The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells.

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factor that may be exploited to develop therapeutic approaches.

The Pro-Oncogenic Protein IF1 Promotes Proliferation of Anoxic Cancer Cells during Re-Oxygenation.

Cancer cells overexpress IF1, the endogenous protein that inhibits the hydrolytic activity of ATP synthase when mitochondrial membrane potential (ΔµH+) falls, as in ischemia. Other roles have been ascribed to IF1, but the associated molecular mechanisms are still under debate. We investigated the ability of IF1 to promote survival and proliferation in osteosarcoma and colon carcinoma cells exposed to conditions mimicking ischemia and reperfusion, as occurs in vivo, particularly in solid tumors. IF1-silenced and parental cells were exposed to the FCCP uncoupler to collapse ΔµH+ and the bioenergetics of cell models were validated. All the uncoupled cells preserved mitochondrial mass, but the implemented mechanisms differed in IF1-expressing and IF1-silenced cells. Indeed, the membrane potential collapse and the energy charge preservation allowed an increase in both mitophagy and mitochondrial biogenesis in IF1-expressing cells only. Interestingly, the presence of IF1 also conferred a proliferative advantage to cells highly dependent on oxidative phosphorylation when the uncoupler was washed out, mimicking cell re-oxygenation. Overall, our results indicate that IF1, by allowing energy preservation and promoting mitochondrial renewal, can favor proliferation of anoxic cells and tumor growth. Therefore, hindering the action of IF1 may be promising for the therapy of tumors that rely on oxidative phosphorylation for energy production.