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Recently, the ectopic expression of myoglobin (MB) was reported in human epithelial cancer cell lines and breast tumor tissues, where MB expression increased with hypoxia. The better prognosis of MB-positive breast cancer patients suggested that the globin exerts a tumor-suppressive role, possibly by impairing mitochondrial activity in hypoxic breast carcinoma cells. To better understand MB gene regulation in cancer, we systematically investigated the architecture of the human MB gene, its transcripts and promoters. In silico analysis of transcriptome data from normal human tissues and cancer cell lines, followed by RACE-PCR verification, revealed seven novel exons in the MB gene region, most of which are untranslated exons located 5′-upstream of the coding DNA sequence (CDS). Sixteen novel alternatively spliced MB transcripts were detected, most of which predominantly occur in tumor tissue or cell lines. Quantitative RT-PCR analyses of MB expression in surgical breast cancer specimen confirmed the preferential usage of a hitherto unknown, tumor-associated MB promoter, which was functionally validated by luciferase reporter gene assays. In line with clinical observations of MB up-regulation in avascular breast tumors, the novel cancer-associated MB splice variants exhibited increased expression in tumor cells subjected to experimental hypoxia. The novel gene regulatory mechanisms unveiled in this study support the idea of a non-canonical role of MB during carcinogenesis

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) 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 O(2) uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O(2) 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 O(2). Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) 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 cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on 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, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.