Oxygen‐independent degradation of HIF‐α via bioengineered VHL tumour suppressor complex
2009; Springer Nature; Volume: 1; Issue: 1 Linguagem: Inglês
10.1002/emmm.200900004
ISSN1757-4684
AutoresRoxana I. Sufan, Eduardo H. Moriyama, Adrian Mariampillai, O. Roche, Andrew Evans, Nehad M. Alajez, I. Alex Vitkin, Victor X. D. Yang, Fei‐Fei Liu, Brian C. Wilson, Michael Ohh,
Tópico(s)Cancer Research and Treatments
ResumoResearch Article26 March 2009Open Access Oxygen-independent degradation of HIF-α via bioengineered VHL tumour suppressor complex Roxana I. Sufan Roxana I. Sufan Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Eduardo H. Moriyama Eduardo H. Moriyama Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Adrian Mariampillai Adrian Mariampillai Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Olga Roche Olga Roche Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andrew J. Evans Andrew J. Evans Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Nehad M. Alajez Nehad M. Alajez Department of Applied Molecular Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author I. Alex Vitkin I. Alex Vitkin Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Radiation Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Victor X. D. Yang Victor X. D. Yang Department of Radiation Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Electrical and Computer Engineering, Ryerson University, Toronto, ON, Canada Search for more papers by this author Fei-Fei Liu Fei-Fei Liu Department of Applied Molecular Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Brian C. Wilson Brian C. Wilson Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Michael Ohh Corresponding Author Michael Ohh [email protected] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Roxana I. Sufan Roxana I. Sufan Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Eduardo H. Moriyama Eduardo H. Moriyama Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Adrian Mariampillai Adrian Mariampillai Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Olga Roche Olga Roche Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Andrew J. Evans Andrew J. Evans Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Nehad M. Alajez Nehad M. Alajez Department of Applied Molecular Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author I. Alex Vitkin I. Alex Vitkin Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Radiation Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Victor X. D. Yang Victor X. D. Yang Department of Radiation Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Department of Electrical and Computer Engineering, Ryerson University, Toronto, ON, Canada Search for more papers by this author Fei-Fei Liu Fei-Fei Liu Department of Applied Molecular Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Brian C. Wilson Brian C. Wilson Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada Search for more papers by this author Michael Ohh Corresponding Author Michael Ohh [email protected] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Author Information Roxana I. Sufan1, Eduardo H. Moriyama2,3, Adrian Mariampillai3, Olga Roche1, Andrew J. Evans1,4, Nehad M. Alajez5, I. Alex Vitkin2,3,6, Victor X. D. Yang6,7, Fei-Fei Liu5, Brian C. Wilson3 and Michael Ohh *,1 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada 2Division of Biophysics and Bioimaging, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada 3Department of Medical Biophysics, University of Toronto, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada 4Department of Pathology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada 5Department of Applied Molecular Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada 6Department of Radiation Oncology, University Health Network, Princess Margaret Hospital, Toronto, ON, Canada 7Department of Electrical and Computer Engineering, Ryerson University, Toronto, ON, Canada *Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Tel: 416 946 7922; Fax: 416 978 5959; EMBO Mol Med (2009)1:66-78https://doi.org/10.1002/emmm.200900004 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-α subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-α is carried out by the von Hippel–Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-α for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL–HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-α