Recent findings indicate that prevalent human carcinomas overexpress the mitochondrial ATPase Inhibitory Factor 1 (IF1). Overexpression of IF1 inhibits the synthase activity of the mitochondrial H(+)-ATP synthase and plays a crucial role in metabolic adaptation of cancer cells to enhanced aerobic glycolysis. Herein, we demonstrate that IF1 overexpression in colon cancer cells triggers mitochondrial hyperpolarization and the subsequent production of superoxide radical, a reactive oxygen species (ROS). ROS are required to promote the transcriptional activation of the NFκB pathway via phosphorylation-dependent IκBα degradation. Activation of NFκB results in a cellular adaptive response that includes proliferation and Bcl-xL mediated resistance to drug-induced cell death. Quenching the mitochondrial production of ROS prevents the activation of NFκB and abolishes the IF1-mediated cellular adaptive response. Overall, our findings provide evidence linking the activity of a mitochondrial protein with retrograde signaling to the nucleus to promote cellular proliferation and survival.

The H؉ -ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1 In oxidative phosphorylation, ATP is synthesized by the mitochondrial ATP synthase, a H ϩ -driven rotatory engine of the inner membrane that utilizes as driving force for ATP synthesis the H ϩ electrochemical gradient generated by the respiratory chain (1-4). The cellular expression level of ␤-F1-ATPase, 2 which is the catalytic subunit of the H ϩ -ATP synthase, is diminished in diverse human pathologies (5), which include cancer (6 -9), affording a relevant marker of disease progression (6, 7, 10 -12) and of the response to chemotherapy (7,(13)(14)(15). Moreover, the down-regulation of ␤-F1-ATPase in lung carcinomas (12) and colon cancer cells (15) also provides a mechanistic explanation to the increased glucose avidity of carcinomas, i.e. to the enhanced aerobic glycolysis of cancer cells (16,17). Interestingly, the quantitative determination of ␤-F1-ATPase relative to the content of glyceraldehyde-3-phosphate dehydrogenase in human tumors has revealed that cancer abolishes the tissue-specific differences in the cellular complement of the bioenergetic ␤-F1-ATPase protein (18).It is well established that when mitochondrial respiration is impaired, the H ϩ -ATP synthase can function in reverse acting as an ATP hydrolase for maintaining the proton motive force (1,19). This process is regulated by an inhibitor peptide called ATPase inhibitory factor 1 or IF1 (19 -21), a highly conserved nuclearly encoded protein. When matrix pH drops, IF1 becomes activated and binds ␤-F1-ATPase, blocking ATP hydrolysis and preventing a useless waste of energy (20). The substitution of histidine 49 in IF1 by a lysine residue renders a mutant form (H49K) that inhibits the ATP hydrolase activity in a pH-insensitive way (22). The structure and in vitro mechanism of action of IF1 has been studied in detail, and its role as an inhibitor of the hydrolase activity of the H ϩ -ATP synthase is well documented (19,20,23). However, the information on IF1 expression in human tissues and its putative contribution to the development of human pathology are unknown. In this study, we demonstrate that IF1 is overexpressed in human carcinomas. Moreover, we document that IF1 plays a regulatory role in controlling cellular energetic metabolism, strongly supporting its participation as an additional molecular switch used by cancer cells to trigger the induction of aerobic glycolysis, i.e. their Warburg phenotype. 2 The abbreviations used are: ␤-F1-ATPase, ␤ catalytic subunit of the H ϩ -ATP synthase; IF1, ATPase inhibitory factor 1; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; siRNA, small interfering RNA; NRK, normal rat kidney. EXPERIMENTAL PROCEDURES