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.