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

Recent findings in colon cancer cells indicate that inhibition of the mitochondrial H+-adenosine triphosphate (ATP) synthase by the ATPase inhibitory factor 1 (IF1) promotes aerobic glycolysis and a reactive oxygen species (ROS)-mediated signal that enhances proliferation and cell survival. Herein, we have studied the expression, biological relevance, mechanism of regulation and potential clinical impact of IF1 in some prevalent human carcinomas. We show that IF1 is highly overexpressed in most (>90%) of the colon (n=64), lung (n=30), breast (n=129) and ovarian (n=10) carcinomas studied as assessed by different approaches in independent cohorts of cancer patients. The expression of IF1 in the corresponding normal tissues is negligible. By contrast, the endometrium, stomach and kidney show high expression of IF1 in the normal tissue revealing subtle differences by carcinogenesis. The overexpression of IF1 also promotes the activation of aerobic glycolysis and a concurrent ROS signal in mitochondria of the lung, breast and ovarian cancer cells mimicking the activity of oligomycin. IF1-mediated ROS signaling activates cell-type specific adaptive responses aimed at preventing death in these cell lines. Remarkably, regulation of IF1 expression in the colon, lung, breast and ovarian carcinomas is exerted at post-transcriptional levels. We demonstrate that IF1 is a short-lived protein (t1/2 ∼100 min) strongly implicating translation and/or protein stabilization as main drivers of metabolic reprogramming and cell survival in these human cancers. Analysis of tumor expression of IF1 in cohorts of breast and colon cancer patients revealed its relevance as a predictive marker for clinical outcome, emphasizing the high potential of IF1 as therapeutic target.