Differential Dependence of Hypoxia-inducible Factors 1α and 2α on mTORC1 and mTORC2<sup><a href="#fn1" rid="fn1" class=" fn">*</a></sup>

Alfredo Toschi

Evan Lee

Noga Gadir

Michael Ohh

David Foster

EXPERIMENTAL PROCEDURES

Cells, Cell Culture Conditions, and Transfection—RCC4 and 786-O cells were obtained from American Type Culture Collection. All cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Transfections were performed using Lipofectamine LTX (Invitrogen) for plasmid transfection and Lipofectamine RNAiMAX (Invitrogen) for small interfering RNA (siRNA) according to the manufacturer's instructions. Transfection efficiency was determined by transfection of pEGFP-C1, which expresses green fluorescent protein. The percentage of green cells was determined microscopically and was routinely in excess of 90%.

Materials—Antibodies against mTOR, Rictor, Raptor, HIF2α, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against pan-Akt, Akt1, Akt2, Akt3, phospho-Akt (Ser^{), S6 kinase (S6K), and phospho-S6K (Thr}^{) were obtained from Cell Signaling (Danvers, MA). The antibody to HIF1α was from BD Biosciences. siRNAs targeting Raptor, Rictor, mTOR, and Akt1 were obtained from Sigma. The siRNA targeting Akt2 was from Cell Signaling, and the siRNA for Akt3 was from Santa Cruz Biotechnology. Rapamycin was obtained from Calbiochem.}

Western Blot Analysis—Extraction of proteins from cultured cells and Western blot analysis of extracted proteins were performed using the ECL system (Amersham Biosciences) as described previously (17).

siRNA—Cells were plated on 12-well plates at 30% confluence in medium containing 10% serum without antibiotics. After 1 day, cells were transfected with siRNA using Lipofectamine RNAiMAX according to the manufacturer's directions. After 24 h, the medium was changed to fresh medium containing 10% serum, and 2 days later, cells were lysed and analyzed by Western blotting.

RESULTS

Differential Effect of Rapamycin on HIF1α and HIF2α—Although several laboratories have reported that the expression of HIF1α is dependent upon mTOR (13–16), there have been no reports implicating mTOR in the expression of HIF2α. However, both HIF1α and HIF2α are dependent upon PLD activity in RCC cells (17). Because PLD activity has been widely implicated in the activation of mTOR (32), we investigated the effect of rapamycin on the expression of HIF2α in VHL-null 786-O RCC cells, which have elevated expression of HIF2α, and in VHL-null RCC4 cells, which express both HIF1α and HIF2α. A 24-h treatment with 20 μm rapamycin partially suppressed the expression of HIF2α in both 786-O and RCC4 cells, but completely suppressed the expression of HIF1α in RCC4 cells (Fig. 1A). Phosphorylation of the mTORC1 substrate S6K was also evaluated to ensure that rapamycin was able to suppress mTORC1. We performed a shorter term kinetic analysis of the effect of rapamycin on HIF1α and HIF2α in the RCC4 cells, in which both HIF1α and HIF2α are expressed, and as shown in Fig. 1B, suppression of HIF1α could be detected by 2 h, whereas there was no detectable drop in HIF2α levels by 8 h. A rapamycin dose-response experiment for HIF2α expression in 786-O cells (Fig. 1C) and for HIF1α and HIF2α expression in RCC4 cells (Fig. 1D) was also performed. The sensitivity of HIF1α corresponded to the sensitivity of S6K phosphorylation, with an IC₅₀ of between 20 and 200 nm, corresponding to an effect of rapamycin on mTORC1, which phosphorylates S6K. These data reveal that despite a similar dependence of HIF1α and HIF2α on PLD activity, there was a clear difference in the sensitivity of HIF1α and HIF2α to rapamycin.

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FIGURE 1.

