Descriptive Proteome Analysis to Investigate Context-Dependent Treatment Responses to OXPHOS Inhibition in Colon Carcinoma Cells Grown as Monolayer and Multicellular Tumor Spheroids

We have previously identified selective upregulation of the mevalonate pathway genes upon inhibition of oxidative phosphorylation (OXPHOS) in quiescent cancer cells. Using mass spectrometry-based proteomics, we here investigated whether these responses are corroborated on the protein level and whether proteomics could yield unique insights into context-dependent biology. HCT116 colon carcinoma cells were cultured as monolayer cultures, proliferative multicellular tumor spheroids (P-MCTS), or quiescent (Q-MCTS) multicellular tumor spheroids and exposed to OXPHOS inhibitors: nitazoxanide, FCCP, oligomycin, and salinomycin or the HMG-CoA-reductase inhibitor simvastatin at two different doses for 6 and 24 h. Samples were processed using an in-depth bottom-up proteomics workflow resulting in a total of 9286 identified protein groups. Gene set enrichment analysis showed profound differences between the three cell systems and confirmed differential enrichment of hypoxia, OXPHOS, and cell cycle progression-related protein responses in P-MCTS and Q-MCTS. Treatment experiments showed that the observed drug-induced alterations in gene expression of metabolically challenged cells are not translated directly to the protein level, but the results reaffirmed OXPHOS as a selective vulnerability of quiescent cancer cells. This work provides rationale for the use of deep proteome profiling to identify context-dependent treatment responses and encourages further studies investigating metabolic processes that could be co-targeted together with OXPHOS to eradicate quiescent cancer cells.