or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-α while preserving its E3 enzymatic activity. Using speckle variance–optical coherence tomography (sv–OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of 'bio-tailored' enzymes in the treatment of HIF-overexpressing tumours. The paper explained PROBLEM: Under normal oxygen tension, von Hippel–Lindau (VHL) tumour suppressor protein promotes the degradation of a key transcription factor called the hypoxia-inducible factor (HIF), which governs cellular adaptation to hypoxia or low oxygen tension. Tumour hypoxia, a common feature of solid tumours, or mutations in the VHL gene cause inappropriate accumulation of HIF, the extent of expression of which correlates with disease progression, poor prognosis and resistance to radiation and chemotherapy. Thus, various drugs designed to block the activity of HIF have been recognized as attractive strategies for cancer therapy, but have shown limited success in the clinic. RESULTS: In the present study, the authors describe the bioengineering of a VHL protein that can recognize HIF and promote its degradation in the absence of oxygen. The study goes further and demonstrates the potency of this engineered VHL on the growth of arguably one of the most resistant tumours to conventional cancer therapy, kidney cancer, in a mouse model system. IMPACT: This report introduces the concept and feasibility of 'bio-tailored' proteins in the treatment of tumours that overexpress HIF and highlights the potential of this novel anti-cancer strategy. INTRODUCTION Tumour growth invariably outstrips its blood supply as the diffusional capacity of oxygen from the nearest blood vessels is surpassed, leading to regions of hypoxia within the tumour mass. In addition, tumour cells close to a blood vessel can experience hypoxia due to disruptions in blood flow, a common characteristic of malformed tumour vasculature (Brown & Wilson, 2004). The transcription factor HIF is activated under hypoxia and triggers the transcription of a large number of genes that promote various adaptive cellular responses ranging from anaerobic metabolism, erythropoiesis and angiogenesis to cell survival. HIF-induced genes are known to drive oncogenesis and as a result, HIF overexpression is frequently associated with increased phenotypic aggressiveness and poor prognosis in numerous tumour types including brain, breast, lung, colon, skin, prostate and kidney cancers (Kim & Kaelin, 2004; Semenza, 2003). HIF is a heterodimeric transcription factor composed of two subunits, HIF-α and aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-α and ARNT are members of the basic-helix–loop–helix (bHLH) Per/ARNT/Sim (PAS) family of transcription factors. The basic domain is essential for DNA binding, whereas the HLH and PAS domains are necessary for heterodimerization and DNA binding. HIF-α contains two transactivation domains (NAD and CAD, located in the amino (N) and carboxy (C)termini, respectively), whereas ARNT contains one transactivation domain (TAD) in the C-terminus. Under low oxygen tension, HIF-α recruits transcriptional co-activators p300/CBP and binds ARNT. The active HIF complex binds to hypoxia-responsive elements (HREs) in the promoters/enhancers of the numerous HIF-target genes such as vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1), transforming growth factor-α (TGF-α) and erythropoietin (EPO) to initiate their transcription (Kim & Kaelin, 2004; Semenza, 1999). Under normal oxygen tension, HIF-α is hydroxylated on conserved proline residues in the oxygen-dependent degradation domain (ODD) by prolyl hydroxylase domain-containing family of enzymes (PHD1-3) (Bruick & McKnight, 2001; Epstein et al, 2001; Ivan et al, 2001; Jaakkola et al, 2001; Masson et al, 2001). This oxygen-dependent modification of HIF-α permits recognition by the VHL tumour suppressor protein, which functions as the substrate-recognition component of an E3 ubiquitin ligase ECV (Elongins BC/Cul2/VHL) that polyubiquitylates HIF-α for subsequent proteasomal degradation (Cockman et al, 2000; Kamura et al, 2000; Maxwell et al, 1999; Ohh et al, 2000; Tanimoto et al, 2000). Unlike HIF-α, ARNT is constitutively expressed and stable, and thus the regulation of HIF is at the level of HIF-α stability (Kim & Kaelin, 2004). VHL protein has two major functional domains: the α domain is required for the nucleation of the Elongins BC/Cul 2/VHL complex and