Ectopic ATP Synthase Blockade Suppresses Lung Adenocarcinoma Growth by Activating the Unfolded Protein Response

Abstract

Ectopic expression of the mitochondrial F 1 F 0 -ATP synthase on the plasma membrane has been reported to occur in cancer, but whether it exerts a functional role in this setting remains unclear. Here we show that ectopic ATP synthase and the electron transfer chain exist on the plasma membrane in a punctuated distribution of lung adenocarcinoma cells, where it is critical to support cancer cell proliferation. Applying ATP synthase inhibitor citreoviridin induced cell cycle arrest and inhibited proliferation and anchorage-independent growth of lung cancer cells. Analysis of protein expression profiles after citreoviridin treatment suggested this compound induced the unfolded protein response (UPR) associated with phosphorylation the translation initiation factor 2a (eIF2a), triggering cell growth inhibition. Citreoviridin-enhanced eIF2a phosphorylation could be reversed by siRNA-mediated attenuation of the UPR kinase PKR-like endoplasmic reticulum kinase (PERK) combined with treatment with the antioxidant N-acetylcysteine, establishing that reactive oxygen species (ROS) boost UPR after citreoviridin treatment. Thus, a coordinate elevation of UPR and ROS initiates a positive feedback loop that convergently blocks cell proliferation. Our findings define a molecular function for ectopic ATP synthase at the plasma membrane in lung cancer cells and they prompt further study of its inhibition as a potential therapeutic approach. Cancer Res; 72(18); 4696-706. Ó2012 AACR.

Temporal Phosphoproteome Dynamics Induced by an ATP Synthase Inhibitor Citreoviridin*

“…Cell Culture-Human lung adenocarcinoma cells CL1-0 and A549 were grown as previously described (10). Briefly, cells were cultured in Dulbecco's modified Eagle's medium (Gibco, New York, NY) containing 10% fetal bovine serum (Gibco) at 37°C and 5% carbon dioxide.…”

“…CL1-0 and A549 cells were treated with citreoviridin (4.6 M for CL1-0 and 1.5 M for A549) or with the same volume of DMSO, as previously described (10). Time points for treatment were 10 s; 1, 5, and 10 min; and 1, 2, 12, 24, 36, and 48 h. Two biological replicates for each time point experiment were performed.…”

“…After citreoviridin treatment on cells with ectopically expressed ATP synthase, there was no change in the mitochondrial membrane potential, suggesting that citreoviridin inhibited the activity of ectopic, but not mitochondrial ATP synthase (10). Furthermore, the inhibition of ecto-ATP synthase by citreoviridin stimulates the unfoldedprotein response and elevates levels of ROS in lung cancer cells (10). Quantitative proteomic analysis of human lung tumor xenografts showed that citreoviridin induces alternations in the expression of glucose metabolism-related enzymes and suggested that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation (20).…”

“…However, recent studies have revealed that ATP synthase is also expressed on the cell surface of keratinocyte, hepatocyte, and adipocytes, as well as endothelial, neuronal, and cancer cells (10 -19). After citreoviridin treatment on cells with ectopically expressed ATP synthase, there was no change in the mitochondrial membrane potential, suggesting that citreoviridin inhibited the activity of ectopic, but not mitochondrial ATP synthase (10). Furthermore, the inhibition of ecto-ATP synthase by citreoviridin stimulates the unfoldedprotein response and elevates levels of ROS in lung cancer cells (10).…”

“…In previous studies, we reported that citreoviridin could specifically kill cancer cells but not normal cells because of ectopically expressed ATP synthase (ecto-ATP synthase) on plasma membrane of cancer cells (10,11). In general, ATP synthase is located in the inner membrane of the mitochondria.…”