Interaction of HIF and USF signaling pathways in human genes flanked by hypoxia-response elements and E-box palindromes.

Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1α and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1α and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O(2)-independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells.

Endogenous myoglobin in breast cancer is hypoxia-inducible by alternative transcription and functions to impair mitochondrial activity: a role in tumor suppression?

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.