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Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis

Abstract

Cancer cells gain a growth advantage through the so-called Warburg effect by shifting glucose metabolism from oxidative phosphorylation to aerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) has been suggested to function in metabolic reprogramming; however, the underlying mechanism has not been fully elucidated. We found that the aberrant expression of wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer, decreased α-ketoglutarate levels and increased the stability and transactivation activity of HIF-1α in cancer cells. The silencing of IDH3α significantly delayed tumor growth by suppressing the HIF-1-mediated Warburg effect and angiogenesis. IDH3α expression was associated with the poor postoperative overall survival of lung and breast cancer patients. These results justify the exploitation of IDH3 as a novel target for the diagnosis and treatment of cancers.

Introduction

The outcome of conventional therapies on cancer patients, such as chemotherapy, radiation therapy and surgery, are known to be affected by various intracellular and extracellular factors in malignant tumors. One of the most influential factors is the activity of the transcription factor, hypoxia-inducible factor 1 (HIF-1).1, 2 HIF-1 is a heterodimer that is composed of an α-subunit (HIF-1α) and β-subunit (HIF-1β), and its activity is mainly dependent on the stability and transactivating activity of HIF-1α, which are strictly regulated by the α-ketoglutarate (α-KG)-dependent enzymes, prolyl-4-hydroxylases (PHDs) and factor-inhibiting HIF-1, respectively.3 PHDs hydroxylate the proline residues, P402 and P564, in the oxygen-dependent degradation domain (ODD domain) of HIF-1α in both an oxygen- and α-KG-dependent manner.4 Prolyl-hydroxylation has been shown to trigger the polyubiquitilation and subsequent proteasomal degradation of HIF-1α. Factor-inhibiting HIF-1 hydroxylates the asparagine residue, N803, in the C-terminal transactivation domain of HIF-1α, resulting in the suppression of its transactivating activity under normoxic conditions.5 HIF-1α is stabilized under hypoxic conditions due to the inactivation of these hydroxylases,5 and then interacts with its binding partner, HIF-1β.6 The resulting heterodimer, HIF-1, binds to its cognate enhancer sequence, the hypoxia-responsive element (HRE), and induces the transcription of various downstream genes related to improving oxygen availability (angiogenesis), adapting cellular metabolism to hypoxia (metabolic reprogramming) and escaping from hypoxic regions (invasion and metastasis of cancer cells).1, 2, 7, 8 Clinical as well as basic research reported a correlation between HIF-1α expression levels and poor prognosis and the incidences of both tumor recurrence and distant tumor metastasis, which justified the targeting of HIF-1 and its upstream activators for cancer therapy.1, 2, 8 However, the molecular mechanisms underlying the activation of HIF-1 have not yet been fully elucidated, which makes developing strategies to eradicate cancers difficult.

In the present study, we demonstrated the importance of exploiting IDH3α as a novel target for the diagnosis and treatment of various types of cancers. We first developed a genetic screening strategy and successfully identified IDH3α as a novel upstream activator of HIF-1. IDH3α was shown to be aberrantly overexpressed in various cancers and functioned in the induction of both the HIF-1-mediated metabolic reprogramming of cancer cells (known as the Warburg effect) and HIF-1-mediated angiogenesis. Loss-of-function studies for IDH3α confirmed the importance of IDH3α for cancer therapy. IDH3α expression levels in human lung and breast cancers were found to be associated with the poor prognosis of these patients.

Results

Aberrant IDH3 activation upregulated HIF-1 activity

We established a new genetic screening method to explore novel genes responsible for the activation of HIF-1 (Supplementary Figure S1A). Using an artificial promoter composed of five repeats of HRE and a human CMV minimal promoter (5HRE promoter: herein designated 5HREp),9, 10 we constructed a plasmid expressing the blasticidin S-resistant gene (bsd) in a HIF-1-dependent manner (5HREp-bsd herein; Supplementary Figure S1A). NIH3T3 cells stably transfected with 5HREp-bsd (NIH3T3/5HREp-bsd) were sensitive to blasticidin S under normoxic conditions because of the insufficient expression of both HIF-1α and BSD (Supplementary Figure S1B). After introducing a human cDNA library into NIH3T3/5HREp-bsd cells, these cells were cultured in blasticidin S-containing medium under normoxic conditions with the expectation that some cDNAs would encode the upstream activators of HIF-1, induce BSD expression, and eventually produce blasticidin S-resistant colonies (Supplementary Figure S1A). Through such a gene screening experiment, we acquired several surviving colonies with higher blasticidin S-resistance than their parental cells. We rescued cDNA from the genomic DNA of one of the colonies by PCR and identified it as wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer composed of two α, one β, and one γ subunits. We performed a luciferase assay using the 5HREp-luc reporter gene, which expressed luciferase under the control of 5HREp9, 10 to examine whether the forced expression of IDH3α induced HIF-1 activity. The overexpression of IDH3α resulted in the upregulation of HIF-1 activity in various cancer cell lines and in the blasticidin S-resistant cells obtained through the screening under both normoxic and hypoxic conditions (Figures 1a and b; Supplementary Figure S1C). The knockdown of endogenous IDH3α, which had no impact on the expression levels of other isoforms of isocitrate dehydrogenases such as IDH1 and IDH2 (Supplementary Figure S2), resulted in the significant suppression of HIF-1 activity regardless of oxygen availability (Figures 1c–e; Supplementary Figure S3). The forced overexpression or knockdown of IDH3α resulted in the significant induction of and reduction in the expression of various HIF-1-downstream genes, respectively (Figures 1f and g; Supplementary Figures S4 and S5). The IDH3α-mediated increase in luciferase bioluminescence from the 5HRE-luc reporter gene was not suppressed by the knockdown of endogenous HIF-2α, which indicated the predominant involvement of HIF-1 in our experimental setting (Supplementary Figure S6). Silencing of the IDH3β subunit significantly suppressed the positive impact of IDH3α overexpression on HIF-1 activity, which indicated that IDH3α functioned, at least in part, as a component of the IDH3 heterotetramer to activate HIF-1 (Figure 1h). Taken together, these results clearly demonstrated that IDH3α overexpression had the potential to positively regulate HIF-1 activity as a component of IDH3.

