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.