Hypoxia down-regulates CCAAT/enhancer binding protein-α expression in breast cancer cells

Abstract

The transcription factor CCAAT/enhancer binding protein-α (C/EBPα) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBPα mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole- 1-β-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBPα mRNA by ∼30%. C/EBPα gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1α, as essential for down-regulation of C/EBPα transcription in hypoxia. Immunocytochemical analysis showed that C/EBPα was localized in the nucleus at 21% O2, but was mostly cytoplasmic under 1% O2. Knockdown of HIF-1α by RNAi restored C/EBPα to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBPα and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBPα expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBPα in hypoxia is mediated by HIF-1. ©2008 American Association for Cancer Research.