Abstract
ID (inhibitor of differentiation/DNA binding) proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis. We have previously demonstrated that hypoxia decreases expression of neuronal marker genes in neuroblastoma, but induces genes expressed in the neural crest, such as ID2. Because of its involvement in normal neural crest development and its ability to inhibit proneuronal bHLH proteins, the hypoxic induction of ID2 was of particular interest. Here we report fast induction kinetics of ID2 expression in hypoxic neuroblastoma cells. The up-regulation of ID2 was abolished by addition of actinomycin D, implicating a hypoxia-driven transcriptional mechanism. Analyzing the ID2 promoter revealed several potential binding sites for hypoxia-inducible factors. Subsequent electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated two functional HIF-1 binding sites within ID2 gene regulatory sequences located at -725 and -1893 relative to the transcriptional initiation point. In transfection assays, DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner. These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. Hypoxia-induced ID2 expression could play a significant role in the previously observed dedifferentiation of hypoxic neuroblastoma cells, which in a clinical setting could lead to less mature and more aggressive tumors.
Highlights
ID proteins, frequently deregulated in advanced human malignancies, can participate in multiple fundamental traits of cancer, such as block of differentiation, increased proliferation, tissue invasiveness, and angiogenesis
We show that the ID2 gene is transcriptionally activated in response to hypoxia through a mechanism that involves binding of HIF-1␣/ARNT complexes to sites located within the promoter region
ID2 expression was up-regulated by hypoxia (1% O2) in three human neuroblastoma and two breast cancer cell lines and in HeLa cells
Summary
DNA constructs of the ID2 promoter, including the functional HIF-1 binding sites, induced luciferase reporter activity in a HIF-1-specific manner These observations demonstrate that ID2 is actively engaged by hypoxia and represents a novel HIF-1 target. The ID (inhibitor of differentiation/DNA binding) proteins (ID1-ID4) are part of the helix-loop-helix (HLH) family They are direct or indirect negative regulators of basic helix-loophelix (bHLH) transcription factors involved in cell lineage specification and in numerous developmental processes [22]. Hypoxiainduced up-regulation of ID2 was seen in HeLa and human breast cancer MCF-7 and T47 cells, suggesting a general hypoxia-driven mechanism These observations, together with a role of ID2 in the specification of neuronal lineages derived from the neural crest, indicated a possible involvement of ID2 in hypoxic neuroblastoma exhibiting neural crest-like features. The activation of ID2 could, in concert with down-regulation of transcription factors involved in neuronal lineage specification, such as dHAND, HASH-1, and E2-2 [19, 20], have a significant role in the dedifferentiation and progression of hypoxic neuroblastoma cells
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