Combinatorial regulation of neuroblastoma tumor progression by N-Myc and hypoxia inducible factor HIF-1alpha

Abstract

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.

Figure 1. MYCN amplified neuroblastoma cells preferentially express HIF-1α

(A) Western blot analysis of HIF-α expression in LAN5 and IMR32 cells cultured at different time points under 5% O₂ and 1.5% O₂ conditions. β-actin was used as a loading control. (B) Western blot analysis of HIF-α expression in SHSY5Y and SK-N-SH cells cultured at different time points under 5% O₂ and 1.5% O₂ conditions. β-actin was used as a loading control. (C) Expression of HIF-α in different neuroblastoma cell lines at normoxia. The mRNA level of HIF-1α or HIF-2α in SHSY5Y cells was arbitrarily set as 1, and the relative expression of the remaining cell lines were calculated as shown. Data are presented as an average of triplicates and normalized to β-actin mRNA. *p< 0.001. (D) Synergistic regulation of HIF-2A expression by DNA methylation and histone deacetylation in MYCN amplified cells. LAN5 cells were either treated with vehicle or 3 µM DAC, 500 nM TSA, as well as DAC and TSA combinations. CASP8 and YWHAZ were used as positive and negative controls, respectively. Data are presented as an average of triplicates. *p< 0.01.

Figure 2. MYCN amplified neuroblastoma tumors preferentially express HIF-1α

Representative HIF-α immunochemical staining of neuroblastoma tumors. Renal clear cell carcinomas were used as controls for specific HIF-α staining. Magnification: 400×. Arrows indicate positive HIF-α staining in nuclei.

Figure 3. Proliferation of MYCN amplified neuroblastoma cells is maintained under hypoxia

(A) Growth of control and N-Myc knockdown LAN5 cells over seven days as measured by serial cell counts at 21% O₂ (N) and 1.5% O₂ (H). (B) Representative FACS plots from LAN5 cells grown at 21% O₂ (N) and 1.5% O₂ (H) for 48 hr. (C) Summary of changes in BrdU incorporation in LAN5 cells after 48 hr hypoxia. Results are averaged from 3 independent experiments. (D) Overexpression of c-Myc rescued proliferation of N-Myc knockdown LAN5 cells. Inset: a) LAN5 cells; b) N-Myc knockdown LAN5 cells; c) N-Myc knockdown LAN5 cells with c-Myc overexpression.

Figure 4. Changes in Myc/Max interaction and subsequent effect on target gene expression in LAN5 and HCT116 cells grown at 21% O₂ (N) and 1.5% O₂ (H)

HCT116 (A) and LAN5 (B) cell lysates from different time points were coprecipitated with Max antibody, and immunoblotted against specific c-Myc, N-Myc and HIF-1α antibodies. (C) Binding of Myc (c-Myc or N-Myc) to target promoters analyzed by ChIP assay. HCT116 and LAN5 cells were grown at 21% O₂ (N) and 1.5% O₂ (H) for 24 hr and then assayed by ChIP with specific Myc antibodies or isotype control IgG. The graphs show the fold difference between Myc IP and IgG control (background) with results obtained from triplicate assays. *p< 0.01. (D) Expression of Myc targets involved in cell cycle progression. HCT116 and LAN5 cells were grown at 21% O₂ (N) and 1.5% O₂ (H) for 24 hr, and relative gene expression was analyzed by QRT-PCR. Results were averaged from triplicates. *p< 0.01.

Figure 5. N-Myc and HIF-1α contribute to the Warburg effect of MYCN amplified neuroblastoma cells

(A) MYCN amplified neuroblastoma cells exhibit the Warburg phenotype. Glucose consumption and lactate production in LAN5, Kelly, NLF, SK-N-DZ and IMR32 cells cultured at normoxia for 24 hr. Data were averaged from triplicates. (B) N-Myc regulates expression of specific genes involved in glycolysis. N-Myc was inhibited by control or specific shRNAs in LAN5 cells. Expression of glycolytic genes was determined by QRT-PCR and shown as an average of triplicates. *p< 0.001. (C) HIF-1α regulates the expression of specific glycolytic genes at normoxia. Expression of glycolytic genes in control and HIF-1α knockdown cells was determined by QRT-PCR and shown as an average of triplicates. *p< 0.005. (D) Effects of simultaneous HIF-1α and N-Myc inhibition on HK2, PDK1 and LDHA expression. N-Myc and HIF-1α were depleted by control or specific shRNAs in LAN5 cells. Expression of HK2, PDK1 and LDHA was determined by QRT-PCR and shown as an average of triplicates. *p< 0.01.

Figure 6. LDHA is a promising therapeutic target for neuroblastoma patients

(A) Relative expression of LDHA in primary neuroblastoma tumors. LDHA levels in fetal brain were used as a control. Tumor numbers: low risk group (28), intermediate (21), MYCN single-copy, high risk (32) and MYCN amplified, high risk (20). (B) Depletion of LDHA expression profoundly inhibits the proliferation of MYCN amplified neuroblastoma cells. The LDHA protein is indicated by arrowhead. *p< 0.01. (C) Depletion of LDHA expression inhibits the tumorigenic capacity of Kelly cells (8 tumors were analyzed in each group). (D) A model depicting N-Myc and HIF-1α cooperation in neuroblastoma tumor progression. See text for details.