Abstract 1073: Cell surface F1-ATPase, a new potential target in colorectal cancer

Abstract

Abstract High concentrations of gastrin precursors have been observed in colon tumors and in blood of patients with colorectal cancer while they are absent in healthy subjects. These precursors are now established as growth factors for colonic epithelial cells and play an important role in colon carcinogenesis. The glycine-extended form of gastrin (G-gly) is the first gastrin precursor for which growth factor properties have been demonstrated. More recently, an in vitro study has also shown that G-gly induces the tubule formation by human vascular endothelial cells (HUVEC) in a similar manner to what is observed with VEGF. Several publications are in favor of the existence of high affinity binding sites for G-gly at the cell surface of gastrointestinal cells. However, the cell surface protein mediating G-gly effects remains unidentified to-date. In the present study, using SPR technology coupled to mass spectrometry, we identify in the solubilized plasma membranes from human colorectal cancer cells, the F1-ATPase as a binding protein for G-gly. This mitochondrial protein was recently found located at the cell surface of lung and prostate tumor cells and vascular endothelial cells, where it plays a role in cell proliferation and angiogenesis. Using purified F1-ATPase we confirmed by SPR a direct interaction between G-gly and this multi-subunits transmembrane protein (Kd in the nanomolar range). In addition by molecular modeling, we identified the motif in the G-gly sequence (EE/DxY) which directly interacts with F1-ATPase as well as the amino-acid residues in the alpha-and beta-subunits of the F1-ATPase which bind this motif. We confirmed this molecular model by mutation of the EE/DxY motif that resulted in a strong decrease of G-gly binding to the F1-ATPase as well as a loss of the proliferative activity of the peptide. Using immunofluorescent approaches and confocal microscopy, we demonstrate for the first time the presence of the F1-ATPase at the cell surface of normal or cancerous colonic epithelial cells in vitro and in vivo. We also found that cell surface F1-ATPase activity is modulated by G-gly on endothelial and colorectal cancer cells. Furthermore, the proliferative effects of G-gly on both cell types, was blocked by a specific inhibitor of the F1-ATPase (IF1) or antibodies targeting the beta- subunit of the enzyme. These results suggest an important contribution of cell surface ATPase in the pro-tumoral action of the gastrin precursor, G-gly. Interestingly, an anti-tumor strategy using monoclonal antibodies against the beta-subunit of ATPase has been previously proposed for hepatocarcinoma since these antibodies have been shown to reduce growth of these tumors. In view of our results, targeting cell surface ATPase might also be a new therapeutic approach for colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1073. doi:1538-7445.AM2012-1073