Figure 1
figure1

Aberrant IDH3 activation upregulated HIF-1 activity (a, b) HeLa/5HRE-Luc (a), A549/5HRE-Luc (b: left), and NCI-H441/5HRE-Luc (b: right) cells were transfected with pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (empty vector: EV), cultured under normoxic or hypoxic conditions for the indicated period, and then subjected to the luciferase assay. The plasmid, pRL-SV40, was used as an internal control to calculate relative HIF-1 activity. (c–e) HeLa (c), HeLa/5HRE-Luc (d), and A549/5HRE-Luc (e) cells were treated with scramble- (Scr) or IDH3α-siRNA (siIDH3α), cultured under normoxic or hypoxic conditions for 24 h, and were then subjected to western blotting using anti-IDH3α and anti-β-actin antibodies (c) and to the luciferase assay through the same method as a and b (d, e). (f, g) HeLa cells were transiently transfected with either pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (EV; f) or with either scramble- (Scr) or IDH3α-siRNA (siIDH3α; g), cultured under normoxic or hypoxic conditions for 24 h, and subjected to qRT-PCR to quantify the mRNA levels of VEGF-A (f left; g) and PDGF-β (f right) genes. (h) HeLa/5HRE-Luc cells were transiently transfected with either pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (EV) and with either Scr or IDH3β-siRNA (si3β-1 or 2), cultured under normoxic or hypoxic conditions for 48 h, and subjected to the luciferase assay through the same method as a and b. All results (except for c) are expressed as mean±s.d. n=3. *P<0.05. **P<0.01 (Student’s t-test).

IDH3 upregulated the stability and transactivating activity of HIF-1α

We next investigated the molecular mechanisms underlying the positive impact of IDH3α on HIF-1 activity. Because HIF-1 activity is known to be mainly regulated through posttranslational modifications and the resultant oxygen-dependent destabilization of the α-subunit (HIF-1α),4, 6, 11 we first examined the influence of IDH3α on HIF-1α protein levels. Immunoblotting clearly demonstrated that the expression of the HIF-1α protein was significantly increased after the overexpression of IDH3α under hypoxic conditions (Figure 2a). On the other hand, it was difficult to observe an increase in the protein under normoxic conditions because basal HIF-1α expression levels were below detectable levels under this experimental condition (Figure 2a). The induction of HIF-1α protein expression under low-oxygen conditions was markedly abrogated when cells were treated with small interfering RNA against the endogenous IDH3α gene (siIDH3α; Figure 2b). To determine how IDH3 increased HIF-1α protein levels, we constructed various kinds of reporter genes and performed luciferase assays. We first employed the SV40p-ODD-Luc reporter gene, in which the constitutively active SV40 promoter was responsible for the expression of a fusion protein composed of an ODD domain (HIF-1α 548–604 a.a.) and luciferase12 (Supplementary Figure S7A). As the stability of the fusion protein is known to be regulated through the same oxygen-dependent mechanism as the HIF-1α protein, we could monitor HIF-1α stability indirectly through luciferase activity. The luciferase assay showed that the overexpression and knockdown of IDH3α significantly increased and decreased luciferase activity from the reporter gene, respectively (Figures 2c and d; Supplementary Figure S7B). Moreover, IDH3α overexpression significantly prolonged the half-life of luciferase bioluminescence from the ODD-Luc fusion protein after the reoxygenation treatment in vitro (Figure 2e). These results suggested that IDH3 had the potential to stabilize the HIF-1α protein. Indeed, the overexpression of IDH3α resulted in the prolonged stability of the HIF-1α protein under reoxygenated conditions (Figure 2f). We then examined whether IDH3α influenced HIF-1α mRNA levels. A luciferase assay using the HIF-1α promoter-luc reporter gene13 and quantitative real-time PCR demonstrated that IDH3α overexpression did not influence the promoter activity or mRNA levels of HIF-1α (Figures 2g and h; Supplementary Figure S8). We next evaluated the influence of IDH3α on the translational initiation of the HIF-1α protein. We employed the HIF-1α 5 UTR-luc reporter gene, in which a pyrimidine tract of HIF-1α 5′UTR influenced the translational initiation of the luciferase gene,13 and confirmed that the forced expression of IDH3α had no impact on the initiation of HIF-1α translation (Figure 2i; Supplementary Figure S9). We then investigated the influence of IDH3α on the transactivating activity of the HIF-1α protein using a plasmid expressing the Gal4 DNA-binding domain (Gal4 DBD) fused to the HIF-1α transactivation domain (TAD: 531-826 a.a.; HIF-1αTAD).5 A proline residue corresponding to P564 of HIF-1α, which should have a role in the ODD of Gal4 DBD-HIF-1αTAD, was substituted for alanine (Gal4 DBD-HIF-1α TAD P564A; Supplementary Figure S10A) to purely quantify the transactivating activity of TAD. The overexpression of IDH3α significantly stimulated the transactivating activity of TAD P564A (Figure 2j). On the other hand, IDH3α-dependent activation was completely abrogated when the asparagine residue, N803, which is important for the factor-inhibiting HIF-1-mediated regulation of transactivating activity,5 was additionally substituted for alanine (Gal4 DBD-HIF-1αTAD P564A and N803A; Figure 2k; Supplementary Figure S10B). Taken together, these results revealed that the aberrant activation of IDH3 upregulated both the stability and transactivating activity of the HIF-1α protein, which resulted in the activation of HIF-1.

