Integrated proteomics and metabolomics reveal phytosesquiterpene lactones inhibit TNBC cell activity by depleting ATP synthesis and reprogramming primary metabolism

Phytosesquiterpene lactones deoxyelephantopin (DET) and its derivative DETD-35 are reported to induce oxidative stress towards inhibiting triple-negative breast cancer (TNBC) cell activities. This study aimed to elucidate how DET and DETD-35 affect mitochondrial function and systemic metabolism in TNBC cells. DET and DETD-35 promoted mitochondrial superoxide production by upregulating expression of SOD1 and SOD2, induced permeability transition pore opening, and attenuated intracellular ATP levels. Neither compound interfered with mitochondrial respiration/bioenergetics in normal mammary MCF-10A cells. Comparative mitochondrial proteome and bioinformatic analyses showed significant deregulation of proteins related to the oxidative phosphorylation, depolarization of mitochondria, and apoptosis signaling in DET- or DETD-35-treated TNBC cells, and primary metabolomics revealed that both compounds deregulated metabolites dynamics and the corresponding metabolic pathways in TNBC cells. Knockdown of the PRKCA gene/protein involved in inducing mitochondrial toxicity in TNBC cells reversed cytotoxicity, apoptosis, and the levels of several metabolites induced by DET or DETD-35 in the cancer cells. Integrated Pearson's correlation and IPA network analyses of differentially expressed proteins and metabolites revealed the networks of ATP synthesis, energy homeostasis, and respiration, depolarization, and transmembrane potential in mitochondria highly correlated to the compound effects. Notable, DET/DETD-35 inhibited mitochondrial ATPase activity, and molecular modeling further predicted the binding sites of either compound with ATP synthase at the subunits α/β and c/a interfaces. The overexpression of ATP synthase-related proteins ATP5A1 and ATP5C1 in the tumor microenvironment of MDA-MB-231 xenograft mice were also significantly suppressed by DET and DETD-35 treatments. In summary, this study identifies DETD-35 and DET as novel ATPase inhibitors which are attributed to disrupting mitochondrial biogenetics and cellular metabolism and networking in TNBC cells.