Del Bufalo1* Abstract Bcl-2 Regulates HIF-1a Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90 Daniela Trisciuoglio1, Chiara Gabellini1,2, Marianna Desideri1, Elio Ziparo2, Gabriella Zupi1, Donatella 1 Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy, 2 Department of Histology and Medical Embryology, Sapienza University, Rome, Italy 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-1a, 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. Methodology/Principal Findings: By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1a 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-1a protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1a 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-1a stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1a degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1a protein. We also showed that bcl-2, HIF-1a and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1a 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- 1a protein during hypoxia, and in particular the isoform HSP90b is the main player in this phenomenon. Conclusions/Significance: We identified the stabilization of HIF-1a protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the b isoform of molecular chaperone HSP90. Citation: Trisciuoglio D, Gabellini C, Desideri M, Ziparo E, Zupi G, et al. (2010) Bcl-2 Regulates HIF-1a Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90. PLoS ONE 5(7): e11772. doi:10.1371/journal.pone.0011772 Editor: Marc Vooijs, UMC Utrecht, Netherlands Received February 9, 2010; Accepted June 29, 2010; Published July 27, 2010 Copyright: 2010 Trisciuoglio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Italian Association for Cancer Research, grant number 08/30/c/91 (D.D.B.) and the Italian Ministry of Health, grant number 08/01/c/48 (D.D.B.) and 08/01/c/1 (G.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: delbufalo@ifo.it Introduction The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) regulates the expression of more than 70 genes involved in tumour angiogenesis, metabolic switch to anaerobic glycolysis, pro- survival, proliferative and apoptotic mechanisms [1]. Overall, the expression of HIF-1 target genes helps cells to adapt to, and thereby survive in, a stressful microenvironment. The activity of HIF-1 dimer, which is composed of a and b subunits, is modulated by the availability of the extremely labile oxygen-sensitive HIF-1a protein subunit. HIF-1 activity depends on the inhibition of the post-transcriptional hydroxylation of the subunit a by prolyl hydroxylases PHD1-3 and Factor Inhibiting HIF-1 (FIH-1). PHDs-mediated hydroxylation targets HIF-1a for proteasomal degradation via the ubiquitination-dependent Von Hippel-Lindau (VHL) complex, while FIH-1-mediated hydroxylation leads to the inhibition of HIF-1 transactivation. The activity of PHD1-3 enzymes is dependent on substrates oxygen and 2-oxoglutarate, a Krebs cycle intermediate, and cofactor Fe2+; thus, under hypoxic conditions, PHDs are less active due to the substrate-limiting conditions. The regulation of HIF-1a stability by an oxygen- independent degradation pathway was also reported: the molec- ular chaperone Heat Shock Protein 90 (HSP90) binds and stabilizes HIF-1a, competing with Receptor of Activated protein Kinase C (RACK1), which mediates prolyl hydroxylase/VHL- independent ubiquitination and proteasomal degradation of HIF- 1a [2]. Other post-translational modifications of HIF-1a, such as acetylation, phosphorylation and nitrosylation, were also reported, despite contradictory results with regard to their effect on HIF-1a protein stability and transcriptional activity [3–6]. Adding to the complexity of HIF-1a regulation, it has recently been shown that the SUMOylation of HIF-1a enables the hydroxylation-indepen- dent binding and subsequent degradation of HIF-1a by the VHL- E3 ligase complex [7]. Although hypoxia is considered the main stimulus that drives HIF-1 function, a number of non-hypoxic stimuli allows the formation of an active HIF-1 complex in many types of human cancers. Effectors implicated in stimulating or suppressing an PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11772