Differential effect of rapamycin on HIF1α and HIF2α.A, 786-O and RCC4 cells were plated at 80% confluence for 24 h in medium containing 10% serum. Cells were then shifted to medium without serum. Rapamycin (Rap) was added at 20 μm, and the levels of HIF1α (RCC4 cells only), HIF2α, phosphorylated S6K (P-S6K), and actin were determined by Western blot analysis 18 h later. B, RCC4 cells were plated and then shifted to medium without serum as described for A. Rapamycin was added at 20 μm, and the levels of HIF1α and HIF2α were determined at the times indicated. C and D, 786-O and RCC4 cells, respectively, were plated at 80% confluence for 24 h in medium containing 10% serum. Cells were then shifted to medium without serum. Rapamycin was added at the indicated concentrations, and the levels of HIF1α, HIF2α, phosphorylated S6K, and S6K were determined by Western blot analysis 18 h later. All data shown are representative of at least three independent experiments.

Sensitivity of HIF1α and HIF2α to Suppressed Expression of mTOR—The partial sensitivity of HIF2α expression upon long-term rapamycin treatment is consistent with recent reports from Sabatini and co-workers (30, 31) indicating that mTORC2 can be suppressed by long-term rapamycin treatment by preventing the formation of a complex between mTOR, Rictor, and other components of mTORC2. To begin to determine whether the partial sensitivity of HIF2α to rapamycin is due to an mTORC2 requirement, we examined the effect of suppressing mTOR expression using siRNA for mTOR. In the 786-O cells, siRNA for mTOR strongly suppressed the expression of mTOR and, notably, also suppressed the expression of HIF2α (Fig. 2A). In the RCC4 cells, siRNA for mTOR suppressed the levels of both HIF1α and HIF2α (Fig. 2B). Depleting cells of mTOR also suppressed phosphorylation of the mTORC1 substrate S6K and phosphorylation of Akt at the mTORC2 site at Ser⁽Fig. 2, A and B) (30). These data indicate that although HIF1α is more sensitive to rapamycin compared with HIF2α, both HIF1α and HIF2α are dependent upon the expression of mTOR.

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FIGURE 2.

Both HIF1α and HIF2α are sensitive to suppression of mTOR expression.A, 786-O cells were plated at 30% confluence. 24 h later, the cells were transfected with mTOR siRNA or a scrambled siRNA as indicated. 24 h later, the cells were treated with fresh medium containing 10% serum for an additional 48 h. The control cells were treated with transfection medium, but without the transfection. The cells were then harvested and analyzed for levels of HIF2α, mTOR, Akt phosphorylated at Ser⁽P-Akt S⁴⁷³), Akt, phosphorylated S6K (P-S6K), and S6K by Western blotting as described in the legend to Fig. 1. B, RCC4 cells were prepared and transfected with mTOR siRNA as described for A. The cells were evaluated by Western blot analysis as described for A except that HIF1α levels were also evaluated. All data shown are representative of at least three independent experiments.

Dependence of HIF1α and HIF2α Expression on Raptor and Rictor—The data showing a differential sensitivity of HIF1α and HIF2α to rapamycin but equal sensitivity to mTOR depletion could be explained if HIF2α were dependent upon mTORC2, which is relatively resistant to rapamycin, and HIF1α were dependent upon mTORC1. To test this hypothesis, the 786-O and RCC4 cells were depleted of Raptor, which is a component of mTORC1, and Rictor, which is a component of mTORC2. As shown in Fig. 3A, HIF2α expression in 786-O cells was sensitive to the depletion of Rictor, but not Raptor. These data are consistent with a dependence of HIF2α expression on mTORC2, but not mTORC1. We also examined the effect of depleting the expression of Raptor and Rictor on HIF1α and HIF2α in RCC4 cells, and as shown in Fig. 3B, suppression of Rictor, but not Raptor, suppressed the expression of HIF2α, as was observed in the 786-O cells. The expression of HIF1α was dependent upon both Raptor and Rictor (Fig. 3B). Consistent with the roles of Raptor and Rictor in regulating mTORC1 and mTORC2, respectively, suppression of Raptor reduced S6K phosphorylation at the mTORC1 site at Thr^{, and suppression of Rictor reduced Akt phosphorylation at the mTORC2 site at Ser}^{. These data indicate that the expression of HIF1α is dependent upon both mTORC1 and mTORC2, whereas the expression of HIF2α is dependent only upon mTORC2. Thus, the differential sensitivity of HIF1α and HIF2α to rapamycin is due to a differential dependence on mTORC1 and mTORC2.}

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FIGURE 3.