Figure 2
figure2

IDH3 upregulated the stability and transactivating activity of HIF-1α. (a– d) HeLa (a, b) and HeLa/ODD-Luc cells (c, d) were transiently transfected with pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (empty vector (EV); a, c), or treated with scramble- (Scr) or IDH3α-siRNA (siIDH3α; b, d), cultured under normoxic or hypoxic conditions for 24 h, and subjected to western blotting using anti-HIF-1α, anti-myc epitope tag (for IDH3α-myc), anti-IDH3α and anti-β-actin antibodies (a, b) and to the luciferase assay (c, d). The plasmid, pRL-SV40, was used in the luciferase assay to calculate relative ODD-Luc stability (c, d). Densitometric analysis of the band intensities of IDH3α (a; right). The same cell lysate as that in Figure 1c was used; therefore, the same images were repeatedly represented (b: IDH3α and β-actin). (e, f) HeLa/ODD-Luc (e) and HeLa (f) cells were transiently transfected with pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (EV), cultured under hypoxic conditions for 24 h, and treated with cyclohexamide (10 μg/ml) under normoxic conditions. After the reoxygenation treatment for the indicated period, cells were harvested for the luciferase assay (e) and for western blotting with anti-HIF-1α, anti-myc epitope tag (for IDH3α-myc) and anti-β-actin antibodies (f). The luciferase activities and signal intensities of western blotting at each time point were compared with those before the reoxygenation treatment to calculate the relative stabilities of ODD-Luc (e) and the HIF-1α protein (f; right), respectively. A representative image from three independent experiments is shown (f). (g– i) HeLa cells were transiently introduced with either pHIF-1α promoter-luc (g), none (h) or pGL3/HIF-1α5′UTR-luc (i), cultured under normoxic or hypoxic conditions for 24 h, and subjected to the luciferase assay with pRL-SV40 as an internal control (g, i) and to qRT-PCR for HIF-1α mRNA levels (h). (j, k) HeLa cells were transiently cotransfected with four kinds of plasmids: pG5H1bLuc containing five Gal4-binding sites upstream of an adenovirus E1b promoter and firefly luciferase CDS,5, 19 pRL-SV40 as an internal control for normalization, either pcDNA6/Gal4 DBD-HIF-1αTAD P564A (j) or pcDNA6/Gal4 DBD-HIF-1αTAD P564A & N803A (k), and either pcDNA4/IDH3α (IDH3α) or its EV. Cells were cultured under normoxic or hypoxic conditions for 24 h and subjected to the luciferase assay to evaluate the transactivation activity of HIF-1α TADs. All results (except for b and f) are expressed as means±s.d. n=3. *P<0.05. **P<0.01. NS, not significant (Student’s t-test).

Aberrant IDH3 activation reduced α-KG levels and suppressed hydroxylation of the HIF-1α protein in cancer cells

Based on a previous study in which the stability and transactivating activity of HIF-1α were shown to be regulated by the α-KG-dependent dioxygenases, PHDs and factor-inhibiting HIF-1, respectively,3 we hypothesized that IDH3 could sequentially affect α-KG levels and the extent of HIF-1α hydroxylation in cancer cells. Fluorometric assays revealed that the forced expression of IDH3α and silencing of endogenous IDH3α resulted in a significant decrease and increase in α-KG levels, respectively (Figures 3a and b; Supplementary Figure S11). A liquid chromatography-mass spectrometry (LC/MS)-based analysis consistently revealed that the silencing of endogenous IDH3α significantly increased α-KG levels (Figure 3c). Hydroxylation of the proline residue P564, which is dependent on both oxygen and PHDs and, therefore, is sensitive to the PHD inhibitor CoCl2 (Supplementary Figure S12), was markedly inhibited by the forced expression of IDH3α (Figure 3d). Taken together, these results indicated that the forced activation of IDH3 reduced the level of intracellular α-KG, suppressed the hydroxylation of the HIF-1α protein, and eventually upregulated its stability and transactivating activity in cancer cells. Consistent with this notion, we confirmed that the cell-permeable form of α-KG, octyl α-KG, abrogated the IDH3-mediated increases in the stability and transactivating activity of HIF-1α in the ODD-Luc assay and TAD assay, respectively (Supplementary Figure S13).

