In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. Because hypoxia contributes to aggressive tumor phenotypes, predominantly via two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, we examined hypoxia responses in MYCN-amplified neuroblastoma cells. We demonstrate here that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN-amplified neuroblastoma cells and primary tumors in comparison to samples without MYCN amplification. Our results showed that interplay between N-Myc and HIF-1α plays critical roles in neuroblastoma. For example, high levels of N-Myc override HIF-1α inhibition of cell cycle progression, enabling continued proliferation under hypoxia. Furthermore, both HIF-1α and N-Myc are essential for the Warburg effect (aerobic glycolysis) in neuroblastomas by activating the transcription of multiple glycolytic genes. Of note, expressions of Phosphoglycerate Kinase 1 (PGK1), Hexokinase 2 (HK2), and Lactate Dehydrogenase A (LDHA) were each significantly higher in MYCN-amplified neuroblastomas than in tumors without MYCN amplification. Interestingly, MYCN-amplified neuroblastoma cells are “addicted” to LDHA enzymatic activity, as its depletion completely inhibits tumorigenesis in vivo. Thus, our results provide mechanistic insights explaining how MYCN-amplified neuroblastoma cells contend with hypoxic stress and paradoxically how hypoxia contributes to neuroblastoma aggressiveness through combinatorial effects of N-Myc and HIF-1α. These results also suggest that LDHA represents a novel, pharmacologically tractable target for neuroblastoma therapeutics. Cancer Res; 70(24); 10351–61. ©2010 AACR.
Neuroblastoma is one of the most frequent solid tumors in infants and children (1). Risk factors indicative of poor prognosis include age greater than 18 months at diagnosis, unfavorable histologic grade, and MYCN amplification (1, 2). Recent studies demonstrate that mutations in the Anaplastic Lymphoma Kinase (ALK) gene cause familial neuroblastoma, whereas several common polymorphisms, such as those in the BRCA1-associated RING Domain-1 (BARD1) gene, influence susceptibility to neuroblastoma (3, 4). Nevertheless, MYCN amplification remains the most important and reliable oncogenic marker, strongly correlating to advanced stages of disease and poor survival. MYCN amplification occurs in 20% to 25% of patients and is consistently associated with high levels of N-Myc protein (1), which is likely to directly contribute to tumor cell behavior (5). Presumably, N-Myc promotes neuroblastoma tumor progression through regulating and/or cooperating with other oncogenic pathways. However, the exact nature of these pathways remains largely unclear.
Hypoxia, a common feature of solid tumors, contributes to aggressive tumor phenotypes. In an adaptive response to O2 deprivation, cells alter their gene expressions primarily through the activation of hypoxia inducible factor (HIF)-1α and HIF-2α (6). HIFs function as heterodimers in which the O2-labile α-subunits form dimeric complexes with a stable β-subunit (also called ARNT) and activate the transcription of genes encoding angiogenic, metabolic, and metastatic factors (6). It is well known that HIF-1α and HIF-2α share both common and unique target genes (7–9). Although both HIF-α subunits cooperatively regulate genes involved in angiogenesis and metastasis (8), HIF-1α selectively activates genes involved in glycolysis and epigenetics and HIF-2α stimulates Oct4 and Erythropoietin (EPO) expressions (7, 10–12). Intriguingly, genome-wide analysis of HIF-1α and HIF-2α binding sites across more than 25,500 human gene promoters demonstrated that despite a large degree of overlap in binding of the 2 HIF-α isoforms there are striking differences in gene regulation by HIF-1α and HIF-2α (13), further highlighting the distinct roles of HIF-α in hypoxic adaptation (13, 14).
MYC family genes are frequently deregulated in numerous types of human cancer. Whereas C-MYC is expressed in a wide variety of human tumors, MYCN expression is mostly restricted to tumors derived from the nervous system, such as neuroblastoma (15). On forming a binary complex with its partner, Max, Myc activates transcription of target genes involved in cell proliferation, angiogenesis, apoptosis, and metabolism (15). “Crosstalk” between the c-Myc and HIF pathways has been clearly documented, but is highly complex and depends on cell type and Myc protein levels (16). In response to hypoxia, cell proliferation decreases through increased expressions of p21 and p27 and decreased expressions of cyclin D2 and E2F1 as a result of HIF-1α–mediated inhibition of c-Myc activity (17, 18). In direct contrast to HIF-1α, HIF-2α appears to enhance c-Myc activity in clear cell renal carcinoma cells (ccRCC) and primary tumors (18, 19). However, in Burkitt's lymphoma cells, in which c-Myc is overexpressed, HIF-1α actually cooperates with, rather than antagonizes, c-Myc to selectively enhance the expressions of Hexokinase 2 (HK2), Pyruvate Dehydrogenase Kinase 1 (PDK1), and Vascular Endothelial Growth Factor (VEGF; ref. 20). It appears that specific target gene promoters, cell types, and hypoxic conditions determine the way HIF-1α and HIF-2α engage the c-Myc pathway, resulting in either increased or decreased target gene expression. The complexity of HIF-α/c-Myc interaction raises important questions: do mechanisms involving HIF-α/c-Myc apply to other tumor microenvironments, including neuroblastoma? A previous study suggested that N-Myc and c-Myc only share approximately 40% of their target genes (21); therefore, do HIF-1α and HIF-2α employ similar mechanisms to interact with other Myc family members, such as N-Myc?
HIF-2α has been previously implicated in promoting an aggressive neuroblastoma phenotype (22–24), with HIF-2α (and not HIF-1α) correlating with an unfavorable clinical outcome (24). These studies underscore the importance of discriminating HIF-1α versus HIF-2α expression in vivo. However, the impact of HIF-α on Myc was not addressed and neuroblastomas were not segregated on the basis of MYCN amplification status. In an effort to investigate how HIF-α and N-Myc regulate neuroblastoma tumor progression, we systematically analyzed the dynamics of HIF-1α and HIF-2α expressions and subsequently evaluated the interaction between HIF-α and N-Myc in vitro and in vivo. Interestingly, we found that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN-amplified neuroblastoma cells and primary tumors in comparison to nonamplified samples. Our data suggest that neuroblastomas can be categorized into 2 groups on the basis of Myc and HIF-α expression patterns, with N-Myc/HIF-1α cooperating in MYCN-amplified neuroblastomas whereas c-Myc/HIF-2α appear to cooperate in MYCN single-copy tumors. Our data also suggest small molecules targeting tumor metabolism may be a promising and effective treatment in neuroblastoma patients. Furthermore, this study, combined with a previous report on Burkitt's lymphoma (20), demonstrate that cooperation of MYC family genes and HIF-1α may play a global role in human tumor progression.
Materials and Methods
HCT116 cells were maintained in Dulbecco's modiﬁed Eagle's medium with 10% FBS, and all the neuroblastoma cells were maintained in RPMI medium containing 10% FBS. For hypoxia treatment, cells were cultured in a hypoxic workstation (Ruskinn Technologies) at 1.5% O2.
Cell cycle analysis
HCT116 or LAN5 cells were cultured at normoxia or hypoxia for 48 hours before analysis. BrdU incorporation was performed following the standard protocol (Becton Dickinson) after a 30 min pulse with 10 μmol/L BrdU. Cells were stained with Alexa 488 anti-BrdU (Invitrogen) and 0.1 mol/L propidium iodide and analyzed in an LSR FACS machine (Becton Dickinson).
Total RNA was extracted from cells with Trizol reagent following the manufacturer's instructions (Invitrogen). cDNA was produced from 1 μg of RNA using Superscript II (Invitrogen) with random hexamer primers (Boehringer Mannheim). Analysis of gene expression was performed in a 7900HT Sequence Detection System by using specific Taqman primers (Applied Biosystems).
Specific shRNAs against human HIF-1α, N-Myc, and LDHA or a control shRNA were obtained from Open Biosystems. After viral transduction, cells were selected with puromycin (Sigma).
Chromatin immunoprecipitation, immunoprecipitation, and Western blot analysis
Chromatin immunoprecipitation (ChIP) was performed following standard protocol from Upstate Biotech. For immunoprecipitation (IP) assays, cells were lysed in 25 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, and 1% Triton X-100 containing Complete protease inhibitors (Roche) and 200 μmol/L DFX. For all other Western blots, cells were lysed in RIPA and 50 μg total cellular proteins were used for each blot. Antibodies were used as follows: human HIF-1α (BD Biosciences), human HIF-2α (Novus NB 100-122), actin (Sigma AC-15), c-Myc (Santa Cruz N-262 and C-33), and N-Myc (Santa Cruz B8.4.B).
Female BALB/C nude mice (Charles River) were subcutaneously injected in both flanks with 3 million Kelly cells (control or LDHA shRNA) diluted in 200 μL PBS containing 50% matrigel (BD Bioscience). Tumor weight was measured at the time of sacrifice.
Sections of primary neuroblastoma tumors were deparaffinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 20 minutes. Slides were incubated with the antibodies against HIF-1α (Labvision HIF-1A67) and HIF-2α (Novus rabbit polyclonal) overnight at 4°C. The remaining steps were performed using the DAKO CSA kit. Sections from clear cell renal carcinoma were used as controls for HIF-α staining.
HIF-1α, but not HIF-2α, is preferentially expressed in advanced-stage, MYCN-amplified neuroblastoma cells
In neuroblastoma cells, HIF-2α was selectively stabilized under physiologic O2 conditions (5% O2) and governed a prolonged hypoxic adaptation under chronic hypoxia (22). In an effort to evaluate the functional interaction between HIF-α (HIF-1α and HIF-2α) and N-Myc, we first examined HIF-α expression in 2 MYCN-amplified cell lines (LAN5 and IMR32) cultured at both 5% O2 and 1.5% O2, respectively, at different time points. We also examined HIF-α levels in 2 MYCN single-copy cell lines (SHSY5Y and SK-N-SH). Interestingly, unlike MYCN single-copy cells, which expressed both HIF-1α and HIF-2α (Fig. 1B), the 2 MYCN-amplified cell lines selectively expressed HIF-1α (Fig. 1A). We expanded our studies of HIF-α expression in a series of MYCN-amplified and single-copy cell lines at 5% O2 (Supplementary Fig. S1). HIF-1α was selectively expressed in MYCN-amplified cells, with the Kelly cell line being the only exception. We then exposed neuroblastoma cell lines to 1.5% O2 for 24, 48, and 72 hours, respectively. Again, we detected abundant HIF-1α protein in all the cell lines tested (Fig. 1A and B; Supplementary Fig. S2); however, HIF-2α was expressed at very low levels in the majority of MYCN-amplified cell lines. Moreover, a single-copy line, SK-N-AS, also exhibited undetectable HIF-2α expression under these conditions. Thus, lack of detectable HIF-2α expression in most MYCN-amplified cells and SK-N-AS cells is largely independent of O2 concentrations. In contrast to a previous study suggesting that HIF-2α levels remain high whereas HIF-1α decays (22), we found that chronic hypoxic stress destabilized both HIF-α proteins with similar kinetics in most cell lines (Fig. 1A and B; Supplementary Fig. S2). In support of our data, several recent studies also independently demonstrated that chronic hypoxia resulted in HIF-1α and HIF-2α degradation with similar kinetics in other contexts (25–27), indicating destabilization of both HIF-α isoforms during chronic hypoxia is a general adaptative mechanism.
The fact that HIF-1α was stabilized whereas HIF-2α was not in most hypoxic MYCN-amplified cells suggested HIF-2α expression is specifically silenced at the transcriptional level. To investigate the potential mechanisms involved, we quantitated HIF-1α and HIF-2α mRNA abundance in 13 neuroblastoma cell lines, and 9 MYCN-amplified and 4 single-copy cell lines, under normoxia. We chose a single-copy cell line, SHSY5Y, as a control because this line showed abundant HIF-1α and HIF-2α expressions under hypoxia. The relative mRNA levels of both HIF-1α and HIF-2α in other neuroblastoma cells were analyzed by quantitative RT-PCR (QRT-PCR). Consistent with protein expression patterns, there was no dramatic difference between HIF-1α mRNA abundance in all the cell lines tested; however, that of HIF-2α was significantly lower in most MYCN-amplified cells and the single-copy cell line, SK-N-AS, suggesting that lack of HIF-2α expression is because of low basal mRNA levels (Fig. 1C). As all the neuroblastoma cell lines currently available were derived from high-risk tumors, we were unable to compare the relative mRNA levels of HIF-α among different risk groups in cell lines. We therefore analyzed microarray data from 101 primary neuroblastoma tumors ranging from low risk to high risk (Supplementary Fig. S3). Interestingly, HIF-1α expression is significantly elevated in the MYCN-amplified group when compared with MYCN single-copy, low-risk tumors (P = 0.0201), whereas that of HIF-2α is lower in MYCN-amplified tumors (P = 0.0611).
Gene function is frequently disrupted by epigenetic alterations. To determine whether HIF2A was epigenetically silenced, we administered 2 different chemicals affecting chromatin structure, 5-aza-2′-deoxycytidine (DAC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, to LAN5 cells in which HIF-2α expression was largely absent (Fig. 1D). Genes encoding Caspase 8 (CASP8) and Tyrosine 3-Monooxygenase/tryptophan 5-Monooxygenase activation protein, zeta polypeptide (YWHAZ) were used as controls, as CASP8 is transcriptionally silenced in most MYCN-amplified cells through a mechanism involving DNA methylation and histone deacetylation (28), whereas YWHAZ is a constitutively expressed “housekeeping” gene. Interestingly, administration of either DAC or TSA significantly increased expressions of both HIF2A and CASP8, and a combination of both chemicals lead to further increases in mRNA levels (Fig. 1D). Of note, YWHAZ expression was largely unchanged with these treatments, arguing against the notion that increased HIF2A expression was because of global activation of transcription. Instead, these data demonstrated that in most MYCN-amplified cells, synergy of DNA methylation and histone deacetylation silenced HIF2A transcription.
HIF-1α, but not HIF-2α, is preferentially expressed in primary MYCN-amplified neuroblastoma tumors
To confirm that our observations were representative of HIF-α status naturally present in human neuroblastomas, we analyzed HIF-α expression by immunochemistry of primary neuroblastoma tumors. We first evaluated the specificity and efficacy of the antibodies in ccRCCs. VHL mutation in ccRCCs leads to constitutive stabilization of HIF-1α and/or HIF-2α (Fig. 2; ref. 14). We then analyzed HIF-α levels in 15 neuroblastoma tumors (13 MYCN-amplified and 2 single-copy; Fig. 2; Supplementary Table S1). Again, HIF-1α, but not HIF-2α, was preferentially expressed in MYCN-amplified tumors. Whereas HIF-2α was distributed to both the cytoplasm and nucleus, HIF-1α was predominantly located in the nucleus (Fig. 2; and data not shown). Taken together, these data demonstrated that HIF-1α, but not HIF-2α, is the primary HIF-α subunit involved in adaptation to hypoxia by MYCN-amplified tumors.
Proliferation of MYCN-amplified neuroblastoma cells in vitro is, paradoxically, unaltered under hypoxia
Moderate levels of hypoxia (approximately 1.5% O2) inhibit proliferation through HIF-1α–mediated inhibition of c-Myc activity (17, 18). As MYCN-amplified cells preferentially expressed HIF-1α, we examined how these cells responded to O2 deprivation. Interestingly, we observed no significant change in proliferation of LAN5 cells when cultured at either 1.5% O2 (Fig. 3A) or 0.5% O2 (Supplementary Fig. S4D). We further analyzed 3 additional MYCN-amplified cell lines, and found that cell numbers were not decreased by low O2 (Supplementary Fig. S4A–C). Importantly, MYCN-amplified neuroblastoma cells are highly dependent on N-Myc activity, as depletion of N-Myc expression significantly decreased their proliferation under either normoxia or hypoxia (Fig. 3A). We then assessed cell cycle progression of LAN5 cells by BrdU incorporation. No obvious changes in either G1 or S phase were observed in LAN5 cells (Fig. 3B and C), in agreement with cell proliferation as measured by serial cell counting (Fig. 3A). Moreover, overexpression of c-Myc rescued proliferation of N-Myc depleted LAN5 cells (Fig. 3D), confirming that MYCN-amplified neuroblastoma cells depend on Myc activity to counteract hypoxic inhibition of cell proliferation.
N-Myc overrides HIF-1α inhibition of cell cycle progression under hypoxia
We hypothesized that high levels of N-Myc counteract HIF-1α inhibition of cell proliferation. Based on this reasoning, we examined the interaction between Myc and its binding partner, Max. We chose HCT116 colon cancer cells for comparison, as it has been previously established that HCT116 cell proliferation is inhibited by hypoxia (17, 18). Furthermore, hypoxic HCT116 cells reproducibly exhibit decreased Myc activity and Myc/Max interaction (17, 18). Note that HCT116 cells express c-Myc, whereas LAN5 cells express N-Myc only. We detected specific binding of Myc (c-Myc or N-Myc) to its partner, Max, via IP using antibodies against Max (Fig. 4A and B, compare lane 2 with lane 1 in IP panel). In HCT116 cells, chronic hypoxia significantly decreased interaction between c-Myc and Max (Fig. 4A, compare lane 4 with lanes 2 and 3 in IP panel). In contrast, interaction of N-Myc with Max did not obviously change in LAN5 cells after 24 hours of hypoxia (Fig. 4B, compare lane 4 with lanes 2 and 3 in IP panel). We did not observe detectable interaction between HIF-1α and Max (Fig. 4A and B), as the Max-specific antibody efficiently coprecipitated Myc (c-Myc or N-Myc) but not HIF-1α in either HCT116 or LAN5 cells. Failure to detect Max/HIF-1α interaction was not due to a lack of HIF-1α proteins in the cell lysates (Fig. 4A and B, input panels). Moreover, reciprocal IP using HIF-1α specific antibody also failed to pull down Max; instead, abundant HIF-1β (the binding partner of HIF-1α) was coprecipitated (data not shown). We have also been unable to observe detectable interaction between c-Myc and HIF-1α using either endogenous or in vitro translated proteins (data not shown). These data indicated that direct disruption of Myc/Max interaction by HIF-1α was unlikely to occur in neuroblastoma cells, as shown for ccRRC (18). Instead, chronic hypoxia decreased c-Myc protein abundance (Fig. 4A, compare lanes 2, 3, and 4 in input panel), whereas that of N-Myc was not obviously changed (Fig. 4B, compare lanes 2, 3, and 4 in input panel). Of note, changes in Myc abundance correlated with those of Myc/Max interaction, suggesting that the Myc protein levels per se determined the stoichiometry of Myc/Max complexes in hypoxic colon carcinoma and neuroblastoma cells.
To confirm whether these in vitro results reflected the situation in vivo, we assessed Myc occupancy of target gene promoters by ChIP assays, and the relative binding was quantified by QRT-PCR. Consistently, binding of c-Myc to target gene promoters in HCT116 cells did not appreciably change at 6 hours of hypoxia (data not shown), but significantly decreased at 24 hours (Fig. 4C). In contrast, hypoxia failed to decrease the binding of N-Myc to target gene promoters at either time point (Fig. 4C; and data not shown). We next tested hypoxic regulation of Myc targets involved in cell cycle progression: p21 and p27 (repressed by Myc), as well as E2F1 and cyclin D2 (activated by Myc). Under the conditions described earlier in the text, we observed increased p21 and p27 mRNA expression and decreased cyclin D2 and E2F1 mRNA level in hypoxic HCT116 cells at 24 hours (Fig. 4D). When the same target genes were tested, hypoxic LAN5 cells exhibited no detectable changes in their mRNA abundance (Fig. 4D). As a positive control, hypoxia significantly increased VEGF expression in either cell line (Fig. 4D). Taken together, these results provided novel mechanistic insights explaining how MYCN-amplified neuroblastoma cells thrive under hypoxic stress.
Both N-Myc and HIF-1α regulate the Warburg effect of neuroblastoma
Neuroblastomas, like all solid tumors, must meet specific metabolic requirements to fuel their deregulated growth and invasion into surrounding tissues. When O2 is abundant, differentiated cells extract energy primarily from glucose by oxidative phosphorylation, whereas most (60%–70%) tumor cells consume glucose more avidly, converting it to lactate (29, 30). This long-observed phenomenon is known as aerobic glycolysis (the Warburg effect). To investigate whether MYCN -amplified neuroblastoma cells exhibit the Warburg phenotype, we measured glucose uptake and consequent lactate production in 5 cell lines at 21% O2 (Fig. 5A). All cell lines predominantly exhibited a glycolytic metabolism, as they released an average of 1.6 molecules lactate for each glucose molecule consumed.
We then sought to investigate the mechanisms whereby neuroblastoma cells promoted aerobic glycolysis. In this regard, immunostaining of primary neuroblastoma tumors showed that HIF-1α is robustly expressed in multiple distinct tumor areas (Fig. 2). One consequence of HIF-1α activation is the stimulation of glycolysis and angiogenesis through increased transcription of glycolytic and angiogenic genes (6). In MYCN-amplified neuroblastomas, amplification of the MYCN gene frequently results in high N-Myc protein levels. Although c-Myc clearly stimulates glycolysis (31), no detailed study has shown that N-Myc acts similarly in this process, as genome-wide analysis of gene expression associated with N-Myc overexpression in either cell lines or primary tumors demonstrated that N-Myc predominantly regulates genes involved in cell cycle progression and ribosome biogenesis (21, 32). Moreover, N-Myc and c-Myc only share approximately 40% of their target genes, none of which included genes involved in glycolysis based on this analysis (21). For this purpose, we depleted the expressions of N-Myc and HIF-1α, respectively, by specific shRNAs in LAN5 cells (Fig. 5B; Supplementary Fig. S5). Knockdown of N-Myc selectively decreased expressions of genes encoding Glucose Transporter 1 (GLUT1), HK2, PDK1, PGK1, Aldolase A Fructose-Bisphosphate (ALDA), and LDHA at normoxia, but had no obvious effect on that of GAPDH (Fig. 5B). Interestingly, HIF-1α depletion (Supplementary Fig. S5) specifically decreased the expressions of GAPDH, HK2, PDK1, ALDA, and LDHA at normoxia (Fig. 5C). Simultaneous knockdown of N-Myc and HIF-1α indicated that they additively regulate HK2 and LDHA expressions, as inhibiting both factors resulted in a further decrease in expression (Fig. 5D). As shown in Figure 4, HIF-1α and N-Myc do not form a physical interaction. However, HK2, PDK1, and LDHA harbor hypoxia response elements (HREs) and E boxes in close proximity to each other (20). Taken together, our data and data from the Dang lab (20) suggest that overexpressed Myc and HIF-1α converge on the promoters of these genes to regulate their transcription, thereby combining in the transactivation of some common targets.
As stated earlier in the text, MYCN-amplified neuroblastoma cells preferentially express HIF-1α, but not HIF-2α. To determine what impact HIF-2α would have on MYCN-amplified neuroblastoma cells, we stably transfected a plasmid expressing wild-type HIF-2α into MYCN-amplified cells to mimic “reactivation” of endogenous HIF-2α (Supplementary Fig. S6A). We first examined the expression of numerous HIF-α target genes at 1.5% O2 (Supplementary Fig. S6B). Expression of HIF-2α further increased GLUT1 expression, but had no effect on other glycolytic genes (Supplementary Fig. S6B), demonstrating that HIF-1α, but not HIF-2α, predominantly controls expression of glycolytic genes in neuroblastoma. We then analyzed glucose consumption (Supplementary Fig. S6C). Interestingly, HIF-2α expression did not increase glucose uptake when compared with controls. We observed that hypoxia significantly increases glucose consumption by both control and HIF-2α-transfected cells, suggesting that HIF-1α is sufficient to maintain the glycolytic phenotype of neuroblastoma cells under hypoxia. In addition, HIF-2α had no detectable effect on cell cycle progression under hypoxia (Supplementary Fig. S6D). Consistent with these in vitro data, HIF-2α expression had no obvious impact on the tumorigenic capacity of SK-N-BE2 cells in a xenograft tumor model (Supplementary Fig. S6E). Taken together, these data suggest that the function of HIF-1α and HIF-2α is redundant in MYCN-amplified neuroblastomas, and that HIF-1α plays the dominant role in these tumors. Even if HIF-2α were “reactivated,” it would not necessarily result in a dramatic phenotype because of the high levels of HIF-1α present.
LDHA is a promising therapeutic target for MYCN-amplified neuroblastomas
Based on the data shown earlier in the text, we analyzed the expressions of glycolytic genes in 101 primary neuroblastoma tumors (33). Interestingly, expressions of LDHA, HK2, and PGK1 were significantly upregulated in MYCN-amplified tumors when compared with MYCN single-copy samples (Fig. 6A; and data not shown). We were particularly interested in LDHA for the following reasons: (1) LDHA expression is significantly upregulated in MYCN-amplified neuroblastoma subclass (Fig. 6A); (2) it was shown to maintain the Warburg phenotype in other tumor contexts (34, 35); and (3) individuals lacking LDHA expression have no obvious phenotype under normal conditions (36). On the basis of this reasoning, we depleted LDHA expression in 2 MYCN -amplified cell lines, LAN5 and Kelly, by means of specific shRNAs (Fig. 6B). Strikingly, both cell lines exhibited addiction to LDHA activity as depletion of its expression significantly inhibited their proliferation at normoxia (Fig. 6B). More importantly, depletion of LDHA completely inhibited the tumorigenic capacity of Kelly cells in vivo (Fig. 6C). Taken together, these data suggest that targeting LDHA may provide an effective, nontoxic approach to neuroblastoma therapy.
In this study, we identified a previously underappreciated role for N-Myc/HIF-1α cooperation in neuroblastoma tumor progression. Based on these data, we propose the following model (Fig. 6D): under normoxia, N-Myc promotes proliferation of neuroblastoma cells by activating genes involved in cell cycle progression. Meanwhile, both N-Myc and low levels of HIF-1α cooperatively contribute to the Warburg effect via regulation of glycolytic genes. Under hypoxia, HIF-1α is further stabilized due to decreased protein degradation. On one hand, high levels of N-Myc protein resulting from genomic amplification override HIF-1α inhibition of cell cycle progression, enabling sustained cell proliferation. On the other hand, stabilized HIF-1α, together with N-Myc, further increases glucose uptake with concomitant lactate production. It should be noted that N-Myc and HIF-1α may also cooperatively contribute to other processes, such as angiogenesis.
The Warburg effect has been inferred in many cancers by fluorodeoxyglucose positron emission tomography (37). Indeed, increased glycolytic capability and overall tumor aggressiveness is being recognized as a common trait of most solid tumors. We demonstrated here that N-Myc and HIF-1α cooperatively contribute to the Warburg effect of neuroblastoma (Figs. 5 and 6). Advanced-stage neuroblastomas with MYCN amplification are often resistant to conventional therapeutic drugs because of aberrations in their apoptotic machinery (38), making the search for novel druggable targets in this tumor type critical. Systemic inhibition of c-Myc in a Ras-induced lung adenocarcinoma mouse model indicated the feasibility of targeting c-Myc, a common downstream conduit for many oncogenic signals, as an efficient and tumor-specific cancer therapy (39); however, small molecules targeting non-kinase oncogenes like MYC have never been achieved. MYCN-amplified neuroblastomas are highly vascular (1). In principle, blocking angiogenesis (e.g., using anti-VEGF agents) may provide an alternative promising therapeutic approach. Nevertheless, recent studies demonstrated that antiangiogenesis agents significantly increased invasion and metastasis in a number of tumor models (40, 41), somewhat decreasing the enthusiasm of targeting angiogenesis for treatment of cancers like neuroblastoma. Many cancer cells avidly take up glucose and generate lactate through LDHA. However, whether LDHA plays a more general role in tumor progression is still largely unknown, given that 30% to 40% human tumors do not exhibit a Warburg phenotype. In this study, we first systematically analyzed a series of N-MYC–amplified neuroblastoma cells and showed that they consistently exhibit a Warburg phenotype. We then demonstrated that targeting LDHA could be another attractive approach in treating neuroblastoma patients with MYCN amplification, given that small molecule inhibitors against LDHA are already available (42). Because MYCN single-copy neuroblastomas frequently express c-Myc (but not N-Myc; ref. 43), conceivably, c-Myc and HIF-2α cooperate as shown for ccRCCs (18). Thus, we propose that neuroblastomas can be categorized into 2 groups based on Myc and HIF-α expression patterns, with N-Myc/HIF-1α cooperating in MYCN-amplified neuroblastomas whereas c-Myc/HIF-2α cooperate in MYCN single-copy tumors. Inhibitors targeting metabolism, either alone or in combination with other chemotherapeutic drugs, should be considered for translation into clinical trials for neuroblastoma patients.
Disclosure of Potential Conflicts of Interest
The authors declared no potential conflicts of interest.
This work was supported by the Howard Hughes Medical Institute, NIH Grant CA104838 (M.C. Simon), NIH Grant CA097323 (J.M. Maris) and NIH F32 Training Grant 1F32CA137988 (G.L. Qing). M.C. Simon is an Investigator of the Howard Hughes Medical Institute.
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.
We thank members of the Simon lab for helpful suggestions.
NOTE: Supplementary material for this article is available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received March 2, 2010.
- Revision received September 18, 2010.
- Accepted October 7, 2010.
- ©2010 American Association for Cancer Research.