Dependence of HIF1α and HIF2α expression on Raptor and Rictor.A, 786-O cells were plated as described in the legend to Fig. 2. 24 h later, the cells were transfected with Raptor or Rictor siRNA or a scrambled siRNA as indicated. 24 h later, the cells were treated with fresh medium containing 10% serum for an additional 48 h. The cells were then harvested and analyzed for levels of HIF2α, mTOR, Akt phosphorylated at Ser⁽P-Akt S⁴⁷³), Akt, phosphorylated S6K (P-S6K), and S6K by Western blotting as described in the legend to Fig. 2. B, RCC4 cells were prepared and transfected with Raptor and Rictor siRNAs as described for A. The cells were evaluated by Western blot analysis as described for A except that HIF1α levels were also evaluated. All data shown are representative of at least three independent experiments.

Dependence of HIF1α and HIF2α on Akt—The data in Fig. 3 indicate that both HIF1α and HIF2α are dependent on mTORC2. It is becoming apparent that a key target of mTORC2 is Akt, which is phosphorylated at Ser⁽30). Consistent with the previous reports, suppression of Rictor, but not Raptor, suppressed Akt phosphorylation at Ser⁽Fig. 3). We therefore examined whether the expression of either HIF1α or HIF2α is dependent on Akt expression. There are three Akt isoforms, Akt1, Akt2, and Akt3. Akt1-deficient mice have developmental defects, Akt2-deficient mice have defects in glucose homeostasis, and Akt3-deficient mice have defects in brain development (33). The 786-O cells were treated with siRNAs for the Akt isoforms, and the levels of HIF2α were evaluated by Western blotting. As shown in Fig. 4A, depleting cells of Akt2, but not Akt1, abolished HIF2α expression. There was no detectable expression of Akt3 in the 786-O cells (Fig. 4A, lower panel). We also examined the effect of depleting cells of the Akt isoforms in the RCC4 cells, which express both HIF1α and HIF2α. In contract with the 786-O cells, the RCC4 cells expressed all three Akt isoforms. As shown in Fig. 4B, depleting cells of Akt2, but not Akt1 or Akt3, suppressed the expression of HIF2α, as was observed in the 786-O cells. Suppression of either Akt1 or Akt2 had no effect on HIF1α expression; however, suppression of Akt3 completely abolished HIF1α in the RCC4 cells (Fig. 4B, lower panel). These data indicate that Akt2 is a critical downstream target of mTORC2 for HIF2α expression and that Akt3 is a critical downstream target of mTORC2 for HIF1α expression.

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FIGURE 4.

Dependence of HIFα expression on Akt. 786-O (A) and RCC4 (B) cells were plated as described in the legend to Fig. 2. 24 h later, the cells were transfected with Akt1, Akt2, or Akt3 siRNA or a scrambled siRNA as indicated. 24 h later, the cells were treated with fresh medium containing 10% serum for an additional 48 h. The cells were then harvested and analyzed for levels of HIF1α, HIF2α, Akt1, Akt2, and Akt3 by Western blotting as described in the legend Fig. 2. The data shown are representative of two independent experiments.

DISCUSSION

We previously reported that the expression of both HIF1α and HIF2α is dependent on PLD activity (17). The PLD metabolite phosphatidic acid has been implicated in the activation of mTOR (26, 32). However, there is a clear differential sensitivity of HIF1α and HIF2α to rapamycin. In this work, we have demonstrated that although there is a differential sensitivity of HIF1α and HIF2α to rapamycin, both HIF1α and HIF2α are dependent upon mTOR. This observation is explained by a dependence of HIF2α expression on the rapamycin-resistant mTORC2, whereas there is a dependence of HIF1α expression on both mTORC1 and mTORC2. This study reveals an mTORC2 requirement for the expression of HIF2α.

Akt2, which is a downstream target of mTORC2, is also required for the expression of HIF2α. Of interest here is that Akt2 has been implicated in the regulation of glycolysis (33), as has HIF2α (10). HIF1α, which is also dependent on mTORC2, is dependent on Akt3, which has been implicated in melanoma and ovarian cancer (34, 35). At this point, it is difficult to determine the link between Akt and HIFα expression in that the regulation is very complicated (33). The impact of phosphorylating Akt at Ser^{by mTORC2 is not clear. Although this phosphorylation leads to increased kinase activity (}36), suppressing mTORC2 activity prevents phosphorylation of the Akt substrate FOXO, but does not prevent phosphorylation of the tuberous sclerosis complex (37, 38). Thus, it has been speculated that phosphorylation of Akt by mTORC2 may influence substrate specificity. Thus, the data presented here only implicate Akt2 and Akt3 in regulating the expression of HIF2α and HIF1α, respectively. The data do not indicate how this is accomplished.