Figure 3
figure3

IDH3-mediated decrease in α-KG levels and HIF-1α hydroxylation in cancer cells. (a, b) HeLa cells were transiently transfected with pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (empty vector (EV); a), or treated with scramble- (Scr) or IDH3α-siRNA (siIDH3α; b), cultured under normoxic or hypoxic conditions for 24 h, and subjected to the fluorometric α-KG assay. (c) Total α-KG levels in HeLa/Scr and HeLa/shIDH3α cells were quantified by a LC/MS-based analysis. (d) HeLa cells were transiently transfected with pcDNA4/IDH3α (IDH3α) or pcDNA4/myc-His A (EV), treated with 30 μm MG132 for 6 h under normoxic conditions, and harvested for western blotting for hydroxy-HIF-1α (Pro564) and HIF-1α. All results (except for c) are expressed as means±s.d. n=3. *P<0.05; **P<0.01 (Student’s t-test).

IDH3-mediated activation of HIF-1 enhanced tumor growth

We then investigated whether the positive impact of IDH3α on the stabilization and activation of HIF-1α influenced tumor growth. We established stable transfectants of HeLa cells with the IDH3α expression vector (HeLa/IDH3α-1, 2, 3) or its empty vector (HeLa/EV-1, 2, 3; Figure 4a). The forced expression of the IDH3α-myc protein increased the stability of the ODD-fused protein and, therefore, induced HIF-1 activity in these stable transfectants (Figures 4b and c; Supplementary Figure S14). The overexpression of IDH3α-myc significantly accelerated the growth of HeLa/IDH3α xenograft tumors in immunodeficient mice (Figure 4d; Supplementary Figure S15); however, this positive effect was not very prominent, and may have been because the stable transfectants expressed particular levels of endogenous IDH3α (Figure 4a; Supplementary Figure S16). Thus, we performed loss-of-function studies by silencing endogenous IDH3α to directly examine the involvement of IDH3α in tumor growth. HeLa cells were stably transfected with plasmids expressing short hairpin RNAs against the IDH3α sequence (shIDH3α) and scramble sequence (Scr) to establish stable IDH3α knockdown cell lines (HeLa/shIDH3α cell lines) and their control counterparts (HeLa/Scr cell lines), respectively (Figure 4e). The knockdown of IDH3α markedly suppressed the induction of HIF-1α expression in HeLa/shIDH3α cells under hypoxic conditions (Figure 4e). Stable knockdown significantly delayed the growth of tumor xenografts in vivo (Figures 4f and g). The extent of the growth delay positively correlated with both the efficiency of IDH3α silencing and resultant extent of the decrease in HIF-1α expression (Figures 4e and g); thorough silencing of IDH3α almost completely inhibited the formation of tumor xenografts (see HeLa/shIDH3α-B1 and B2 in Figures 4e and g). We used an inhibitor of HIF-1, YC-1, to directly examine the involvement of HIF-1 in the promotion of tumor growth by IDH3α overexpression. Daily administrations with relatively low dose of YC-1 from days 0–2 significantly suppressed the IDH3α-mediated promotion of tumor growth (Figure 4h). These results collectively indicated that the IDH3α-mediated activation of HIF-1 in cancer cells enhanced the growth of malignant tumors, and, therefore, can be exploited as a rational target for cancer therapy.

Figure 4
figure4

IDH3-mediated activation of HIF-1 enhanced tumor growth. (a–c) HeLa/IDH3α-1, 2, 3 and HeLa/EV-1, 2, 3 cells were transiently transfected with a plasmid encoding the SV40p-ODD-luc (b) or 5HRE-luc (c) reporter gene, cultured under normoxic or hypoxic conditions for 24 h, and subjected to western blotting using anti-HIF-1α, anti-myc epitope tag (for IDH3α-myc), anti-IDH3α and anti-β-actin antibodies (a), and to the luciferase assay (b, c; n=3). The plasmid, pRL-SV40, was used as an internal control for the luciferase assay. Means±s.d. (d) Growth curves of tumor xenografts after transplanting HeLa/IDH3α-1, 2, 3 and HeLa/EV-1, 2, 3 cells into six nude mice per one cell line. Means±s.d. (e) HeLa/shIDH3α and HeLa/Scr cells were cultured under normoxic or hypoxic conditions for 24 h and subjected to western blotting using anti-IDH3α, anti-HIF-1α and anti-β-actin antibodies. (f, g) Growth curves of tumor xenografts after transplanting HeLa/shIDH3α and HeLa/Scr cell lines into six nude mice per one cell line. Means±s.d. (f). Representative images of tumor-bearing mice 46 days after transplantation (g). (h) Tumor-bearing mice with HeLa/EV or HeLa/IDH3α (six mice in each group) were treated with (+) or without (−) the HIF-1 inhibitor, YC-1 (30 mg/kg daily) between days 0 and 2. All results (except for a, e and g) are expressed as means±s.d. n=3. *P<0.05; **P<0.01 (Student’s t-test).

