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. 2010 Mar 9;107(10):4675-80.
doi: 10.1073/pnas.0907705107. Epub 2010 Feb 22.

Negative feedback control of HIF-1 through REDD1-regulated ROS suppresses tumorigenesis

Peter Horak  1 , Andrew R Crawford, Douangsone D Vadysirisack, Zachary M Nash, M Phillip DeYoung, Dennis Sgroi, Leif W Ellisen
Affiliations

Negative feedback control of HIF-1 through REDD1-regulated ROS suppresses tumorigenesis

Peter Horak et al. Proc Natl Acad Sci U S A. .

Abstract

The HIF family of hypoxia-inducible transcription factors are key mediators of the physiologic response to hypoxia, whose dysregulation promotes tumorigenesis. One important HIF-1 effector is the REDD1 protein, which is induced by HIF-1 and which functions as an essential regulator of TOR complex 1 (TORC1) activity in Drosophila and mammalian cells. Here we demonstrate a negative feedback loop for regulation of HIF-1 by REDD1, which plays a key role in tumor suppression. Genetic loss of REDD1 dramatically increases HIF-1 levels and HIF-regulated target gene expression in vitro and confers tumorigenicity in vivo. Increased HIF-1 in REDD1(-/-) cells induces a shift to glycolytic metabolism and provides a growth advantage under hypoxic conditions, and HIF-1 knockdown abrogates this advantage and suppresses tumorigenesis. Surprisingly, however, HIF-1 up-regulation in REDD1(-/-) cells is largely independent of mTORC1 activity. Instead, loss of REDD1 induces HIF-1 stabilization and tumorigenesis through a reactive oxygen species (ROS) -dependent mechanism. REDD1(-/-) cells demonstrate a substantial elevation of mitochondrial ROS, and antioxidant treatment is sufficient to normalize HIF-1 levels and inhibit REDD1-dependent tumor formation. REDD1 likely functions as a direct regulator of mitochondrial metabolism, as endogenous REDD1 localizes to the mitochondria, and this localization is required for REDD1 to reduce ROS production. Finally, human primary breast cancers that have silenced REDD1 exhibit evidence of HIF activation. Together, these findings uncover a specific genetic mechanism for HIF induction through loss of REDD1. Furthermore, they define REDD1 as a key metabolic regulator that suppresses tumorigenesis through distinct effects on mTORC1 activity and mitochondrial function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
REDD1 loss confers tumorigenicity and activates a HIF transcriptional program. (A) REDD1−/− cells are tumorigenic. Immortalized MEFs were transduced with a control retroviral vector, and then injected (4 × 106 cells) into the flanks of nude mice (n = 6 per genotype). P value by repeated-measures ANOVA. (B) Relative overexpression of HIF-1α protein in tumorigenic REDD1−/− MEFs is not affected by rapamycin (50 nM, 24 h). (Upper) HIF-1α was detected by immunoprecipitation (IP)/western analysis under normoxia. Immunoglobulin heavy chain (HC) is shown as a loading control. (Lower) Western analysis for phosphorylated S6 (P-S6, S235/236) indicates mTORC1 activity. (C) Rapamycin (50 nM, 24 h) abolishes elevated HIF-1α expression in TSC2−/− MEFs. Cells were treated under hypoxia (1% O2) before IP/western analysis. (D) Induction of HIF-1α target genes in REDD1−/− MEFs described in A, assessed by quantitative real-time RT-PCR (qRT-PCR). (E) Increased HIF-1α and HIF-2α in REDD1−/− immortalized primary keratinocytes exposed to hypoxia (1% O2, 4 h), followed by IP/Western (Upper) or Western (Lower) analysis. (F) Increased glucose uptake in REDD1−/− MEFs. Cells were cultured for 72 h in normoxia or hypoxia (1% O2), followed by direct measurement of residual glucose in the medium (***P < 0.001; **P < 0.01 by Student's t test). (G) Increased lactate production in REDD1−/− MEFs cultured as in F (***P < 0.001).
Fig. 2.
Fig. 2.
HIF-1α drives tumorigenesis induced by loss of REDD1. (A) Knockdown of HIF-1α in tumorigenic REDD1−/− MEFs by lentiviral HIF-1α-directed shRNA (shHIF-1α) or control vector (V), assessed by IP/Western analysis. Right lanes: HIF-1α levels postknockdown approximate those in wild-type cells (compare lanes 3 and 4, 5 and 6). Ig heavy chain (HC) serves as a loading control. (B) HIF-1α knockdown decreases proliferation of REDD1−/− cells under hypoxia. Equal numbers of the indicated REDD1−/− MEFs were plated in normoxia or hypoxia (1% O2), and cell numbers were determined at the times shown (***P < 0.001; **P < 0.01). (C) HIF-1α knockdown inhibits tumorigenesis of REDD1−/− cells. Vector or HIF-1α knockdown (shHIF-1α) REDD1−/− MEFs (4 × 106 cells) were injected into opposite flanks of nude mice (n = 6). P value by repeated-measures