Although there is a well established connection between PLD activity and mTORC1 (26, 32), there has been no connection between PLD activity and mTORC2. Because PLD activity is required for the expression of HIF2α (17), which is dependent on mTORC2, this study also suggests that PLD is required for the activation of mTORC2 as well as mTORC1, and we now have preliminary data indicating that the PLD metabolite phosphatidic acid is required for the assembly of active mTORC1 and mTORC2 complexes with Raptor and Rictor, respectively.³ Thus, the PLD requirement for the expression of HIF2α reported previously (17) and the dependence of HIF2α expression on mTORC2 reported here are consistent with our preliminary study indicating a role for PLD in the activation of mTORC2.

The dependence of HIF2α on mTORC2 is significant in that HIF2α expression has been shown to be critical for tumorigenesis (11, 12). Rapamycin and rapamycin derivatives have been widely employed in clinical trials, with mostly disappointing results (25). A recent clinical study (39) focused on glioblastoma, in which there are commonly defects in PTEN. Defective PTEN increases signals that lead to increased activation of mTORC1, but a role for PTEN in the regulation of mTORC2 is not clear (27). This clinical study indicated that there is cell cycle arrest in response to rapamycin as well as effects on S6K phosphorylation, implicating mTORC1. However, mTORC1 is much more sensitive to rapamycin compared with mTORC2. As described here, the expression of HIF2α in renal cancer cells is dependent on mTORC2, which is much more resistant to rapamycin compared with mTORC1. Although suppression of mTORC1 with rapamycin is achievable, it may be that to effectively target mTOR in cancer, targeting mTORC2 may be more important because HIF2α is likely more critical (11, 12). It will be important to develop strategies that can suppress mTORC2 and HIF2α. PLD is required for the expression of both HIF1α and HIF2α (17), suggesting that PLD is required for the activation of both mTORC1 and mTORC2. Thus, targeting PLD or the signals that activate PLD may represent a viable therapeutic strategy for suppressing HIF2α in RCC and other cancers in which HIF2α has been implicated. The dependence of HIF2α on mTORC2 indicates that targeting mTORC1 with rapamycin will likely have limited therapeutic effects given the apparent significance of HIF2α in RCC.

^{Department of Biological Sciences, Hunter College, The City University of New York, New York, New York 10021 and the}^{Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Ontario M5S 1A8, Canada}

^{To who correspondence should be addressed: Dept. of Biological Sciences, Hunter College, The City University of New York, 695 Park Ave., New York, NY 10021. Tel.: 212-772-4075; Fax: 212-772-5227; E-mail:}ude.ynuc.retnuh.rtceneg@retsof.

Received 2008 Aug 25; Revised 2008 Oct 21

Abstract

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1α and HIF2α has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2α expression is the more important contributor. Expression of HIF1α is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2α expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1α and HIF2α to rapamycin, both HIF1α and HIF2α expression is dependent on mTOR. HIF1α expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2α was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1α is dependent on both mTORC1 and mTORC2, HIF2α is dependent only on mTORC2. We also examined the dependence of HIFα expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2α was dependent on Akt2, whereas that of HIF1α was dependent on Akt3. Because HIF2α is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2α has been implicated.