IDH3-mediated activation of HIF-1 was responsible for metabolic reprogramming and angiogenesis in cancer

Accumulating evidence has demonstrated that HIF-1 induces the transcription of various genes that function in the metabolic reprogramming of cancer cells from oxidative phosphorylation to aerobic glycolysis, and tumor angiogenesis. Therefore, we hypothesized that the aberrant expression of IDH3α may have a role in accelerated tumor growth by inducing HIF-1-mediated malignant phenotypes (Figure 4). To examine this possibility, we analyzed the involvement of IDH3α in the metabolic reprogramming of cancer cells. We treated HeLa/shRNA-Scr and HeLa/shIDH3α cells with 13C6-D-glucose for 10 min and then extracted metabolites for metabolome analysis using LC/MS (Figure 5a). 13C-labeled lactate (13C3-lactate) levels were markedly decreased, whereas 13C-labeled metabolite levels at the beginning of the tricarboxylic acid cycle, such as 13C2-citrate and 13C2-isocitrate, were markedly increased by the knockdown of the IDH3α gene. The same results were obtained in human lung adenocarcinoma cell line (Supplementary Figure S17). These results clearly showed that IDH3α overexpression was responsible for the reprogramming of glucose metabolism from oxidative phosphorylation to glycolysis in cancer cells. We then performed immunohistochemical analyses of tumor xenografts with an antibody against a marker of endothelial cells, CD31, to analyze the impact of IDH3α overexpression on tumor angiogenesis (Figures 5b and c). The knockdown of IDH3α resulted in a significant decrease in microvessel density, which suggested the important role of the IDH3-HIF-1 axis in tumor angiogenesis. Interestingly, IDH3α expression and HIF-1 activity were confirmed to be upregulated as a result of cellular immortalization and transformation by the human papilloma virus 18 E6 and E7 genes and K-Ras G12V gene, respectively (Supplementary Figure S18A). Moreover, the transformation-related increase in HIF-1 activity was significantly abrogated by the silencing of IDH3α in the transformed cells (Supplementary Figure S18B). Collectively, these results suggest that the aberrant activation of IDH3 accelerated tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis in cancers.

Figure 5
figure5

Induction of metabolic reprogramming and angiogenesis by the IDH3-HIF-1 pathway in cancers. (a) LC/MS-based metabolome analysis of the levels of the indicated metabolites after labeling HeLa/Scr and HeLa/shIDH3α cells with 13C6-D-glucose for 10 min. A representative data set is shown. (b, c) The tumor xenografts of HeLa/Scr and HeLa/shIDH3α were subjected to immunohistochemical staining with the anti-CD31 antibody (b), and microvessel density was quantified (c; six fields in each of the six representative xenografts, n=36). Results are expressed as means±s.d. n=3. *P<0.05. **P<0.01. NS, not significant (Student’s t-test).

Importance of IDH3α as a novel target for the diagnosis and treatment of cancers

We analyzed the relationship between IDH3α expression levels and prognosis in human lung and breast cancers using the PrognoScan database to validate our results in human tumors.14 We confirmed that IDH3α expression levels positively correlated with poor overall survival in various human cancers (Figures 6a–c). The RefEx analysis, which enables the expression levels of any genes of interest to be quantified in 10 major groups of normal tissues based on a public data set of microarrays (GeneChip), revealed that the expression levels of IDH3α in normal tissues were lower than those of VEGF-A, one of the most important target molecules for cancer therapy (Figures 6d–g). These results highlighted the importance of IDH3α expression levels as a prognostic marker as well as a therapeutic target.

Figure 6
figure6

Importance of IDH3α as a novel diagnostic marker and therapeutic target for cancers. (a–c) PrognoScan database14-based Kaplan–Meier analysis of the overall survival of 117 lung (a), 198 breast (b), and 159 breast (c) cancer patients after the removal of primary tumors stratified by high (red) and low (blue) IDH3α levels (a, high: n=29, low: n=88; b, high: n=61, low: n=137; and c, high: n=31, low: n=128). This analysis was based on the PrognoScan database (http://www.prognoscan.org/) using the publicly available Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) with the accession numbers GSE 1321329 (a), GSE 739030 (b), and GSE 145631 (c). (d– g) RefEx-based quantification of the mRNA expression levels of the VEGF-A (d, e) gene and IDH3α (f, g) gene in the indicated 10 major groups of normal tissues.

