Abstract A2-55: Identification of evolutionarily conserved hypoxia-induced genomic pathways responsible for aggressive neuroblastoma phenotypes
Authors: Applebaum Mark A. , Jha Aashish R. , Chlenski Alexandre , Hernandez Kyle , Mariani Christopher J. , Stranger Barbara E. , Cohn Susan L. 
Published: November 2015

Abstract
Background: In cancer, hypoxia leads to a clinically aggressive phenotype by inducing angiogenesis, altering metabolism, and promoting cell infiltration, invasion and metastasis. Hypoxia also results in specific adaptive genetic changes in Drosophila melanogaster and many other species. We hypothesized that an evolutionarily conserved transcriptional program exists in Drosophila, neuroblastoma cell lines, and primary neuroblastoma tumors conferring both a survival advantage in Drosophila bred in hypoxia and aggressive tumor behavior.
Methods: RNA-seq data from neuroblastoma cells grown in normoxia or hypoxia were analyzed to identify differentially expressed genes (DEG). These data were compared to previously identified human orthologous DEG from Drospohila bred in normoxia or hypoxia. The analysis was validated using public microarray expression data from 11 additional neuroblastoma cell lines cultured in normal or hypoxic conditions (GEO accession GSE17714). In order to evaluate these genes in patients, gene expression microarray data from 479 primary neuroblastoma tumors (EMBL accession E-MTAB-179) were analyzed with respect to patient survival status.
Results: Pathway analysis of genes differentially expressed in Drosophila bred in hypoxia revealed enrichment for genes of the citric acid cycle (p = 13.1x10-17), pyruvate metabolism (p = 5.4x10-10), and glycolysis (p = 4.1x10-7). Analysis of neuroblastoma cells grown in hypoxia identified DEG in steroid biosynthesis (p = 1.3x10-13), HIF-1α transcription network (p = 3.3x10-13), and glycolysis (p = 3.2x10-6). 222 common DEG in neuroblastoma cells and Drosophila were enriched for glycolysis (p = 2.1x10-8), gluconeogenesis (p = 1.2x10-5) and the HIF-1α transcription network (p = 7.2x10-5). Up-regulation of glycolysis genes was also detected in the 11 additional neuroblastoma cell lines cultured in hypoxia. These cell lines showed additional differential expression in Ataxia telangiectasia and Rad3 (ATR) DNA damage sensing genes and general cell cycle related genes (p = 2.9x10-9 and p = 1.5x10-5, respectively). Of the 479 primary tumor samples with median follow up time of 3.75 years, there were 119 stage 1, 80 stage 2, 69 stage 3, 148 stage 4, and 62 stage 4S patients of whom 91 are deceased. In the analysis of primary tumors, decreased expression of cell cycle and metabolism genes (p = 4.8x10-13 and p = 1.6x10-12, respectively) was associated with survival. Thirty-nine of the DEG from patients, including multiple metabolism genes such as PGK1, GPI, and ACSS2, were common for both Drosophila and cell lines exposed to hypoxic conditions.
Conclusions: Hypoxia alters metabolism in Drosophila, and neuroblastoma cell lines, and similar changes are associated with clinically aggressive phenotype in primary neuroblastoma tumors. Changes in cell cycle gene expression were also common in neuroblastoma cell lines grown in hypoxia and in clinically aggressive primary tumors. Further analysis of these pathways and their regulation will allow us to identify patients at high risk and provide insight to personalize therapy.
Citation Format: Mark A. Applebaum, Aashish R. Jha, Alexandre Chlenski, Kyle Hernandez, Christopher J. Mariani, Barbara E. Stranger, Susan L. Cohn. Identification of evolutionarily conserved hypoxia-induced genomic pathways responsible for aggressive neuroblastoma phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-55.