-40 increased cell survival against the treatments. Therefore, adding detached cells to the expression proteomics analysis of drug-treated cells can significantly increase the analytical value of the approach. The data have been deposited to the ProteomeXchange with identifier PXD007686.
Highlights
Chemotherapeutics cause the detachment and death of adherent cancer cells
Besides the drug target identification, the benefit of Functional Identification of Target by Expression Proteomics (FITExP) is that the proteins with largest abundance changes and highest specificity could be mapped on known protein networks to reveal the drug MOA
Considering only proteins with nonzero abundance values in all three replicates, the final data set contained 1950 proteins. This number was found sufficient for our purposes, as we have previously shown that the abundances of even several hundred most abundant proteins may truthfully reflect the state of the dying cells [13]
Summary
Chemotherapeutics cause the detachment and death of adherent cancer cells. When studying the proteome changes to determine the protein target and mechanism of action of anticancer drugs, the still-attached cells are normally used, whereas the detached cells are usually ignored. To assist deconvolution of protein targets and MOA of small molecule drugs, we have recently devised a mass-spectrometry-based method called Functional Identification of Target by Expression Proteomics (FITExP) [7]. This method is based on the observation that in late stages of cancer cell death, the abundance change of the protein target of a small molecule is exceptionally strong. Proteomics of Cell Death/Survival (PCTL), doxorubicin, 5-fluorouracil (5-FU), methotrexate (MTX), raltitrexed and camptothecin, among more than 4000 proteins This method is based on deduction of drug target solely from proteomic data, by sorting all proteins with respect to their regulation upon treatment and specificity of that regulation for a treatment. Besides the drug target identification, the benefit of FITExP is that the proteins with largest abundance changes and highest specificity could be mapped on known protein networks to reveal the drug MOA
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