Discussion

In the present study, we identified IDH3α as an upstream activator of HIF-1. We found that IDH3α was aberrantly expressed in various cancer cells, and increased the stability and transactivating activity of HIF-1α by modulating α-KG levels as a component of the IDH3 heterotetramer. We also revealed that activation of the IDH3-HIF-1 pathway lead to the metabolic reprogramming of cancer cells and angiogenesis in malignant tumors. We confirmed that IDH3 blockade resulted in significant delays in tumor growth and also that IDH3α expression levels correlated with the poor prognosis of various cancer patients, which provides opportunities for the treatment and diagnosis of cancers.

IDHs are enzymes that were originally shown to catalyze the oxidative decarboxylation of isocitrate to α-KG.15 The NADP+-dependent isoforms, IDH1 and IDH2, have recently received considerable attention in basic and clinical cancer research as they are frequently mutated in multiple types of human cancers at their active sites (R132 of IDH1 and its corresponding amino acid R140, and R172 of IDH2) and promote tumorigenesis and neoplastic growth by producing the so-called oncometabolite, the (R)-enantiomer of 2-hydroxyglutarate (R-2HG)16, 17 from α-KG.16, 18 On the other hand, the function of the NAD+-dependent isoform, IDH3, has not yet been determined in malignant progression. Under such a background, the gene we screened as a novel HIF-1-activating enzyme in the present study was wild-type IDH3α. Although the reason for the functional difference among these isoforms remains unclear, it may be due to a difference in their electron acceptors, NADP+ and NAD+, or intracellular localization. Although we also raised the possibility that the overexpression of IDH3α upregulated both the stability and transactivating activity of HIF-1α by modulating α-KG levels, the mechanism underlying the change in α-KG levels has yet to be fully elucidated. Further studies, such as more detailed metabolome analysis, would help to address these questions.

Our metabolome analysis showed that the level of 13C2-α-KG converted from 13C6-glucose through glycolysis was unaffected by the knockdown of IDH3α (Figure 5a). On the other hand, both the fluorometric α-KG assay and LC/MS analysis demonstrated that total intracellular α-KG levels were increased by the silencing of IDH3α (Figures 3a and b). These results collectively suggested the existence of an IDH3-dependent and glycolysis-independent metabolic pathway that could influence α-KG levels in cancer cells. One of the possible processes is glutamine metabolism. Based on this point of view, it appears reasonable to assume that overexpression of IDH3α may trigger the sequential carboxylation of α-KG to isocitrate and then to citrate through the so-called reductive tricarboxylic acid cycle in order to provide substrates for the syntheses of lipids and phospholipids for the enhanced proliferation of cancer cells (Supplementary Figure S19). If this is the case, the antitumor effects of IDH3α blockade may be attributed not only to the inhibition of IDH3-HIF-1-dependent angiogenesis (Figures 5b and c), but also to perturbations in IDH3-HIF-1-mediated metabolic reprogramming (Figure 5a; Supplementary Figure S19).

Western blotting demonstrated that the basal expression level of IDH3α was high in various cancer cell lines. Retrospective microarray analyses also revealed that the increase observed in IDH3α expression levels was associated with the shorter overall survival of various cancer patients. These results strengthen the importance of IDH3α in the diagnosis of cancer patients. Moreover, data obtained from the RefEx analysis, in which the expression levels of IDH3α in normal tissues were lower than those of VEGF-A, indicated that the side effects associated with IDH3α-targeted therapy may be weaker than those of conventional anti-VEGF therapy. However, the molecular mechanisms responsible for the enhanced expression of IDH3α need to be elucidated in more detail before IDH3α can be exploited as a therapeutic target in the future. Answers to these questions should enable the development of novel strategies for cancer therapy.

Materials and methods

Cell culture and reagents

The mouse embryonic fibroblast cell line (NIH3T3), human cervical epithelial adenocarcinoma cell line (HeLa), human lung carcinoma cell line (A549) and human lung adenocarcinoma cell line (NCI-H441) were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in 10% fetal bovine serum-Dulbecco’s modified Eagle’s medium. Cells were incubated in a well-humidified incubator with 5% CO2 and 95% air at 37 °C for the normoxic incubation. Cells were incubated in the Bactron Anaerobic Chamber, BACLITE-2 (Sheldon Manufacturing, Cornelius, OR, USA) for the hypoxic incubation (<0.02% O2). A stock solution of cycloheximide (Nacalai Tesque, Kyoto, Japan) was prepared in dimethyl sulfoxide (DMSO) (10 mg/ml). Double strand RNAs for the transient silencing of IDH3α and IDH3β and also for the negative control were purchased from Invitrogen (Carlsbad, CA, USA; Cat number: 1299001-HSS105187-HSS105189, 1299001-HSS105190-HSS105192, and 12935-300, respectively).