Abstract

Hypoxia-inducible factor (HIF)² is a critical transcriptional regulator of cellular responses to a variety of stressful conditions (1, 2). Under non-stressful conditions, HIFα is ubiquitinated by the von Hippel-Lindau (VHL) gene product pVHL, a substrate-conferring component of a ubiquitin-protein isopeptide ligase that targets HIFα for degradation by the proteasome (3). Loss of the VHL gene results in a variety of pathologies, most significantly renal cell carcinoma (RCC) (4–6). In the absence of pVHL, there is an up-regulation of HIFα, and elevated expression of HIFα has been strongly implicated in VHL disease and RCC (4–6). HIFα dimerizes with HIFβ to form a transcription factor HIF that stimulates the transcription of genes that regulate angiogenesis and other factors important for responding to hypoxic and other stressful conditions such as vascular endothelial growth factor and glycolytic enzymes (2, 7, 8). There are several distinct α-subunits, but it is the expression of HIF1α and HIF2α that is most frequently elevated in human cancers (4, 9). Whereas HIF1α has both pro- and anti-proliferative properties, HIF2α lacks the anti-proliferative properties and is more strongly implicated in tumorigenesis (10). The somewhat overlapping and antagonistic effects of HIF1α and HIF2α are poorly understood, but it is clear that in RCC, HIF2α is a critical factor in that suppression of HIF2α blocks tumor formation by renal cancer cells (11, 12). It is believed that the elevated expression of HIF2α contributes to the survival signals in renal cancer cells that protect against apoptosis and facilitate angiogenesis (10).

There have been several reports that HIF1α is sensitive to rapamycin (13–16), indicating that HIF1α expression is dependent upon mTORC1. In contrast, HIF2α expression has not been linked to either mTORC1 or mTORC2. We recently reported that elevated expression of both HIF1α and HIF2α in VHL-deficient RCC cell lines is dependent on phospholipase D (PLD) (17). Like HIFα, PLD has been implicated in stress responses (18) and has been shown to provide a survival signal in several human cancer cell lines (17–21). Notably, the PLD metabolite phosphatidic acid has been reported to interact with the mammalian target of rapamycin (mTOR) in a manner that is competitive with rapamycin in association with FKBP12 (FK506-binding protein-12) (23, 24). Consistent with reports that suppression of HIF2α blocks tumor formation by renal cancer cells (11, 12), suppression of PLD activity in RCC cell lines leads to apoptosis when the cells are deprived of serum (17). Thus, like HIF2α, PLD is able to provide a “survival signal” that suppresses apoptosis in RCC cells.

A common node for survival signals in cancer cells is mTOR (25–28). mTOR exists in two distinct complexes, mTORC1 and mTORC2 (27, 28), that differ in their subunit composition and sensitivity to rapamycin. mTORC1 consists of a complex that includes mTOR and a protein known as Raptor (regulatory-associated protein of mTOR), whereas mTORC2 consists of a complex that includes mTOR and a protein known as Rictor (rapamycin-insensitive companion of mTOR) (27). Although there have been several reports linking mTOR and HIF1α expression, there has been no link made between mTOR and HIF2α expression. The link between mTOR and HIF1α is based largely on the sensitivity of HIF1α to rapamycin (13–26). mTORC1 is highly sensitive to rapamycin, whereas mTORC2 is relatively insensitive to rapamycin (28). However, it was recently reported that long-term exposure to rapamycin prevents the formation of mTORC2 complexes and blocks the phosphorylation of the mTORC2 substrate Akt at Ser⁽29–31).

We report here that HIF2α expression is dependent on mTORC2, whereas HIF1α expression is dependent upon both mTORC1 and mTORC2. Because HIF2α expression is critical for RCC tumorigenesis, this study indicates that targeting mTORC2 signals represents a viable therapeutic strategy in RCC and perhaps other cancers in which HIF2α is critical for tumorigenesis.

Notes

^{This work was supported, in whole or in part, by National Institutes of Health Grant}CA46677 from NCI and SCORE Grant GM60654 (to D. A. F.) and by Research Centers in Minority Institutions Award RR-03037 from the National Center for Research Resources of the National Institutes of Health, which supports infrastructure and instrumentation in the Department of Biological Sciences at Hunter College. This work was also supported by grants from the Canadian Cancer Society of the National Cancer Institute of Canada (to M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Notes

Footnotes

^{The abbreviations used are: HIF, hypoxia-inducible factor; VHL, von Hippel-Lindau; RCC, renal cell carcinoma; PLD, phospholipase D; mTOR, mammalian target of rapamycin; siRNA, small interfering RNA; S6K, S6 kinase.}

^{A. Toschi, E. Lee, N. Gadir, M. Ohh, and D. A. Foster, manuscript submitted for publication.}

Footnotes

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