Comparative proteomics of dying and surviving cancer cells improves the identification of drug targets and sheds light on cell life/death decisions

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, while the detached cells are usually ignored. Here we tested the hypothesis that proteomes of detached cells contain valuable information and therefore separately analyzed the proteomes of detached and attached HCT-116, A375 and RKO cells obtained 48 h after treatment with 5-fluorouracil, methotrexate and paclitaxel. Combined proteome data provided a more accurate identification of drug targets. Six proteins consistently up- or down-regulated in the detached vs attached cells regardless of the drug and cell type were targeted by siRNA. Knocking down USP11, CTTN, ACAA2 and EIF4H had anti-proliferative effects, targeting UHRF1 additionally sensitized the cells to the anticancer drugs, while knocking down RNF-40 increased cell survival against the treatments. Therefore, these proteins are likely to be involved in general cell death and survival decisions. Adding detached cells to the analysis could become a standard practice in expression proteomics of drug-treated cells.