Hexokinase 2 is a determinant of neuroblastoma metastasis

Abstract

Background: Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients.

Methods: Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma.

Results: Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells.

Conclusions: Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

Figure 1

Activity levels of HK2 are higher in neuroblastoma metastatic variants than in local variants. Total HK activity in neuroblastoma variants was determined by measuring microunit enzyme per ml (μU ml⁻¹). Data are means of three independent experiments+s.d. Significance was evaluated using Student's t-test. *P<0.05.

Figure 2

Hypoxic conditions induce high levels of HK2 expression in neuroblastoma metastatic variants. Local and metastatic neuroblastoma variants were treated with 0, 25 and 50 μM Deferoxamine (A) or incubated in a hypoxia chamber (1% oxygen) (B). Following treatment, the expression level of HIF-1α and HK2 was measured by western blot analysis. Shown are the results of one representative blot out of four independent experiments.

Figure 3

Metastatic neuroblastoma variants are more resistant than local variants to growth inhibition mediated by the HK2 inhibitor 3-BrPa. (A) Viability of local and metastatic neuroblastoma variants was determined by XTT-based viability assays following treatment with 25, 50 and 100 μM 3-BrPa. (B) The activity of all HK isoforms in neuroblastoma variants was determined following treatment with 50 μM 3-BrPa by measuring microunit enzyme per ml (μU ml⁻¹) normalised to control untreated cells. Data are means of three independent experiments+s.d. Significance was evaluated using Student's t-test. *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001.

Figure 4

Sh-HK2 reduces HK2 expression level and total HK activity and induces sensitivity to the HK2 inhibitor 3-BrPa. (A) Whole-cell lysates of neuroblastoma sh-HK2 or sh-Con cells were subjected to western blot analysis and immunostaining. The expression level was measured 1 week or 2 weeks following plasmid activation. (B) Total HK activity in neuroblastoma sh-Con or sh-HK2 cells was determined by measuring microunit enzyme per ml (μU ml⁻¹). (C) Viability of sh-HK2 and sh-Con cells was determined by XTT-based viability assay following treatment with 25, 50 and 100 μM 3-BrPa. Data are means of three independent experiments+s.d. The blots shown are of a representative experiment out of three. Significance was evaluated using Student's t-test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 and *****P<0.0005.

Figure 5

Downregulation of HK2 expression reduces the malignant phenotype of neuroblastoma cells. (A) Sh-Con and Sh-HK2 cell viability was examined using XTT-based viability assays 24, 48 and 72 h post plasmid activation. (B) Sh-Con and sh-HK2 cells were activated (day 0) and fluorescent pictures demonstrating cell proliferation and morphology were taken on days 0, 7, 14 and 17 post plasmid activation. (C) Fluorescence photomicrographs of scratched monolayers in a wound healing assay of sh-Con and sh-HK2 cells. The scratches were performed 1 week after plasmid activation (0 h). Pictures were taken at 0, 24, 48 and 72 h after the scratch was performed. (D) Percentage of the development of local adrenal tumours in mice inoculated with sh-Con and sh-HK2 cells. (E) Volume measurements of local adrenal tumours in mice inoculated with sh-Con and sh-HK2 cells. (F) Quantification of human mRNA in mouse lungs using qRT–PCR reactions indicating lung metastasis of human origin in mice inoculated with sh-Con and sh-HK2 cells. Data are means of three independent experiments+s.d. (A–C) or of mice in each group (n=14, 7 mice in each group)+s.d. (D–F). Significance was evaluated using Student's t-test (A–C) or Wilcoxon rank test (E and F). *P<0.05, **P>0.01 and ***P<0.005.