ANOVA. (D) Representative photograph, day 30. (E) Down-regulation of HIF-1α and its target genes by HIF-1α knockdown in REDD1−/− tumors. Equal microgram amounts of RNA from tumors generated in C were pooled for analysis by qRT-PCR (**P < 0.01). (F) Up-regulation of Glut-1 in primary human tumors that have silenced REDD1. (Upper) Frozen primary microdissected breast cancer specimens were analyzed for REDD1 and Glut-1 mRNA by qRT-PCR, and the mean Glut-1 expression was compared in tumors with silenced versus nonsilenced REDD1 (see text and Methods). P value by Student's t test. (Lower) RNA in situ hybridization for REDD1 (α-sense probe, blue staining) in representative breast tumors. Sense probe is shown as a specificity control.
Fig. 3.
Fig. 3.
REDD1 regulates HIF-1α stability. (A) REDD1 reconstitution down-regulates HIF-1α target genes. Tumorigenic REDD1−/− MEFs were infected with a retrovirus expressing REDD1 or a control vector, and then exposed to 1% O2 (4 h); RNA analysis was carried out by qRT-PCR (***P < 0.001; **P < 0.01). (B) HIF-1α synthesis is not affected in REDD1−/− cells. Wild-type or matched tumorigenic REDD1−/− MEFs were pulse-labeled with 35S (1 h, 1% O2) followed by IP for HIF-1α. (C) Proteasome inhibition demonstrates increased baseline HIF-1α stability in REDD1−/− cells (compare lanes 1 and 2, 5 and 6). Cells were untreated (U) or treated with MG132 (100 μM) followed by IP/Western analysis. Loading control (cont.) is total AKT. (D) Deferoxamine (DFO) treatment (150 μM, 4 h) preferentially affects unstable HIF-1α in wild-type versus REDD1−/− cells. (E) HIF-1α half-life is prolonged in REDD1−/− cells. Cells were treated with cycloheximide (CHX; 10 μg/mL) under normoxia for the indicated times before IP/Western analysis.
Fig. 4.
Fig. 4.
Increased mitochondrial ROS induced by loss of REDD1. (A) Increased mitochondrial ROS in REDD1−/− cells. Mitochondria were isolated from matched wild-type or tumorigenic REDD1−/− cells, and ROS were measured by relative fluorescence units (RFU) after staining for superoxide (O2 −) with dihydroethidium (DHE; Left) or for peroxide (H2O2) with Amplex Red (Right) as described in Methods (***P < 0.001; **P < 0.01). (B) Increased ROS (DHE) in whole REDD1−/− versus wild-type cells (***P < 0.001). (C) Retroviral REDD1 reconstitution reduces ROS (DHE) in REDD1−/− cells (***P < 0.001). (D) Endogenous REDD1 is a mitochondrial protein. Cellular fractionation of 293T cells was carried out before Western analysis for endogenous REDD1 in the cytosol (C) and mitochondria (M). (E) Confocal imaging demonstrates colocalization of REDD1 and mitochondria. REDD1−/− MEFs were reconstituted with control retroviral vector (pLPC) or REDD1, and then costained with anti-REDD1 antibody and MitoTracker Red (MITO). Arrows show colocalization. Nuclei were stained with Hoechst dye. (F) Reconstituted wild-type (WT) REDD1, but not the K3R3 mutant, exhibits physiological mitochondrial (M) localization. (G) Mitochondrial localization is required for REDD1-mediated suppression of ROS. Mitochondrial preparations shown in F were used for measurement of ROS (DHE) (***P < 0.001). (H) HIF-1α target gene suppression by REDD1 is associated with ROS suppression and mitochondrial localization. Relative mRNA expression was measured by qRT-PCR in triplicate samples (**P < 0.01; ***P < 0.001). Legend as shown in G.
Fig. 5.
Fig. 5.
Antioxidant treatment blocks HIF-1α and tumorigenesis induced by REDD1 loss. (A) Antioxidant treatment (ascorbate, 100 μM, 4 h) normalizes HIF-1α levels in tumorigenic REDD1−/− MEFs. (B) Antioxidant treatment (ascorbate, 100 μM) selectively inhibits proliferation of REDD1−/− MEFs under hypoxia (1% O2). Note little or no effect of ascorbate in wild-type cells (**P = 0.01). (C) Antioxidant treatment (ascorbate, 100 μM, 4 h) inhibits mitochondrial ROS, measured by relative fluorescence units (RFU) after staining with DHE (***P < 0.001). (D) Antioxidant treatment inhibits tumorigenic growth of REDD1−/− cells in vivo. Tumorigenic REDD1−/− MEFs (4 × 106 cells) were injected into nude mice treated with ascorbate (5 g/L) in drinking water. P value by repeated-measures ANOVA. (E) Schematic diagram of proposed roles for REDD1 in mTORC1-dependent and mitochondrial ROS-dependent tumor suppression. REDD1 loss induces mTORC1 dysregulation under hypoxia, which promotes tumorigenesis. REDD1 also plays a distinct role in mitochondria to control ROS production, as REDD1 loss induces mitochondrial ROS, which promotes HIF-1α stabilization and HIF-dependent metabolic adaptations that drive tumorigenesis.
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