the β domain is required for prolyl-hydroxylated HIF-α recognition (Stebbins et al, 1999). Furthermore, functional inactivation of the VHL protein is the cause of hereditary VHL cancer syndrome—characterized by the development of hypervascular tumours in multiple organs including the brain, spine, retina and kidney, and is also associated with the development of the vast majority of sporadic clear-cell renal cell carcinoma (CCRCC), which is the most common form of kidney cancer (Kim & Kaelin, 2004). Notably, all CCRCC-causing VHL mutants tested-to-date have shown a failure in either assembling into an ECV complex or binding to HIF-α (Kim & Kaelin, 2004). Concordantly, tumour cells including CCRCC devoid of functional VHL protein have enhanced expression of HIF-target genes irrespective of oxygen tension. In addition, growth factors binding to their cognate receptor tyrosine kinases (RTKs) and ensuing activation of the phosphoinositide 3-kinase and MAPK signalling pathways can regulate HIF-1α protein levels in an oxygen-independent fashion (Maynard & Ohh, 2007; Semenza, 2003). Both pathways can activate mTOR-mediated cap-dependent translation of HIF-1α mRNA, and PI3K can also increase translation of HIF-1α mRNA through an internal ribosomal entry site (IRES)-dependent mechanism (Maynard & Ohh, 2007). Thus, tumour-associated mutations impinging on the PI3K or MAPK pathways including gain-of-function mutations in RTKs and Ras or loss-of-function mutations in phosphatase and tensin homolog and tumour suppressor complex 1/2 tumour suppressor genes, increase HIF-1α synthesis. In addition to mutations in various oncogenes and tumour suppressor genes, the most common mechanism of HIF-α stabilization in cancer arguably involves the general oxygen-sensing pathway in regions of tumour hypoxia, in which, for example, a functional VHL protein would be rendered ineffectual in negatively regulating HIF-α stability. Overexpression of HIF-1α or HIF-2α has been strongly associated with tumour progression and resistance to therapy, implicating HIF-1α and HIF-2α as compelling therapeautic targets for anti-cancer therapy (Kondo et al, 2002; Semenza, 2003). Currently, there are a number of compounds either in clinical development or approved by the Food and Drug Administration that have been shown to block HIF-1α activity. For example, gefitinib and erlotinib, trastuzumab, and everolimus and temsirolimus have been shown to inhibit HIF-1α synthesis by blocking upstream oncogenic epidermal growth factor receptor, human epidermal growth factor receptor 2/Neu and mammalian target of rapamycin signalling pathways, respectively (Melillo, 2007). A topoisomerase I inhibitor topotecan and a microtubule polymerization inhibitor 2ME2 have also been found to interfere with HIF-1α mRNA translation. DNA-binding molecule echinomycin interrupts the DNA binding of HIF-1α, whereas HIF-1α-mediated transcription is reduced by the proteasome inhibitor velcade and the antifungal agent amphotericin B. In addition, inhibition of the chaperone Hsp90 by 17-AAG and 17-DMAG, as well as inhibition of HDAC by LAQ824, has been found to induce HIF-1α protein degradation (Melillo, 2007; Semenza, 2007). However, none of the above agents directly targets HIF-1α and each drug has multiple functions other than blocking HIF-1α. Moreover, the inhibitory effect of these agents on HIF-2α is largely unknown, despite an increasing evidence supporting an important role of HIF-2α in tumourigenesis. For example, HIF2 has recently been shown to transactivate Oct-4, a transcription factor essential for maintaining stem cell pluripotency, and angiopoietin-1 receptor, Tie-2 and VEGFR2 have been established as HIF2-target gene products (Covello et al, 2006; Duan et al, 2005; Elvert et al, 2003; Tian et al, 1997). HIF2 also has a higher transactivation activity than HIF1 on the promoters of VEGF, TGF-α and EPO (Gunaratnam et al, 2003; Warnecke et al, 2004; Wiesener et al, 1998). In addition, several lines of evidence have shown the stabilization of HIF-2α, but not HIF-1α, to be the critical oncogenic event upon the loss of VHL protein in CCRCC (Kondo et al, 2002, 2003). In the present study, we demonstrate that a bioengineered VHL protein can engage and degrade HIF-1α and HIF-2α irrespective of oxygen tension, eliminating the necessity for prolyl-hydroxylation of HIF-α for degradation. We further show that adenovirus-mediated delivery of a bioengineered VHL protein dramatically inhibits angiogenesis and regresses CCRCC xenografts in vivo. This is the first report illustrating the feasibility of an E3 ubiquitin ligase designed to remove the oxygen constraint as an alternative mode to directly and constitutively target and destroy HIF-α for rational anti-cancer therapy. RESULTS Unlike binding to VHL protein, prolyl-hydroxylation of HIF-α is not required for binding ARNT since heterodimerization occurs under hypoxic conditions. Thus, we sought to generate a VHL–ARNT chimaera containing the minimal region of ARNT required for binding HIF-α fused to the α domain of VHL known to bind elongin C, which bridges the VHL protein to the rest of the ECV complex (Fig 1A). A prediction is that the VHL–ARNT chimaera would bind HIF-α irrespective of oxygen to promote its degradation. Figure 1. VHL–ARNT fusion proteins bind HIF-1α in vitro. A.. Schematic diagram of a model depicting VHL–ARNT binding HIF-α independent of its prolyl-hydroxylation status and promoting HIF-α polyubiquitylation via ECV (see text for details). B/C, Elongins BC; bHP, basic-helix–loop–helix and PAS; HRE, hypoxia-responsive element; Ub, ubiquitin. B.. Schematic diagram of the various T7-tagged ARNT truncation mutants generated for defining minimal regions required for binding HIF-1α. FL ARNT, full-length ARNT; b, basic; HLH, helix–loop–helix; PAS, Per-ARNT-Sim; PAC, PAS-associated C-terminal domain. C.. 35S-labelled in vitro translated HA-HIF-1α was mixed with the indicated 35S-labelled in vitro translated T7-ARNT truncation mutants. Sample mixtures were immunoprecipitated with anti-T7 antibody, resolved by SDS–PAGE and visualized by autoradiography. The image shown was generated from one autoradiograph. D.. Schematic diagram of the VHL α domain fused to the indicated ARNT truncation mutants with or without the flexible Gly(6) linker. E.. 35S-labelled in vitro translated HA-HIF-1α was mixed with the indicated 35S-labelled in vitro translated T7-tagged VHL–ARNT fusion proteins. Sample mixtures were immunoprecipitated with anti-T7 or anti-HA antibodies. Bound proteins were resolved by SDS–PAGE and visualized by autoradiography. The image shown was generated from one autoradiograph. IP, immunoprecipitation. Download figure Download PowerPoint VHL–ARNT fusion proteins bind HIF-α and form an ECV complex The bHLH, PAS A and PAS B domains of ARNT are required for dimerization with HIF-1α and HIF-2α. C-terminal to the PAS B domain is the PAS C-terminal domain (PAC) that is less well-described, but has been proposed to play a similar role in heterodimerization (Maynard & Ohh, 2004). To define the minimal region of ARNT required for strong dimerization with HIF-α, we generated T7-tagged truncation plasmids encoding the following domains: HLH, PAS A, PAS B (T7-HPAS, residues 103–419); bHLH, PAS A, PAS B (T7-bHPAS, residues 90–419); HLH, PAS A, PAS B, PAC (T7-HPAC, residues 103–467); and bHLH, PAS A, PAS B, PAC (T7-bHPAC, residues 90–467) (Fig 1B). An in vitro binding assay was performed with 35S-labelled in vitro translated HA-HIF-1α mixed with 35S-labelled in vitro translated T7-tagged ARNT truncation mutants. The reaction mixtures were immunoprecipitated with an anti-T7 antibody, resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and visualized by autoradiography (Fig 1C). The ARNT truncation mutants containing the PAC domain showed increased binding to HIF-1α (Fig 1C, compare lanes 5 and 8 with lanes 11 and 14). Based on these findings, T7-HPAC and T7-bHPAC were used to generate the VHL–ARNT chimaera. VHL–ARNT chimaeras were generated by fusing the VHL α domain (residues 151–194) C-terminal to T7-HPAC and T7-bHPAC with or without a 6-Glycine flexible linker between the two heterologous protein fragments, giving rise to the following constructs: T7-HPACV, T7-HPACGV, T7-bHPACV and T7-bHPACGV (Fig 1D). We next tested the ability of the fusion proteins to bind HIF-1α by performing an analogous in vitro binding assay (Fig 1E). The addition of VHL α domain did not diminish the ability of ARNT truncation mutants to bind HIF-1α, and T7-HPACV and T7-HPACGV chimaeras displayed stronger interaction with HIF-1α than T7-bHPACV or T7-bHPACGV containing the basic DNA binding sequences (Fig 1E, compare lanes 9 and 12 with lanes 18 and 21). We explored whether the VHL–ARNT fusion proteins bound HIF-1α in vivo. HEK293A cells were transiently co-transfected with the mammalian expression plasmids encoding HA-HIF-1α and empty plasmid (MOCK) or T7-HPACV, T7-HPACGV, T7-bHPACV or T7-bHPACGV. Cells were treated with the proteasome inhibitor MG132 to stabilize the oxygen-labile HIF-1α. Cells were then lysed, immunoprecipitated with an anti-HA antibody, bound proteins resolved by SDS–PAGE and immunoblotted with anti-HA and anti-T7 antibodies. All VHL–ARNT chimaeras co-precipitated HA-HIF-1α (Fig 2A). Moreover, VHL–ARNT chimaeras likewise bound HA-HIF-2α (data not shown). We next examined whether the VHL–ARNT chimaeras formed an ECV complex. HEK293A cells were transiently transfected with an empty plasmid, T7-VHL, T7-bHPAC, T7-HPACV, T7-HPACGV, T7-bHPACV or T7-bHPACGV. Cells were then lysed, immunoprecipitated with anti-T7 antibody, bound proteins resolved by SDS–PAGE and immunoblotted with anti-Cul2 and anti-T7 antibodies (Fig 2B). T7-VHL served as a positive control showing co-precipitation of the scaffold component Cul2 (Fig 2B, lane 3), while the ARNT truncation mutant T7-bHPAC lacking the VHL α domain served as a negative control showing a failure in co-precipitating Cul2 (Fig 2B, lane 4). VHL–ARNT chimaeras, when normalized for expression, exhibited Cul2 binding with efficiency comparable to that of wild-type VHL protein, indicating their ability to form an ECV complex. Figure 2. VHL–ARNT fusion proteins bind HIF-1α and form ECV complexes in vivo. A.. HEK293A cells transfected with the indicated plasmids were treated with the proteasome inhibitor MG132, lysed, immunoprecipitated with anti-HA antibody and visualized by immunoblotting. B.. HEK293A cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-T7 antibody, resolved by SDS–PAGE and immunoblotted with anti-Cul2 and anti-T7 antibodies. WCE, whole cell extract; IP, immunoprecipitation; IB, immunoblot; IgG-H, immunoglobulin heavy chain. Download figure Download PowerPoint VHL–ARNT chimaeras promote HIF-α degradation and inhibit HRE-mediated transcription under hypoxia We asked whether T7-HPACV or T7-HPACGV could degrade HIF-1α under hypoxia. HEK293A cells were transiently co-transfected with plasmids encoding HA-HIF-1α and empty plasmid or T7-VHL, T7-HPACV or T7-HPACGV. Prior to lysis, cells were treated with or without proteasome inhibitor MG132 and maintained under hypoxia (1% oxygen). Cells were lysed and equal amounts of the whole cell lysates were resolved by SDS–PAGE and immunoblotted with an anti-HIF-1α antibody (Fig 3A). As expected, co-transfection of T7-VHL had negligible effect on HIF-1α protein levels, evidenced by the similar amounts of HIF-1α detected with or without MG132 (Fig 3A, compare lanes 3 and 4). Notably, the endogenous VHL protein in HEK293A cells had likewise no discernable effect on HA-HIF-1α expression under hypoxia (Fig 3A, lane 2). In contrast, the expression of either T7-HPACV or T7-HPACGV caused dramatic attenuation of HIF-1α levels in the absence of MG132 (Fig 3A, lanes 6 and 8). These results indicate that both T7-HPACV and T7-HPACGV effectively promote HIF-1α for proteasome-dependent degradation under hypoxia. Consistent with this notion, non-hydroxylated HIF-1α(Pro564Ala) (Ivan et al, 2001; Jaakkola et al, 2001), which has been shown to be stable in the presence of wild-type VHL protein under normoxia, was effectively degraded by T7-HPACGV in a proteasome-dependent manner (Fig S1 of Supporting Information). Figure 3. HPACV and HPACGV bind and degrade HIF-1α under hypoxia and decrease HRE-mediated transcription. A, B.. HEK293A cells transfected with the indicated plasmids were treated with (+) or without (−) MG132 for 4 h and maintained at 1% oxygen. Cells were lysed and whole cell extracts (A) or anti-HA and anti-T7 immunoprecipitated proteins (B) were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. C.. Dual-luciferase assay was performed in HEK293A cells transfected with (HRE)5-Luc in combination with the indicated plasmids. SV40-driven renilla luciferase was transfected as internal control. Cells were maintained at 21 or 1% oxygen for 16 h prior to lysis and dual-luciferase assay was performed. Experiments and transfections were performed in triplicates with one representative experiment shown. Error bars represent standard deviations. RLU, relative light units. Download figure Download PowerPoint To assess the binding of VHL–ARNT chimaeras to HIF-1α under hypoxia, an analogous experiment in HEK293A cells was performed with anti-HA and anti-T7 immunoprecipitations of the whole cell lysates. Bound proteins were resolved by SDS–PAGE and immunoblotted with anti-HIF-1α and anti-T7 antibodies (Fig 3B). As expected, T7-VHL failed to co-precipitate HA-HIF-1α under hypoxia even in the presence of MG132 (Fig 3B, lane 3). In contrast, both T7-HPACV and T7-HPACGV co-precipitated HA-HIF-1α in the presence of MG132 (Fig 3B, lanes 5 and 7). These results strongly suggest that VHL–ARNT chimaeras bind HIF-1α under hypoxia to promote its proteasome-dependent degradation. AhRryl Hydrocarbon Receptor (AhR) is another well-characterized ARNT binding partner ubiquitously expressed and involved in endo- and xenobiotic metabolism (Kewley et al, 2004). AhR as expected bound ARNT, but failed to bind HPACGV in an in vitro binding assay (Fig S2A and S2B of Supporting Information). Accordingly, HPACGV failed to downregulate the expression of AhR in vitro or in vivo (Fig S2C and D of Supporting Information). Thus HPACGV, which was generated and optimized for binding and degrading HIF-α, does not interact with arguably the next best-characterized ANRT-binding partner AhR. Under hypoxia, HIF-α dimerizes with ARNT to form an active transcription factor HIF, which engages HREs in the promoters of a myriad of hypoxia-inducible genes to initiate their transcription. We sought to determine the effect of T7-HPACV and T7-HPACGV on HRE-driven transcription by performing a dual-luciferase assay using a firefly luciferase reporter driven by five contiguous HRE elements from the phosphoglycerate kinase-1 promoter (Fig 3C). HEK293A cells were transiently co-transfected with plasmids encoding (HRE)5-Luc and T7-VHL, T7-HPACV or T7-HPACGV. Cells were maintained at either normoxia (21% oxygen) or hypoxia (1% oxygen) for 16 h prior to lysis. The transactivation activity from the HRE promoter was markedly higher under hypoxia than normoxia, as expected. Also, the ectopic expression of T7-VHL did not influence HRE-driven transactivation under hypoxia, since a VHL protein is ineffective in targeting HIF-α for destruction under hypoxia. In contrast, T7-HPACV and T7-HPACGV significantly reduced the transactivation from the HRE promoter under hypoxia (Fig 3C), indicating a marked loss of endogenous HIF function. Notably, T7-HPACGV was reproducibly more potent in attenuating HIF-mediated transcription than T7-HPACV (Fig 3C), and thus the T7-HPACGV chimaera was selected for subsequent experimentation. Furthermore, HPAC in the absence of a VHL protein diminished HIF-driven transcription under hypoxia by forming an inactive transcriptional complex, whereas HPACGV in comparison, dramatically attenuated HIF-driven transcription (Fig S3 of Supporting Information), suggesting that the potency of HPACGV is due to the rapid E3 enzymatic activity causing the degradation of HIF-α upon binding. Adenoviral delivery of T7-HPACGV attenuates HIF-α and HIF-target gene expression independent of oxygen tension A cardinal feature of CCRCC is the overexpression of hypoxia-inducible genes. This is principally due to the inactivation of VHL protein that is observed in the vast majority of CCRCC. Interestingly, CCRCC harbouring wild-type VHL protein also displays strong hypoxic signatures and several lines of evidence suggest the stabilization of HIF-2α to be a critical oncogenic event in the pathogenesis of CCRCC (Kondo et al, 2002). We generated recombinant adenoviruses expressing enhanced green fluorescence protein (EGFP) alone or in combination with T7-VHL (Ad-EGFP-T7-VHL) or T7-HPACGV (Ad-EGFP-T7-HPACGV) and tested their ability to form an ECV complex in the CCRCC cell line 786-O (VHL−/−; HIF-1α−/−), a widely used cell system for CCRCC with constitutive activation of HIF-2α. Upon high-efficiency infection of 786-O cells with the indicated adenoviruses (>95%, as determined by EGFP fluorescence; data not shown), cells were lysed and immunoprecipitated with anti-T7 antibody. Bound proteins were resolved by SDS–PAGE and immunoblotted with anti-T7, anti-Cul2 and anti-Elongin B antibodies (Fig 4A). Both Ad-EGFP-T7-VHL and Ad-EGFP-T7-HPACGV, but not Ad-EGFP, co-precipitated Cul2 and Elongin B to comparable levels, suggesting equal ability of ECV complex formation (Fig 4A). Figure 4. Ad-EGFP-T7-HPACGV forms an ECV complex and attenuates HIF-α l
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