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. 2013 Jun 27;32(26):3147-55.
doi: 10.1038/onc.2012.333. Epub 2012 Aug 6.

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation

B K Nayak  1 , D Feliers, S Sudarshan, W E Friedrichs, R T Day, D D New, J P Fitzgerald, A Eid, T Denapoli, D J Parekh, Y Gorin, K Block
Affiliations

Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation

B K Nayak et al. Oncogene. .

Abstract

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

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Figures

Figure 1
Figure 1
Effects of mTORC1 and mTORC2 inhibition on eIF4E binding to mRNA repressor, 4E-BP1. (a): VHL-deficient RCC 786-O cells were incubated with buffer alone (−), mTORC1 inhibitor (CCI-779), or the mTORC1/mTORC2 inhibitor (pp242) for 24 hours. Cellular lysates were analyzed by Western blot analysis for HIF-2alpha expression. GAPDH was used as a loading control. (b): In parallel to A, RNA was extracted and HIF-2alpha mRNA was examined by quantitative RT-PCR. (c): Cellular lysates were prepared as described in materials and methods from RCC 786-O cells treated with buffer alone (−), CCI-779, or pp242 for 24 hrs and eIF4E and its associated proteins were affinity purified using 7-methyl-GTP sepharose beads followed by Western blot analysis for total eIF4E and 4E-BP1. The data are representative of three independent experiments. (d): Polysomal fractions were prepared by passing cytosolic lysates from RCC 786-O cells treated with buffer treated (−), CCI-779, or pp242, over sucrose gradients as described in experimental methods. HIF-2alpha and GAPDH mRNAs were detected in each fraction by RT-PCR on RNA extracted from each fraction. Sedimentation analysis was examined in parallel of RCC 786-O cells by measuring optical density at 254 nm. (e): Quantitative RT-PCR was performed as outlined in materials and methods to examine the mRNA expression levels HIF-responsive genes (VEGF and TGF-alpha) in RCC 786-O cells treated with buffer alone (−), CCI-779, and pp242. The data is representative of three independent experiments and are expressed as fold control where the ratio of the buffer treated control cells was defined as 1. Values are the means +/− S.E, * p<0.05 and # p<0.05 compared to CCI-779 alone.
Figure 2
Figure 2
Rictor mediates eIF4E binding to mRNA translational repressor, 4E-BP1. (a): Equivalent amounts of cell lysates were analyzed for known downstream targets of Rictor, HIF-2alpha, pAkt (S473) by Western blot analysis in RCC 786-O cells silenced of Rictor using small inhibitory RNA (siRNA) or scrambled control (scr) as described in materials and methods. Total Akt and GAPDH were used as loading controls. (b): Steady state levels of HIF-2alpha mRNA levels were examined in RCC 786-O cells silenced of Rictor (siRictor) or scrambled control (scr). (c): eIF4E association with 4E-BP1 was examined as outlined in Fig. 1C using 7-methyl-GTP sepharose beads from RCC 786-O cells silenced for Rictor using siRNA (siRictor) or scrambled control (scr). The data are representative of at least three independent experiments. (d): Polysomal fractions were prepared by passing cytosolic lysates from RCC 786-O cells silenced of Rictor (siRictor) or scrambled control (scr), over sucrose gradients as described in experimental methods. HIF-2alpha and GAPDH mRNAs were detected in each fraction by RT-PCR on RNA extracted from each fraction. (e): Quantitative RT-PCR was performed as outlined in materials and methods to examine the mRNA expression levels HIF-responsive genes (VEGF and TGF-alpha) after siRNA down-regulation of Rictor or scrambled control. The data is representative of three independent experiments and are expressed as fold control where the ratio of the scrambled control was defined as 1. Values are the means +/− S.E, ** p<0.01.
Figure 3
Figure 3
