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Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this non-muscle context. The positive correlation with hypoxia-inducible factor 2 and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/-2 dependent transactivation. The hypoxia-induced MB mRNA originated from a novel, alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O2 uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to the mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O2 consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O2. Hypothetically, myoglobins mitochondria-impairing role in hypoxic cancer cells is part of a novel tumor-suppressive function.

The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel-Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions.