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,
Ping Gao,1
Yen-Chun Liu,2
Gregg L. Semenza,1,3,4,5 and
Chi V. Dang1,2,3*
Division of Hematology, Department of Medicine,1 Graduate Program of Pathobiology,2 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,3 Institute for Cell Engineering,4 Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 212055
Received 14 March 2007/ Returned for modification 24 April 2007/ Accepted 24 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These HIF-1-mediated mechanisms are likely to be crucial for normal development where c-Myc is expressed normally and subjected to homeostatic regulation by HIF-1. However, in cancers where MYC is frequently dysregulated, it is not known whether these normal homeostatic mechanisms are bypassed, permitting the cooperation between dysregulated c-Myc and HIF-1. As such, we seek to determine here how dysregulated c-Myc might cooperate with HIF-1 to regulate genes that alter metabolism and recruit new blood vessels. In particular, we focused on HK2, PDK1, and VEGF, all of which have been previously shown to be HIF-1 targets as well as being bound or regulated by c-Myc (3, 16, 37, 38, 47, 54, 61, 77).
Among three isoforms (HIF-1 to -3), HIF-1 has been mainly implicated in tumor progression (20, 25, 69, 72, 74). HIF-1 is a heterodimeric transcription factor composed of two subunits, HIF-1
and HIF-1ß. Oxygen regulation of HIF-1 levels depends on the HIF-1 oxygen-dependent domain, which contains two prolyl residues (402 and 564), which are hydroxylated by prolyl hydroxylase in the presence of oxygen (4, 33, 34, 52, 70, 73). Recognition by the von Hippel-Lindau (VHL) protein targets hydroxylated HIF-1 for ubiquitination and subsequent proteasomal degradation (30, 56). Hence, in the tumor microenvironment, the activation of HIF-1 is critical for tumor cell survival and proliferation.
In contrast to HIF-1, which responds to hypoxia or specific signal transduction pathways, the c-Myc transcription factor is expressed in nonhypoxic proliferating normal cells and is frequently dysregulated in human cancers. The transcriptional network of c-Myc, a basic helix-loop-helix transcription factor that heterodimerizes with Max, has been linked to many biological processes including development, cellular proliferation and growth, cell cycle, apoptosis, and energy metabolism (1, 8, 12, 27, 58). Oncogenic activation through dysregulated expression of c-Myc contributes to tumorigenesis of various tissues in transgenic mice and many types of human cancers including lymphomas (14, 32, 57, 65, 66). Little is known, however, about the role of dysregulated c-Myc expression, compared with its physiological expression, in HIF-1-mediated responses to hypoxia.
Here, we report the use of the human P493-6 B cells that conditionally overexpress human MYC upon tetracycline withdrawal (71). The P493-6 model recapitulates human Burkitt's lymphoma as well as responding to hypoxia in vitro and in vivo with hypoxic activation of HIF-1. Furthermore, HIF-2 is not expressed in the P493 system, and hence, this system is not confounded by another hypoxia-responsive factor (17). As such, we utilized the P493-6 system to study transcriptional regulation by HIF-1 and dysregulated c-Myc. In this study, we provide evidence that HIF-1 cooperates with dysregulated c-Myc to promote lymphomagenesis. Our study further demonstrates that c-Myc and HIF-1 enhance transcription of key switches of glucose metabolism such as HK2 and PDK1, which stimulate glycolysis. HK2 catalyzes the first step of glycolysis by phosphorylating glucose to form glucose-6-phosphate, which is further catabolized by glycolysis. PDK1, which is induced by hypoxia, phosphorylates and inactivates pyruvate dehydrogenase, which is required for the conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) (29). PDK1, therefore, inhibits mitochondrial respiration by limiting the availability of acetyl-CoA for mitochondrial oxidative phosphorylation (37, 61). We also demonstrate that induction of VEGF, a proangiogenic factor, is stimulated cooperatively by c-Myc and HIF-1. Our study uncovers the cooperation between dysregulated c-Myc and hypoxia-induced HIF-1 in promoting glucose metabolism and angiogenesis, which confer an adaptive advantage for tumor progression under the hypoxic tumor microenvironment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(CA5-HIF-1) or empty vector (EV) were generated by retroviral infections and selected with 800 µg/ml G418 as described previously (35). The CA5-HIF-1 mutant derived from adenoviral vector was inserted into retroviral vector pQCXIN (Clontech). P493-HIF-1 short hairpin RNA (shRNA) stably transduced cells were generated by retrovirus infection and G418 selection (800 µg/ml) as described previously (45). Transformed parental rat fibroblasts (TGR), cells null for Myc (HO15.19), and HO15.19 cells overexpressing human MYC (HO15.19-c-Myc) were maintained in high-glucose Dulbecco modified Eagle medium with 1 mM sodium pyruvate, 10% fetal bovine serum, and 1% streptomycin and penicillin. Incubation of P493-6 cells in 0.1 µg/ml tetracycline for 48 h led to significant repression of MYC. Removal of tetracycline induces MYC overexpression as described previously (38, 71). For HIF-1 induction, cells were exposed to hypoxia (0.1% O2 for P493-6 cells or 1% O2 for HO15.19 cells) or 100 µM CoCl2. Nonhypoxic cells (20% O2) were maintained at 37°C in a 5% CO2-95% air incubator. Hypoxic cells were maintained in a control atmosphere chamber (Plas-Labs) at 37°C. Oxygen tension was monitored with a calibrated Series 200 Percent oxygen analyzer (Alpha Omega Instruments).
ChIP assay.
The chromatin immunoprecipitation (ChIP) assay and real-time PCR quantification were performed as described previously (38). Briefly, P493-6 cells not treated or treated with tetracycline for 48 h under conditions of hypoxia (0.1% O2) or normoxia (20% O2) were cross-linked by formaldehyde. Fragmented chromatin was immunoprecipitated using rabbit polyclonal MYC (sc-764; Santa Cruz Biotechnology) and polyclonal human HIF-1 antibodies (NB100-134; Novus). Rabbit immunoglobulin G (IgG) antibody (Zymed) was used as a control. The total input was the supernatant from the no-antibody control (see Table S1 in the supplemental material for the primer sequences for the ChIP assay).
RNA interference. Short interfering RNAs (siRNAs) targeting human HK2 and human PDK1 were purchased from Dharmacon Research Inc. P493-6 cells (3 x 106) were electroporated with 100 nM siRNAs targeting PDK1 (5'-CUACAUGAGUCGCAUUUCAdTdT-3'), HK2 (siGENOME SMART POOL), or scrambled control siRNA (5'-CACGCUCGGUCAAAAGGUUdTdT-3') using the Amaxa Nucleofection Kit according to the manufacturer's instructions. On the following day, 5 x 105 viable cells were subjected to hypoxic exposure (0.1% O2) or CoCl2 (100 µM) treatment. At indicated times, cells were subjected to glucose uptake and immunoblot assays. Conditioned media were used for lactate measurement.
RNA analysis. Human HK2 and VEGF mRNA levels were determined by Northern blot analysis or quantitative real-time reverse transcription-PCR (RT-PCR). Total RNA was isolated from P493-6 cells using TRIzol (Invitrogen). Five micrograms of RNA was used in Northern blot analysis. RNA was subjected to 1.2% agarose gel electrophoresis and transferred to a nylon membrane (Nytran). The membrane was probed with human HK2, which was labeled with 32P using a random prime labeling kit (Stratagene). An ethidium bromide-stained agarose gel of 18S rRNA was used as a loading control. Quantitative real-time RT-PCR was performed using the TaqMan One Step RT-PCR Master Mix kit (PE Applied Biosystems) with probes and primers (see Table S2 in the supplemental material for the probe and primer sequences for the real-time RT-PCR). The expression level of human 18S rRNA was determined with a predeveloped mixture of TaqMan probe and primers (PE Applied Biosystems) and used for normalization. All PCRs were performed in triplicate.
Western blotting.
Equal amounts of protein extracted from cells or tumor xenografts were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels. Monoclonal anti-c-Myc (Oncogene Research Products), monoclonal anti-HIF-1 (Affinity Bioreagent), polyclonal anti-HK2 (Santa Cruz), polyclonal anti-PDK1 (Stressgen Bioreagents), anti-HK1 (Santa Cruz), polyclonal anti-enolase 1 (Santa Cruz), polyclonal anti-lactate dehydrogenase (anti-LDHA; Abcam), and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPD; Advanced ImmunoChemical Inc.) antibodies were used for immunoblotting. Monoclonal antiactin (Sigma), antitubulin (Calbiochem), and polyclonal anti-topoisomerase I (Santa Cruz) antibodies were used as loading controls. CoCl2-treated Cos7 nuclear extract was used for positive control for HIF-1 (active motif). Quantification of Western blotting data was performed using Epi Chemi II Darkroom (UVP Laboratory Products). Optical densities of each band for HK2 or PDK1 were normalized by those of actin bands.
Hexokinase enzyme activity. Hexokinase enzyme activity was determined by the formation of NADH at 340 nm using a spectrophotometer (Du 800 BeckMan) as described previously (62). NADH production is coupled to the formation of glucose-6-phosphate from glucose by glucose-6-phosphate dehydrogenase. Total cell lysates from P493-6 cells were incubated with the assay medium containing 25 mM triethylamino-Cl (Sigma), 15 mM MgCl2 (Sigma), 5 mM ATP (Amersham), 1 mM NADP+ (Sigma), 1 mM dithiothreitol (Sigma), 0.5 mM glucose (Sigma), 0.45 mM KCN (Sigma), 13 µg/ml oligomycin (Sigma), and 1.2 units/ml glucose-6-phosphate dehydrogenase (Roche). The rate of NADH formation was normalized to total protein concentration.
Lactate production. Accumulation of lactate in the culture medium was determined using a lactate assay kit (Biomedical Research Service, State University of New York at Buffalo) or the Yellow Springs Instruments 2300 STAT glucose/lactate analyzer (Yellow Springs Instruments) assay. The lactate assay kit is based on the reduction of the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride in an NADH-coupled enzymatic reaction to formazan, which is water soluble and exhibits an absorption maximum at 492 nm. Total viable cell number was used for normalization.
Glucose uptake. Glucose uptake was determined by intracellular 2-[3H]deoxyglucose level (59). P493-6 cells were incubated in medium containing 0.1 mM 2-deoxyglucose and 10 nM 2-[3H]deoxyglucose for 10 min. Cells were washed with cold phosphate-buffered saline and lysed by the addition of 10 mM NaOH containing 0.1% Triton X-100. Lysates were subjected to a liquid scintillation counting for 3H level. Total protein concentration was used for normalization.
ELISA for VEGF production. The culture medium of P493-6 cells was collected, and 100-µl aliquots were used to determine VEGF levels. Immunoreactive VEGF was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA; Quantikine VEGF immunoassay kit; R & D Systems) according to the manufacturer's recommended protocol.
In vivo xenograft model. Cells (3 x 107) were injected subcutaneously into SCID mice. In vivo suppression of MYC expression in the P493-6 xenograft was achieved by treatment with 0.01% doxycycline in drinking water. Tumor volumes were measured every 4 days.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression by shRNA dramatically inhibited c-Myc-mediated tumorigenesis of P493-6 cells (17). These studies indicate that HIF-1 is necessary for P493-6 cell tumorigenesis in vivo. To determine whether HIF-1 could synergize with c-Myc in P493-6 tumorigenesis, we introduced a constitutively active form of HIF-1 (CA5-HIF-1) with the oxygen-dependent domain deleted and several prolines mutated as previously described (35) into P493-6 cells and found that tumor growth is significantly enhanced compared with control (Fig. 1A). However, no increase in proliferation or viability was observed in CA5-HIF-1 P493-6 cells in vitro (data not shown). These results indicate that HIF-1 contributes to c-Myc-mediated tumorigenesis in vivo.
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-overexpressing P493-6 cells after the induction of c-Myc by tetracycline withdrawal (Fig. 1B and C). We noted that Myc was slightly increased and HK2 and PDK1 protein levels were both higher in P493-6 cells expressing CA5-HIF-1 than in those expressing control EV, suggesting that both HK2 and PDK1 are induced cooperatively by c-Myc and hypoxia. Despite the high levels of endogenous HIF-1 induced by the hypoxic microenvironment of tumor xenografts, additional ectopic CA5-HIF-1 expression further induced HK2 and PDK1 (1.6- and 1.7-fold higher than EV control, respectively) (Fig. 1B, bottom). In particular, while HK2 is induced by c-Myc in both conditions, the expression of PDK1 appears responsive to c-Myc only in the presence of stabilized HIF-1 (CA5-HIF-1) activity (Fig. 1C). Intriguingly, CA5-HIF-1 appears to be induced by c-Myc. We surmise that the effect of c-Myc on ectopic CA5-HIF-1 expression may be transcriptional or posttranscriptional, which has not yet been further investigated. Nonetheless, the enhanced inductions of PDK1 and HK2 correlate with ectopic CA5-HIF-1 levels, supporting the idea that ectopic CA5-HIF-1 indeed contributes to HK2 and PDK1 expression. Enhanced expression of these proteins through c-Myc and HIF-1 was also observed in the rat fibroblasts whose Myc is nullified (HO15.19) or reconstituted with human MYC (HO15.19-c-Myc) (46, 53). It is notable that under hypoxia, HO15.19 cells displayed minimal induction of HK2 compared to HO15.19-c-Myc or TGR cells that expressed higher levels of HK2, suggesting that c-Myc is necessary for significant induction of HK2 (see Fig. S1 in the supplemental material). However, PDK1 induction appears to be mainly regulated by HIF-1 or hypoxia in rat fibroblasts. These studies further support the cooperative effects of c-Myc and HIF-1 in another system (see Fig. S1 in the supplemental material).
c-Myc and HIF-1 cooperate to enhance glucose metabolism by inducing HK2.
Since both gain-of-function and loss-of-function studies demonstrate the importance of HIF-1 in c-Myc-mediated tumorigenesis of P493-6 cells and the expression of HK2 protein is increased cooperatively by c-Myc and HIF-1, we sought to determine whether HIF-1 and dysregulated c-Myc could cooperatively alter glucose catabolism and transcriptionally regulate HK2. P493-6 cells were treated with tetracycline and/or hypoxia to modulate the levels of c-Myc and HIF-1. As illustrated in Fig. 2A, in the presence of tetracycline (0.1 µg/ml), MYC expression was virtually completely repressed. MYC is rapidly induced by removal of tetracycline from P493 cells, which were previously treated with tetracycline for 48 h. To induce HIF-1 expression, P493-6 cells were exposed to hypoxia (0.1% O2) or 100 µM of CoCl2. HIF-1 expression peaked by 29 h of hypoxia or CoCl2 treatment. In these conditions, P493-6 cells were viable up to 72 h and displayed no sign of apoptosis. Cells stopped proliferating by hypoxia or c-Myc suppression (tetracycline treatment) or both. Only nonhypoxic P493-6 cells with high c-Myc expression were proliferating (see Fig. S2 in the supplemental material).
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compared with P493-6 cells expressing either c-Myc or HIF-1 alone. Consistent with mRNA and protein levels, hexokinase enzymatic activity was also enhanced by c-Myc and HIF-1 (Fig. 3C). We also examined the levels of other glycolytic enzymes including HK1, ENO1, GAPD, and LDHA, known to be regulated by either c-Myc, HIF-1, or both (38, 75). No increase of other glycolytic enzyme genes including the ENO1, GAPD, HK1, and LDHA genes by c-Myc and HIF-1 was observed in P493 cells, supporting the major roles of HK2 in enhanced glycolytic metabolism by c-Myc and HIF-1 (see Fig. S3B in the supplemental material). HK2 expression, in contrast to that of the other enzymes, was also increased in P493-6 xenografts as well as P493-6 cells bearing constitutively active HIF-1 (CA5-HIF-1) in nonhypoxic conditions (Fig. 1B and C).
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under nonhypoxic conditions, we employed P493-6 cells expressing shRNA targeting HIF-1 to further confirm that cooperative induction of HK2 under hypoxia is mediated by HIF-1 and not by non-HIF-1 pathways (Fig. 4A). As illustrated in Fig. 4B, induction of HK2 was significantly attenuated in HIF-1-knockdown P493-6 cells under high c-Myc and hypoxic conditions. It should be noted that c-Myc levels were not elevated by reduced HIF-1 expression (Fig. 4A).
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induced by either hypoxia or CoCl2, c-Myc further enhances PDK1 expression (Fig. 7A and B). Similarly, ectopic expression of constitutively active CA5-HIF-1 permits c-Myc induction of PDK1 expression in P493-6 cells and tumor xenografts (Fig. 1B and C). Furthermore, HIF-1 knockdown by shRNA reduced PDK1 induction under hypoxic conditions (Fig. 4B). However, we note a variation in the PDK1 expression in the control cells upon Myc induction under normoxic conditions compared with results shown in Fig. 1C. We surmise that this results from fluctuation of the relatively low expression of PDK1 in nonhypoxic conditions. These observations nonetheless suggest that the increased conversion of pyruvate into lactate observed in hypoxic tumor cells may result from the cooperative induction of PDK1 by c-Myc and HIF-1.
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, c-Myc synergistically increased lactate production while lactate production was not increased by c-Myc alone. Inhibition of PDK1 by siRNA transfection (Fig. 7C) or treatment with the PDK inhibitor dichloroacetate results in significantly reduced lactate production (Fig. 7D and E), confirming a role for PDK1 in the generation of lactate mediated by c-Myc and HIF-1. However, CoCl2 treatment is known to stabilize HIF-1, which in turn activates target glycolytic enzyme genes such as LDHA. In this regard, CoCl2 treatment presumably increases lactate production not only by PDK1 induction but also through elevated LDHA. Since LDHA elevated by CoCl2 is likely to increase basal lactate production, which is blunted by the inhibition of PDK1, complete reduction of lactate production by PDK1 inhibition would not be expected. (Fig. 7D). DNA binding of c-Myc and HIF-1 in the regulatory regions of the PDK1 gene. To determine the nature of c-Myc and HIF-1 collaboration in stimulating PDK1 expression, we performed a scanning ChIP to pinpoint the binding regions of these two factors. We previously reported that HIF-1-associated chromatin fragments enriched by immunoprecipitation cover the boundary of exon 1 and intron 1, which contains a cluster of consensus HIF-1 binding sites (37). To further pinpoint HIF-1 binding sites, phylogenetic footprinting was employed to identify conserved consensus HIF-1 binding sites. As illustrated in Fig. 8, highly conserved consensus sites are located in exon 1 (5'-G/ACGTG-3') that are significantly bound by HIF-1, while those in intron 1 are canonical c-Myc binding sites (E boxes, 5'-CACGTG-3') and mainly bound by c-Myc. The specificity of c-Myc and HIF-1 binding was verified by control experiments with no antibody and control IgG samples (see Fig. S5 in the supplemental material). As similarly seen in the HK2 gene, ChIP suggests that c-Myc and HIF-1 bind nonoverlapping genomic regions.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is essential for c-Myc-mediated tumorigenesis (17). Moreover, our in vivo xenograft assay indicates that gain-of-function via a stabilized HIF-1 mutant enhances the c-Myc-mediated tumorigenic potential of P493-6 cells (Fig. 1). Proliferation of P493-6 cells, however, slowed considerably in hypoxia in vitro (see Fig. S2 in the supplemental material and see also below), while in vivo tumor growth was accelerated by HIF-1 and c-Myc. It is notable that a large number of P493-6 cells are needed for in vivo tumorigenesis, implying that there are additional factors involved in tumorigenesis (17). However, the in vitro model that we use in this study allows for an experimental system to study HIF-Myc interactions, which could be modulated further in vivo. Notwithstanding this caveat, we demonstrate here that hypoxic adaptation, which includes increased glucose uptake, enhanced glycolysis, and elevated VEGF levels, is stimulated by the cooperation of HIF-1 with dysregulated c-Myc expression.
Various recent studies suggest that HIF-1 negatively regulates the transcriptional activity of c-Myc and suppresses c-Myc's physiological roles (9, 24, 42-44, 55, 80). It stands to reason that in a normal cell, which expresses c-Myc when cell proliferative cues are received to augment MYC expression, such as in wound healing, hypoxia should signal diminished blood flow and limitation of nutrients to a tissue. Hence, hypoxia via the induction of HIF-1 would be expected to trigger cell growth arrest, except in endothelial cells, and altered cellular metabolism. We have, in fact, found that hypoxia causes cell growth arrest and a decrease in endogenous MYC expression in vitro (Fig. 2A; see also Fig. S2 in the supplemental material) (18). However, no p27 expression was observed in P493-6 cells even under hypoxic conditions, which is consistent with a previously reported study (60). Very low levels of p21 were detected; however, we did not observe an increase in p21 under hypoxic conditions (Fig. 4B). Rather, p21 appears to be decreased in hypoxia by an unknown mechanism.
The observation in Fig. 1 that constitutive HIF-1 expression augments Myc-mediated P493-6 tumorigenesis in vivo appears to contradict the inability of P493-6 cells in vitro to proliferate in hypoxia as shown in Fig. 2A and in Fig. S2 in the supplemental material. How then could HIF-1 increase Myc-mediated tumorigenesis? We surmise that among the large number of P493-6 cells needed for in vivo tumorigenesis, a subpopulation acquires additional factors that are permissive for establishing a tumor mass that is dependent on both Myc and HIF. Although our attempt to study Myc-HIF interactions in vitro is nuanced by these differences, we believe that insights gleaned from our studies are necessary to further understand the interplay between Myc and HIF in vivo (17).
These earlier studies do not, however, address the interaction of HIF-1 with c-Myc when MYC expression is dysregulated, as one would expect to find in a tumor that may result from the activation of the MYC oncogene. Studies with various model systems revealed that c-Myc function is an essential contributor to cellular proliferation and normal embryonic development through modulating its target genes involved in cellular adhesion, proliferation, growth, and metabolism (12, 27, 84). Previously, we have also reported that c-Myc promotes mitochondrial biogenesis and respiration, in addition to its induction of glycolytic enzyme genes (38, 46, 59, 78). In the current study, we found that P493-6 cells bearing dysregulated c-Myc consume more glucose in nonhypoxic conditions but that lactate production is not increased, since c-Myc promotes mitochondrial biogenesis and respiration as reported previously (Fig. 2B and C). These activities of c-Myc suggest that it could couple the cell growth and cell cycle machinery with the cellular energy production system in normal cells. Because many cancer cells display the Warburg effect or have the propensity to take up glucose and convert glucose to lactate, rather than metabolizing glucose through oxidative phosphorylation (2, 7, 15, 19, 36, 76, 82, 83), we sought to understand how dysregulated expression of c-Myc might interact with HIF-1 in hypoxia to mediate the Warburg effect and adaptation to the tumor microenvironment.
Using the P493-6 system, we determined whether enforced overexpression of c-Myc could liberate c-Myc from homeostatic regulation by HIF-1. HIF-1 is known to activate transcription of genes encoding glycolytic enzymes and PDK1, which appears to be a key hypoxic adaptation through a metabolic switch from oxidative phosphorylation into glycolysis under a hypoxic microenvironment (37, 61). Previously, we comprehensively dissected the transcriptional network of c-Myc and its target glycolytic enzyme genes, demonstrating that c-Myc transactivates virtually all glycolytic enzyme genes, many of which are also activated by HIF-1 in hypoxia (38, 75). In contrast to previous studies, HIF-1 induced by hypoxia or CoCl2 further accelerates glucose uptake and lactate production in P493-6 cells. This indicates that HIF-1-mediated shutdown of mitochondrial function by the HIF-1 target PDK1 is required for converting pyruvate into lactate (Fig. 7). Scanning ChIP assays for DNA binding of c-Myc and HIF-1 in HK2 and PDK1 genes demonstrate that c-Myc and HIF-1 independently bind different genomic regions (Fig. 6 and 8). These observations suggest that in the context of dysregulated c-Myc, HIF-1 no longer inhibits c-Myc activity but rather the two cooperate to transactivate certain common target genes, such as HK2, PDK1, and VEGF.
It is intriguing that c-Myc and HIF-1 could cooperatively activate HK2 and PDK1, which are the critical regulators of glycolytic metabolism and mitochondrial function. Particularly, in addition to its rate-limiting role in glucose metabolism, HK2 is overexpressed in most human cancers and suppresses proapoptotic molecules including Bad and Bax at the mitochondrial outer membrane, by which HK2 functions as a key coordinator by integrating glucose metabolism with apoptosis (13, 23, 26, 49, 63, 64, 68). The PDK1 gene, recently identified as a HIF-1 direct target gene, inactivates mitochondrial tricarboxylic acid cycle and respiration through inhibiting the mitochondrial pyruvate dehydrogenase complex that converts pyruvate into acetyl-CoA (37, 61). Inhibition of PDK by dichloroacetate was recently shown to be antitumorigenic, suggesting that PDK activity is critically important for in vivo tumorigenesis (5). c-Myc binding in PDK promoters has been reported in a ChIP study (47), and our scanning ChIP assay verified that c-Myc binds the intron 1 region of the PDK1 gene even under nonhypoxic conditions (data not shown) and even when PDK1 expression is not induced. These observations are compatible with our finding in the P493-6 system that c-Myc is frequently bound to genes which are not induced at the steady-state mRNA level (84). Other signals, such as hypoxia, would provide additional transcription factors, such as HIF-1, to induce genes bound by c-Myc. Our study suggests that hypoxia via HIF-1 is necessary for the full induction of PDK1 that presumably counteracts c-Myc-mediated activation of mitochondrial functions in nonhypoxic conditions. Conversely, HIF-1 minimally induces PDK1 with low c-Myc expression (Fig. 7A and B). These observations suggest that the Warburg effect could well result from the cooperation between HIF-1 and dysregulated c-Myc.
In addition to the efficient metabolic switch to glycolysis, development of new blood vessels or angiogenesis is a required adaptive process to improve delivery of oxygen and nutrients into hypoxic tumor regions. According to the data from various in vivo model systems, c-Myc has been suggested to be a potent angiogenesis inducer during embryonic development and tumorigenesis (3, 77). A recent study demonstrated that hypoxia further increases c-Myc-mediated VEGF expression and angiogenesis in an in vivo transgenic model, suggesting that HIF-1 cooperates with c-Myc to induce VEGF expression and angiogenic phenotypes (40). These studies, however, did not examine the mechanism by which c-Myc and HIF-1 could cooperate to activate VEGF expression. Similarly to HK2 and PDK1, VEGF is induced by the cooperation between HIF-1 and dysregulated c-Myc (Fig. 9A). Further, we identified strong DNA binding of c-Myc and HIF-1 in a genomic region that contains a cluster of conserved putative binding sites for HIF-1 (Fig. 9B). Our study supports the molecular mechanism underlying enhanced induction of VEGF by c-Myc and HIF-1 and suggests that cooperative roles of c-Myc and HIF-1 may contribute to a broader range of oncogenic processes.
The effects of HIF-2 can be excluded since HIF-2 is not detectable at both mRNA and protein levels in P493-6 cells. However, there is emerging evidence that HIF-2 may play more prominent roles in tumorigenesis (10, 11, 39, 41, 51, 79). Although recent studies reported that adaptive responses to hypoxia such as induction of glycolytic enzyme genes are predominantly mediated by HIF-1 (25, 31), additional studies are necessary to address the effects of HIF-2 on transcriptional and oncogenic activities of c-Myc.
It is intriguing that no significant enhanced expression of other glycolytic enzyme proteins including HK1, LDHA, ENO, and GAPD was observed in the P493-6 cells under conditions with both HIF-1 and c-Myc expressed (see Fig. S3B in the supplemental material). The differences between these enzymes and HK2, PDK1, and VEGF will require further studies. It is conceivable, however, that expression of glycolytic enzymes is also regulated at the posttranscriptional level, since c-Myc binds to and induces mRNA levels of LDHA, ENO, and GAPD (38). Further studies will be needed to address how c-Myc and HIF-1 work together at the transcriptional level. It will be of great interest to ask whether unique or additional coactivators are recruited to the regulatory regions when the factors are both bound.
In summary, we report here that the tumorigenic potential of P493 cells is increased by the cooperation of dysregulated c-Myc with HIF-1, and we demonstrate that this cooperation accelerates glycolytic metabolism and angiogenesis through the induction of key proteins, HK2, PDK1, and VEGF. Our ChIP studies pinpoint the binding of c-Myc and HIF-1 to these critical genes, whose levels are significantly elevated by both transcription factors in a cooperative manner. In particular, our study underscores the importance of distinguishing the effects of HIF-1 on the normal roles of proto-oncogenes from those of dysregulated oncogenes that escape from normal homeostatic mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 4 September 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Department of Anatomy, University of California, San Francisco, HSW 1301, 513 Parnassus Ave., San Francisco, CA 94131.
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