Plasmid constructs

To construct pcDNA4/IDH3α, the cDNA encoding the human IDH3α gene was obtained from the human placenta cDNA library (Clontech, Mountain View, CA, USA; HL8000BB) and inserted between the EcoRI and XhoI sites of pcDNA4/myc-His A (Invitrogen). To construct pEF/SV40p-ODD-Luc, the DNA fragment encoding the SV40p-ODD-luc reporter gene was prepared by digesting pGL3/ODD-Luc12 with KpnI and XbaI, and was then inserted between the corresponding sites of pEF/myc/cyto (Invitrogen). To construct p5HRE-BSD, the luciferase coding sequence between the NcoI and XbaI sites of p5HRE-Luc1 was substituted for the bsd coding fragment. To construct pHIF-1α promoter-luc and pGL3/HIF-1α5′UTR-luc, the DNA fragments of the HIF-1α promoter (−572 to +32) and HIF-1α 5′UTR (+1 to +284) were amplified from the genomic DNA of HeLa cells and inserted between the BglII and HindIII sites and also between the HindIII and NcoI sites of the pGL3 promoter vector (Promega, Madison, WI, USA), respectively. To construct pcDNA6/Gal4 DBD-HIF-1αTAD P564A, which expresses a fusion protein of Gal4 DBD and HIF-1α TAD with the P564A mutation, a DNA fragment encoding Gal4 DBD prepared from the GalA expression vector5, 19 and that encoding HIF-1α CTAD P564A (HIF-1α 531–826 a.a. with P564A mutation) were inserted into the HindIII and EcoRI sites and between the EcoRI and XhoI sites of pcDNA6/V5-His A (Invitrogen), respectively. The N803A mutation was additionally introduced into pcDNA6/Gal4 DBD-HIF-1αTAD P564A to construct pcDNA6/Gal4 DBD-HIF-1αTAD P564A and N803A. The plasmid pG5H1bLuc, containing five Gal4-binding sites upstream of the adenovirus E1b promoter and firefly luciferase coding sequence (CDS) was described previously.5, 19

Stable transfectant

NIH3T3, HeLa, A549 or NIH-H441 cells were transfected with p5HRE-BSD (for NIH3T3/5HRE-BSD), pEF/SV40p-ODD-Luc (for HeLa/ODD-Luc), p5HRE-Luc9 (for A549/5HRE-Luc and NIH-H441/5HRE-Luc), pcDNA4/myc-His A (Invitrogen; for HeLa/EV), pcDNA4/IDH3α (for HeLa/IDH3α), the SureSilencing short hairpin RNA plasmid for human IDH3A (SABiosciences, Frederick, MD, USA, Cat number: KH15075P; for HeLa/shIDH3α), or the SureSilencing short hairpin RNA plasmid for the negative control (for HeLa/shRNA-Scr) by the calcium phosphate method.20 Cells were then cultured for 10–14 days in culture medium containing the corresponding antibiotic to select antibiotic-resistant stable transfectants. The resultant colonies were isolated and established as clones. Representative clones showing the expected and reasonable activities were used in the present study.

Functional screening of the upstream activators of HIF-1

HEK-293-based packaging cells, EcoPack2-293 (BD Biosciences, San Jose, CA, USA; 2 × 106 cells/φ100 mm dish), were transiently transfected with 20 μg of plasmids encoding the human placenta cDNA library (Clontech, HL8000BB) for virus production. NIH3T3/5HRE-BSD cells were infected with the resultant retroviruses, and cultured in the presence of 5 μg/ml blasticidin S for 10 days. DNA fragments encoding the potential activators of HIF-1 were rescued by PCR using 5′ and 3′ LIB primers (Clontech) from the genomic DNA of antibiotic-resistant colonies, and were then subjected to the sequencing analysis.

Luciferase assay and western blotting

Twenty-four hours after cells (1 × 104 cells per well in a 24-well plate) were transfected with the indicated plasmid(s), they were treated under normoxic or hypoxic conditions for the indicated period, and harvested in 100 μl passive lysis buffer (Promega) for the luciferase assay or 100 μl cell lytic buffer (Sigma, St Louis, MO, USA) for western blotting. The luciferase assay was performed using the dual luciferase assay kit (Promega) according to the manufacturer’s instructions. Western blotting was performed using anti-HIF-1α (BD Biosciences), anti-IDH3α (Abcam, Cambridge, UK), anti-myc epitope tag (Cell Signaling, Danvers, MA, USA), anti-hydroxy-HIF-1α (Pro564; Cell Signaling) and anti-β-actin (BioVision Research Products, Mountain View, CA, USA) antibodies as the primary antibodies, anti-mouse and anti-rabbit Immunoglobulin G horseradish peroxidase-linked whole antibodies (GE Healthcare Bioscience, Piscataway, NJ, USA) as the secondary antibodies, and the ECL-PLUS system (GE Healthcare Bioscience) according to the manufacturer’s instructions.

Quantitative real-time PCR

After cells (2 × 105 cells per well in a six-well plate) were treated as described in the Figure legends, total RNA was extracted using the Fast-Pure RNA Kit (Takara Bio Inc., Otsu, Japan) according to the manufacturer’s instructions. RNA (1 μg) was then subjected to reverse transcription using RNA LA PCR Kit (AMV) version 1.1 (Takara Bio). The mRNA level of the indicated gene was quantified by the quantitative real-time PCR technique using the Thermal Cycler Dice Real Time System (TP-800; Takara Bio) with the SYBR Premix Ex Taq kit (Takara Bio) and commercial primers (TaKaRa Primer Set ID HA074624 for HIF-1α mRNA, HA160790 for VEGF-A mRNA, HA154269 for PDGF-β mRNA, HA147875 for IDH3α, HA202978 for IDH1, HA128098 for IDH2, HA176356 for CA9, HA035991 for FGF2 and HA142949 for LDHA) according to the manufacturer’s instructions.

