A Novel HIF-1α-Myc Pathway Regulating Hypoxia-induced Cell-cycle Arrest
olid tumors harbor hypoxic regions that are not only critical for tumor development and progression, but are also associated with resistance to chemotherapy and radiation therapy. Hypoxia-inducible factor 1α (HIF-1α), a basic helix-loop-helix (bHLH) transcription factor of the PAS protein family, plays an essential role in the transcriptional activation of genes involved in angiogenesis and glycolysis, which are required for tumor development and progression. In many human cancers, HIF-1α and HIF-2α, a close member of the HIF-α family, are overexpressed, and their expression levels are correlated with the degree of malignancy. Moreover, genetic studies have shown that Hif1α-null tumors grow much slower in a poor vascular environment, as compared with their Hif1α wild-type counterparts.
HIF-1α expression is regulated primarily by posttranslational stabilization, resulting from inhibition of the ubiquitin-proteasome pathway that targets the oxygen-dependent degradation domain (ODD) of HIF-1α. HIF prolyl 4-hydroxylases function as oxygen sensors to modify two proline residues within the ODD, thereby enabling the VHL E3 ubiquitin ligase to bind specifically to the hydroxyprolines for HIF-1α polyubiquitination. Accordingly, deletion of the ODD renders HIF-1α stable and capable of binding the hypoxia-responsive element (HRE) and activating the downstream target genes.
Interestingly, apart from stimulation of angiogenesis and glycolysis for cell proliferation and survival, hypoxia also induces cell-cycle arrest, apparently against tumor development. Although the results from Hif1α-null cells indicate that HIF-1α is required for hypoxia-induced upregulation of p21cip1, a key cyclin-dependent kinase inhibitor that controls the G1 checkpoint, the role of HIF-1α in the cell cycle remained controversial. Moreover, it remained obscure how HIF-1α transcriptionally activates p21cip1 due to the lack of HIF-1α-bound HRE in the promoter.
...we validated that the N-terminal HIF-1α is critical for cell-cycle arrest, indicating a novel mechanism for HIF-1α function.
To provide direct evidence that HIF-1α controls the cell cycle, we took advantage of an ODD-deficient HIF-1α and demonstrated that expression of HIF-1α in normoxia is sufficient to induce G1 arrest. As expected, HIF-1α activates p21cip1 expression, and conversely, HIF-1α–induced cell-cycle arrest is p21cip1 dependent. Therefore, HIF-1α induces G1 arrest via the activation of p21cip1.
To understand the mechanism underlying p21cip1 activation in hypoxia, we created two functional mutations that inactivate HIF-1α DNA-binding and transcriptional activation, respectively. To our surprise, both mutants were still able to activate p21cip1 and to cause G1 arrest, despite their inability to upregulate known HIF-1α target genes, such as VEGF. Thus, neither HIF-1 transcriptional activity nor its DNA binding is required for p21cip1 activation, implying a novel HIF-1α function in regulating gene expression.
In pursuit of the distinct function of HIF-1α, we hypothesized that HIF-1α upregulates p21cip1 by virtue of functionally counteracting Myc, a known repressor that binds the transcription activator Miz-1 of p21cip1. Consistently, hypoxic treatment or HIF-1α expression in normoxia overrode Myc-targeted gene expression; hypoxia/HIF-1α not only upregulated Myc-repressed gene p21cip1, but also downregulated Myc-activated genes, such as TERT and BRCA1. RNA silencing experiments demonstrated a critical role for Myc in HIF-1α action. Moreover, chromatin immunoprecipitation analysis of the p21cip1 promoter showed that Myc binding was markedly weakened by hypoxia or HIF-1α, suggesting that the HIF-1α action is mediated by the displacement of Myc from the p21cip1 promoter.
We showed further that HIF-1α forms a weak complex with Myc. The protein-protein interaction is mediated by an HIF-1α N-terminal region consisting of bHLH and PAS domains. Finally, we validated that the N-terminal HIF-1α is critical for cell-cycle arrest, indicating a novel mechanism for HIF-1α function.
This study has demonstrated that HIF-1α employs at least two mechanisms for regulating gene expression: in addition to the classical mode of action involving binding to the HRE plus transactivation via transcriptional activation domains, HIF-1α functionally antagonizes Myc via its N-terminal region to override the expression of Myc-targeted genes that lack a canonical HRE. This new pathway may signify a new set of hypoxia-responsive genes that cannot be accounted for by the previously identified mechanisms. In addition, the independent action of HIF-1α N-terminal indicates that HIF-1α polypeptide, devoid of transactivation domains and DNA binding activity, is in fact functional; HIF-1α mutants in this nature are still able to regulate hypoxia-responsive genes that lack HREs. Therefore, the interpretation of effects of such “dominant-negative” mutants may need to be reevaluated.