Aprelikova Olga, Wood Matthew, Tackett Sean, Chandramouli Gadisetti V R, Barrett J Carl
Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Bethesda, Maryland, USA.
Cancer Res. 2006 Jun 1;66(11):5641-7. doi: 10.1158/0008-5472.CAN-05-3345.
Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.
肿瘤缺氧通常与侵袭性表型、转移进展以及化疗耐药直接相关。两种转录因子[缺氧诱导因子-1α（HIF-1α）和HIF-2α]在缺氧区域显著诱导表达，并调节肿瘤适应低氧条件所需基因的表达；然而，这些因子的相对作用仍存在争议。我们利用RNA干扰介导的HIF-1α或HIF-2α失活，随后进行微阵列分析，以鉴定在缺氧条件下由HIF-1或HIF-2特异性调控的基因。我们发现，在MCF7细胞系中，绝大多数缺氧反应基因（>80%）依赖于HIF-1α的存在。然而，一小部分基因优先受HIF-2α调控。对这组基因的启动子分析表明，它们都具有ETS家族转录因子的推定结合位点，并且11个HIF-2α依赖基因中的10个在ETS转录因子结合位点附近至少有一个潜在的缺氧反应元件（HRE）。敲低ETS家族中最常出现的成员ELK-1，可显著降低HIF-2α依赖基因的缺氧诱导。这两种蛋白的共免疫沉淀、使用CITED2启动子的荧光素酶报告基因检测以及ELK-1蛋白与CITED2和WISP2基因启动子在HRE附近的结合，均支持了ELK-1与HIF-2α之间的物理和功能相互作用。这些数据表明，HIF-1或HIF-2对靶基因的选择取决于HIF与其他识别其启动子中同源元件的因子的可用性和合作。