Fluorometric α-KG assay

Twenty-four hours after cells (2 × 106 cells per well in a six-well plate) were transfected with the indicated small interfering RNA, they were cultured under normoxic or hypoxic conditions for an additional 24 h. Cell lysates were harvested in conventional 500 μl NP-40 cell lysis buffer, de-proteinized using the Deproteinizing Sample Preparation kit (BioVision), and subjected to the α-Ketoglutarate Colorimetric/Fluorometric Assay Kit (BioVision) according to the manufacturer’s instruction.

Growth delay assay

Cancer cell suspensions (100 μl of 3 × 105 cells/ml in phosphate-buffered saline) were subcutaneously transplanted into the right hind legs of athymic nude mice (BALB/c nu/nu; Japan SLC. Inc., Hamamatsu, Japan). The tumor volume was calculated as 0.5 × length × width2, and compared with the initial value to calculate the relative tumor volume. Representative images of tumor-bearing mice were taken with the IVIS Spectrum in vivo imaging system (Caliper, Alameda, CA, USA) as described previously.9, 21, 22, 23, 24, 25

LC/MS-based metabolome analysis

Cancer cells were labeled with 13C6-D-glucose for 10 min by exchanging with a glucose-free complete culture medium supplemented with labeled glucose at 3.0 g/l. Metabolites were then extracted as described previously26 and analyzed using an LC/MS (Thermo Scientific, Waltham, MA, USA), as described previously.27 Intracellular levels of metabolites were calculated as nmol/g protein.

Immunohistochemical analysis

Frozen sections of tumor xenografts were treated with the purified rat anti-mouse CD31 antibody (BD Pharmingen, San Diego, CA, USA; × 100 dilution) and Alexa Fluor 594 goat anti-rat IgG (Invitrogen, × 2000 dilution) as described previously.28

Prognoscan analysis

The relationship between IDH3α expression levels and the overall survival of lung and breast cancer patients after the removal of primary tumors was evaluated by the minimum P-value approach using the PrognoScan database.14 Briefly, patients were divided into two groups according to IDH3α expression levels in their tumors at all possible cutoff points. The risk differences of any two groups were then calculated by the log-rank test. The cutoff point giving the most significant P-value was selected and demonstrated in the present study.

RefEx analysis

IDH3α and VEGF-A mRNA expression levels in 10 major groups of normal tissues: the brain, blood, connective tissue, reproductive tissue, muscle, digestive tract, liver, lung, kidney and urinary tract, were quantitatively analyzed using RefEx (Reference Expression data set; http://refex.dbcls.jp/) based on microarray data sets for humans. Please see http://refex.dbcls.jp/gene_info.php?lang=en&db=human&geneID=3419&refseq=NM_005530&unigene=Hs.591110&probe=202069_s_at for the expression levels of IDH3α mRNA and http://refex.dbcls.jp/gene_info.php?lang=en&db=human&geneID=7422&refseq=NM_001025368&unigene=Hs.73793&probe=210512_s_at for those of VEGF-A.

Statistical analyses

The significance of differences was determined using the Student’s t-test. A P-value<0.05 was considered to be significant.

Ethics of animal experiments

All animal experiments were approved by the Animal Research Committee of Kyoto University, and performed according to guidelines governing animal care in Japan.

Accession codes

Accessions

Gene Expression Omnibus

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Acknowledgements

We thank Dr M Suematsu, Dr YA Minamishima, and Dr N Hayakawa for critical discussion. This study was supported by the Funding Program for NEXT Generation World-Leading Researchers (NEXT Program) from the Japan Society for the Promotion of Science (JSPS), Japan to HH (No. LS071), by the program for Precursory Research for Embryonic Science and Technology (PRESTO) from Japan Science and Technology Agency (JST) to HH, by the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to HH, and by Grants-in-Aids for Scientific Research (B) to HH (No. 26293276), for Scientific Research (C) to AM (No. 26461886), for challenging Exploratory Research to HH (No. 26670558) and to MH (No. 26670555), and for Young Scientists (B) to MK (No. 25861088), MY (No. 24791293) and SI (No. 24659563) from MEXT, Japan, by the Sagawa Foundation for the Promotion of Cancer Research to HH, by the Kobayashi Foundation for Cancer Research to HH, by the Takeda Science Foundation to HH, by the Mochida Memorial Foundation for Medical and Pharmaceutical Research to HH, and by the International Science and Technology Cooperation Project of China and Japan to ZL and HH (No. 2010DFA31900).

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Zeng, L., Morinibu, A., Kobayashi, M. et al. Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis. Oncogene 34, 4758–4766 (2015). https://doi.org/10.1038/onc.2014.411

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