Up-regulation of the ATPase Inhibitory Factor 1 (IF1) of the Mitochondrial H+-ATP Synthase in Human Tumors Mediates the Metabolic Shift of Cancer Cells to a Warburg Phenotype

Abstract

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

Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

“…This profound effect of IF1 on mitochondrial morphology remains controversial (19,20). Reports have indicated that overexpression of IF1 decreased the magnitude of pmf and increased the rate of respiration (9), whereas the opposite results have also been reported (8) …”

“…It was found that IF1 is overexpressed in various human cancer cells, and it has been proposed that one role of IF1 is as an additional molecular switch for the metabolic shift from oxidative phosphorylation to glycolysis in tumor cells (8). When cells are deprived of oxygen and glucose, IF1-suppressed cells lose cellular ATP and die more rapidly than control cells, suggesting a role of IF1 in maintaining ATP concentration ([ATP]) in energy crises (9).…”

Phosphatidylethanolamine Deficiency in Mammalian Mitochondria Impairs Oxidative Phosphorylation and Alters Mitochondrial Morphology

“…The idea that mtPE deficiency uncouples ETC function and ATP synthesis is consistent with the lower maximum rate of oxygen consumption in mtPE-de- pleted cells compared with control cells. Thus, organization of ETC complexes and ATP generation are both compromised, perhaps because of limited availability of ADP for ATP production or because CV is bound to an inhibitor (84).…”

Metabolic control of cancer cell stemness: Lessons from iPS cells

“…Also, iPS cells drastically limit the activity and cellular content of the mitochondrial H C -ATPase synthase, a molecular feature that correlates directly with OXPHOS activity and inversely with the rate of glucose utilization by aerobic glycolysis in tumor tissues. [51][52][53][54][55][56][57][58][59] Somatic cell reprogramming involves a dramatic downregulation of the catalytic b1-F1-ATPase subunit and a significant increase in the expression of ATPase inhibitor factor 1 (IF1), a marker that is expressed in adult stem cells (ASC) for maintaining the activity of aerobic glycolysis, but is not expressed in equivalent differentiated cells. 60 Although little is known about the bioenergetic resetting of CSC, it appears that analogous to iPS cells, a direct link might exist between the occurrence of a metabolic switch from OXPHOS to aerobic glycolysis and the occurrence and maintenance of CSC cellular states.…”