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Haeme-responsive gene (HRG)-1 encodes a 16-kDa transmembrane protein that is induced by insulin-like growth factor-1 (IGF-1) and associates with the vacuolar-(H(+)) ATPase (V-ATPase). We previously reported that HRG-1 is essential for V-ATPase activity in endosomal acidification and receptor trafficking. Here, we show that in highly invasive and migratory cancer cell lines, HRG-1 and the V-ATPase are co-expressed at the plasma membrane, whereas in less invasive cell lines and non-transformed cells HRG-1 over-expression remains confined to intracellular compartments. Stable suppression of HRG-1 in invasive breast cancer MDA-MB-231 cells decreases extracellular pH, cell growth, migration and invasion. Ectopic expression of HRG-1 in non-invasive MCF-7 cells enhances V-ATPase activity, lowers the extracellular pH and increases the pH-dependent activity of MMP2 and MMP9 matrix metalloproteinases. HRG-1 enhances trafficking of the glucose transporter-1 (GLUT-1) with a concomitant increase in glucose uptake and lactate production. HRG-1 also promotes trafficking of the insulin-like growth factor I receptor (IGF-1R), β1-integrin and IGF-1 signalling. Taken together, our findings indicate that HRG-1 expression at the plasma membrane enhances V-ATPase activity, drives glycolytic flux and facilitates cancer cell growth, migration and invasion. Thus, HRG-1 may represent a novel target for selectively disrupting V-ATPase activity and the metastatic potential of cancer cells.

Cancer cells are dependent on a continuous supply of nutrients to maintain cell proliferation and migration. Nutrients are acquired through transporters whose transcription, trafficking, and degradation are tightly regulated by growth factors, especially through activity of the Insulin/IGF-I-activated PI3K/mTOR signalling pathway. The intracellular trafficking of nutrient transporters requires an acidic endosomal pH that is maintained by the Vacuolar H+ ATPase (V-ATPase) proton pump. Recent studies have indicated a role for V-ATPase activity in cancer cells, but how this is regulated is not known. We recently isolated a series of new IGF-I-regulated genes whose expression is associated with cellular transformation (1, 2). Among these was a previously uncharacterized endosomal protein, which we determined associates with the V-ATPase, and which we called EVA. This protein was separately identified in C. elegans as a member of a family of heme transporters (HRG-1) that control heme homeostasis (3). We found that EVA is present throughout the endosome compartments; in early, recycling, and late endosomes, but not in lysosomes. Upon nutrient withdrawal EVA traffics to the plasma membrane. EVA interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Suppression of EVA expression increases endosomal pH and reduces V-ATPase holoenzyme assembly. This is accompanied by decreased migration, decreased transferrin receptor trafficking, decreased cellular heme uptake, and cell death. Over-expressed EVA enhances cellular heme uptake in a V-ATPase-dependent manner. We conclude that EVA/HRG-1 regulates V-ATPase-dependent acidification of endosomes necessary for trafficking of heme receptors as well as for heme transport within endosomes. Our data suggest that IGF-I induces expression of this micro-nutrient transporter to facilitate the enhanced metabolic requirements of cancer cells. Citation Information: Cancer Res 2009;69(23 Suppl):C18.

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