Tumor hypoxia is well recognized as a major driving factor for tumor growth and resistance to therapy (1
). In addition to promoting tumor cell survival during hypoxic stress by shifting cells toward anerobic metabolism, neovascularization, and resistance to apoptosis, the hypoxia response may drive other responses that contribute to tumor aggressiveness such as increased genetic instability, invasion, metastasis, and an undifferentiated phenotype (35
). Hence hypoxia, rather than acting as a simple on-off switch for the hypoxia response as once thought, initiates a complex cellular response that involves multiple players, including the HIFs, depending on the duration and intensity of the hypoxia. Indeed, it has been suggested that HIF-1α, because of its rapid induction and negative feedback regulation by prolonged hypoxia (16
), provides a swift response to acute or transient hypoxia. On the other hand, prolonged or chronic hypoxia seems to favor activation of HIF-2α in most cell types, possibly because of differential affinities for specific regulators of stability and activity, such as the prolyl hydroxlases, factor inhibiting HIF, and also Hsp70, which promotes the ubiquitination and proteasomal degradation of HIF-1α but not HIF-2α (7
). Intriguingly, chronic hypoxia has been implicated as a causal factor for the increased aggressiveness of tumors that develop resistance to antiangiogenic therapy such as to VEGF inhibition (40
), underscoring the need for a clearer understanding of the mechanisms that regulate the responses of the chronic versus acute hypoxia.
We have previously reported that the HAF C-terminus (residues 654–800) binds and ubiquitinates HIF-1α within residues 298–400, targeting HIF-1α for proteasomal degradation (25
). We now report the ability of HAF to bind differentially to the 2 HIF-α isoforms resulting in HIF-1α degradation and promoting HIF-2α transactivation. In this regard, HAF (residues 300–500) binds to HIF-2α within residues 604–750 to increase HIF-2α transactivation. Thus, HAF plays a critical role in hypoxia signaling by turning off the HIF-1α response and turning on the HIF-2α response (). Indeed, HAF knockdown inhibits HIF-2α–dependent HRE activity in 786-0 cells, suggesting that HAF is necessary and sufficient for HIF-2α activity. Furthermore, HAF overexpression inhibits the transcription of HIF-1α–specific target genes during acute hypoxia (16 hours) but promotes the transcription of the HIF-2α–dependent genes after prolonged hypoxia (72 hours).
HAF overexpression in cells grown in 3D culture resulted in the formation of colonies with a more diffuse, invasive phenotype compared with vector control cells. This phenotype is consistent with HIF-1α knockdown, which abrogated colony formation, and with increased HIF-2α, which stimulated invasiveness in hypoxia. Intriguingly, long-term knockdown of HAF decreased the size of colonies, both in normoxia and hypoxia, which may be due to previously reported roles of HAF in pre-mRNA splicing and mitosis (42
). Significantly, HAF overexpression was associated with the increased incidence and morbidity of intracranial U87 tumor xenografts, possibly because of the involvement of HAF in promoting self-renewal, which enabled tumor formation from cell numbers that in the vector control cells were insufficient to form tumors. The possibility exists that the availability of HAF may be the limiting factor for the full activation of HIF-2α and further work will show whether HAF levels may be a potential biomarker of highly undifferentiated, HIF-2α–driven tumors. Indeed, the unique hypoxia-dependent modulation of HAF levels may be a determinant of HIF-2α activation in response to varying intensities and durations of hypoxia ().
The differences in specific HIF-1α and HIF-2α downstream targets, when compounded with the genetic aberrations present within the diverse tumor population, may determine which HIF-α isoform may provide the greatest growth advantage to a tumor as a whole. Many tumor cells develop resistance to apoptosis, which could counteract the proapoptotic factors induced by HIF-1α, effectively harnessing protumorigenic outcomes of HIF-1α, while negating its anti-tumorigenic effects (44
). Hence, HAF overexpression inhibits the growth of HT29 tumor xenografts that express high HIF-1α and low HIF-2α (25
). In contrast, the unique ability of HIF-2α to collaborate with oncogenes such as c-Myc
, and K-Ras
may provide a growth advantage to tumor cells with deregulation of these pathways (14
). Our present finding that HAF overexpression in the intracranial GBM model promotes poor survival provides a context in which HIF-1α inhibition and HIF-2α activation may promote tumor progression.
In conclusion, this study characterizes a new mechanism for the differential hypoxic regulation of HIF-1α and HIF-2α by HAF. HAF is overexpressed in many cancers (22
), and its availability may be critical for HIF-2α–driven tumor progression. The HIF-binding domains of HAF which are able to differentially bind to HIF-1/2α may provide novel avenues for modulation of the HIF-1/2α balance for therapeutic benefit.