Effects of p22phox inhibition on eIF4E-dependent mRNA translation. (a): p22phox was stably knocked down using lentiviral short hairpin loop RNA (shp22phox) or vector control (shVector) as described in materials and methods. Western blot analysis (left panel) was carried out to confirm p22phox downregulation in indicated independent single cell shp22phox clones. GAPDH was used as loading control and quantitative RT-PCR (right panel) was carried out to confirm p22phox mRNA down regulation (b): NADPH oxidase activity was measured in parallel. (c): eIF4E association with 4E-BP1 was examined as outlined in Fig. 1C using 7-methyl-GTP sepharose beads from RCC 786-O cells stably silenced for p22phox (shp22phox) or Vector control (shVector). The data are representative of at least three independent experiments. (d): Polysomal analysis was performed from RCC 786-O cells stably silenced for p22phox (shp22phox) or Vector control (shVector) as outlined in 2D and as described in experimental methods.
Figure 4
Figure 4
Redox regulation of mTORC complexes. (a): Association of Rictor and Raptor with mTORC complexes were examined in stable shVector or shp22phox RCC 786-O independent single cell clones by immunoprecipitating mTOR with anti-mTOR antibodies or equivalent amounts of rabbit IgG for control from cell lysates prepared in mTOR lysis buffer as described in materials and methods followed by Western blot analysis for Rictor, Raptor, and mTOR. Histogram (lower panel) represents the ratio of the intensity of the Rictor or Raptor bands to the mTOR band in shp22phox knock down clones compared to vector controls (Fold change control) quantified by densitometry. Values are the means ± S.E. (n =3), * p<0.05. (b): Cellular lysates were prepared from stable shVector or shp22phox RCC 786-O independent single cell clones and analyzed by Western blot analysis for Rictor, mSin1, pAkt473, and p22phox expression. GAPDH was used as a loading control.
Figure 5
Figure 5
HIF2α dependent transcriptional activity and gene regulation is regulated by p22phox. (a): Left panel. Transcriptional activity of a HIF-responsive gene promoter (EPO) was measured in RCC 786-O cells transiently transfected with scrambled control (scr) or siRNA against p22phox (sip22phox) as described in materials and methods. RCC 786-O renal carcinoma cell lines containing wild-type VHL (786-O + VHL) were used as a negative control for HIF-transcriptional activity. Right panel. In parallel to 5A, cell lysates were prepared and protein expression of HIF-2α and p22phox were analyzed by Western blot analysis. β-tubulin was used as a loading control. (b): Quantitative RT-PCR was performed to examine the mRNA expression levels of HIF-responsive genes (VEGF, TGFα) after siRNA down-regulation of the p22phox gene. The data is representative of three independent experiments and are expressed as percent control where the ratio of the scrambled control was defined as 100%. Values are the means +/− S.E, ** p<0.01. (c): Nude mice were injected with stable shVector or shp22phox RCC 786-O independent single cell clones as outlined in materials and methods. Animals were sacrificed at 7 weeks and tumor volumes measured using calipers. There were 10 animals in each individual group and are expressed as fold control (shVector) where the ratio of the shVector control was defined as 1. Values are the means +/− S.E, ** p<0.01.
Figure 6
Figure 6
Effects of mTORC1/mTORC2 inhibitor on eIF4E complex formation in ex vivo RCC cells. (a,b): Left Panel, Steady state mRNA of HIF-2alpha was examined in two independent RCC tumor tissues or normal adjacent control tissues. Right Panel, Analysis of HIF-2 alpha mRNA in polysomal fractions as described in Fig. 1D in RCC tumors and normal adjacent controls. C, Examination of eIF4E and its associated proteins using 7-methyl-GTP sepharose beads in ex vivo cultured renal tumor cells treated with buffer alone, CCI-779, or pp242.
Figure 7
Figure 7
Proposed mechanism of mTORC2 regulation of HIF-2alpha protein expression through p22phox- based Nox oxidase-dependent regulation of eIF4E-dependent mRNA translation in RCC.
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