Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90.
BACKGROUND
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.


RESULTS
By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.


CONCLUSIONS
We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90.
Trisciuoglio
Daniela
D
Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy.
Gabellini
Chiara
C
Desideri
Marianna
M
Ziparo
Elio
E
Zupi
Gabriella
G
Del Bufalo
Donatella
D
D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D016227:Benzoquinones; C497442:HIF1A protein, human; D018841:HSP90 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2; D025801:Ubiquitin; C112765:tanespimycin