Targeting ornithine decarboxylase impairs development of MYCN-amplified neuroblastoma

Abstract

Neuroblastoma is a pediatric malignancy that arises from the neural crest, and patients with high-risk neuroblastoma, which typically harbor amplifications of MYCN, have an extremely poor prognosis. The tyrosine hydroxylase (TH) promoter-driven TH-MYCN transgenic mouse model faithfully recapitulates many hallmarks of human MYCN-amplified neuroblastoma. A key downstream target of Myc oncoproteins in tumorigenesis is ornithine decarboxylase (Odc), the rate-limiting enzyme of polyamine biosynthesis. Indeed, sustained treatment with the Odc suicide inhibitor alpha-difluoromethylornithine (DFMO) or Odc heterozygosity markedly impairs lymphoma development in Emicro-Myc transgenic mice, and these effects are linked to the induction of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1), which is normally repressed by Myc. Here, we report that DFMO treatment, but not Odc heterozygosity, impairs MYCN-induced neuroblastoma and that, in this malignancy, transient DFMO treatment is sufficient to confer protection. The selective anticancer effects of DFMO on mouse and human MYCN-amplified neuroblastoma also rely on its ability to disable the proliferative response of Myc, yet in this tumor context, DFMO targets the expression of the p21(Cip1) Cdk inhibitor, which is also suppressed by Myc oncoproteins. These findings suggest that agents, such as DFMO, that target the polyamine pathway may show efficacy in high-risk, MYCN-amplified neuroblastoma.

Figure 1

Pediatric neuroblastoma with MYCN amplification express elevated levels of ornithine decarboxylase (ODC). A, Hierarchical clustering of MYCN-amplified and non-amplified 101 primary human neuroblastoma samples from Gene Expression Omnibus Series GSE3960. This clustering differentiated MYCN-expressing and non-expressing tumors. Genes within the polyamine pathway present in the Affymetrix microarray data are shown. Genes that are marked with an asterisk (*) are significantly different (p<0.05) between the MYCN-amplified and non-MYCN-amplified groups. B, Real-Time PCR analysis comparing the mRNA expression of MYCN and polyamine pathway genes ODC and SMS in human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134. The relative expression level of each gene is set at 1.0 for the SK-N-MC cell line and levels of mRNA are standardized to the expression of ubiquitin, which is not regulated by Myc oncoproteins. Standard error bars are provided for each cell line. The qRT-PCR analysis for other polyamine genes is provided in Supplementary Fig. S2. C, Western blot analysis of MYCN, ODC, and Actin in human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134.

Figure 2

DFMO selectively impairs the proliferation of MYCN-amplified human neuroblastoma. A, The change in cell cycle distribution of the human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134 upon DFMO treatment for 72 hours is shown. Cells were labeled with BrdU, harvested, and analyzed by FACS. The graph represents the difference in the average of two mock-treated samples and two DFMO-treated samples for each cell line (see also Supplementary Fig. S3). B, Human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134 were either mock-treated (solid line) or were treated with DFMO (5 mM, dashed lines) and cell number was determined daily. The graph shown represents the average cell count relative for three mock-treated samples and three DFMO-treated samples from each cell line. Standard error bars are provided.

Figure 3

DFMO-treatment suppresses neuroblastoma development in TH-MYCN transgenic mice. A, Survival curve of TH-MYCN transgenic mice untreated (H₂O), DFMO-treated, or DFMO-treated from weaning until 120 days of age. The blue arrow above the graph indicates the time (120 days of age) when a cohort of DFMO-treated mice was taken off the drug. Comparison of the survival curves by the Mantel-Cox Log-rank Test shows that the curves are significantly different (p=0.0050). B, Survival Curve of TH-MYCN;Odc^+/+ mice versus TH-MYCN;Odc^+/- littermates. Comparison of the survival curves by the Mantel-Cox Log-rank Test shows that the curves are not significantly different (p=0.6241).

Figure 4

DFMO-induced growth arrest of MYCN-amplified neuroblastoma is associated with the selective induction of p21^Cip1. A, qRT-PCR analysis comparing the ODC, p21^CIP1, and p27^KIP1 mRNA levels of in human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134 following DFMO treatment for 72 hr. The graph represents the average relative expression for two mock-treated samples and two DFMO-treated samples from each cell line. The relative expression level of each gene is set at 1.0 for the mock-treated samples of each cell line and levels of mRNA are standardized to the expression of ubiquitin, which is not regulated by Myc oncoproteins. Standard error bars are provided for each cell line. B, Western blot analysis of MYCN, p21^Cip1, p27^Kip1, p53, and Actin in human neuroblastoma cell lines SK-N-MC, SK-N-SH, IMR-32, and CHP-134 either mock-treated or DFMO-treated for 72 hr.

Figure 5

Neuroblastomas that develop in DFMO-treated TH-MYCN mice have elevated levels of p21^Cip1. A, Western blot analysis of MYCN, ODC, p21^Cip1, p27^Kip1, and tubulin levels in individual neuroblastoma arising in untreated or DFMO-treated TH-MYCN transgenic mice. B, qRT-PCR analysis comparing the levels of MYCN, ODC, p21^Cip1, and p27^Kip1 mRNAs in neuroblastoma that arose in either untreated (H₂O) or DFMO-treated TH-MYCN transgenic mice. The relative expression in individual tumor samples is indicated by the individual marks (circle for untreated; squares for DFMO-treated) on the scatter blot and the average relative expression for the group is indicated by the bar. The average relative expression level of each gene is set at 1.0 for the untreated group and levels of mRNA are standardized to the expression of ubiquitin, which is not regulated by N-Myc. qRT-PCR analysis shows the expression of other genes in the polyamine pathway was unaffected in the DFMO-treated cohort of neuroblastoma (Supplementary Fig. S6).