Abstract A6: Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis
Authors: Baker Amanda F. , Malm Scott , Laughren Cindy , K Setsuko 
Published: August 2014

Ovarian cancer is characterized by genomic and microenvironmental heterogeneity, both of which contribute to tumor progression, metastasis, and drug resistance. Hypoxia, or low oxygen tension, is one microenvironmental factor that influences genomic instability and promotes angiogenesis and metastasis. We investigated the gene expression levels of hypoxia-regulated genes in matched ovarian serious carcinoma primary and peritoneal metastasis from five patients. The hypoxia gene set used was based on a published metagene hypoxia signature established using breast and head and neck cancer samples by Buffa et al, 2010. To ensure that the genes selected were also hypoxia-regulated in ovarian tumor cells, we grew the Hey and Ovcar-3 ovarian cancer cell lines under normoxia, 1.0% and 0.2% oxygen for 4, 16, and 48 hours. RNA was extracted and cDNA prepared for quantitative real-time polymerase chain reaction (qRT-PCR). Each sample was analyzed in triplicate and normalized for loading using ribosomal protein, large, PO (RPLP0). Relative gene expression was calculated by comparing hypoxia treated samples to the normoxic control grown for the same length of time. The Hey cells were also grown in an intraperitoneal xenograft model and tumor tissue harvested following 22 days of growth. Primary tumor gene expression was normalized to the Hey cell line grown in normoxia. The metastatic tissue was also compared relative to each matched primary sample. From an initial list of 16 genes, we identified 9 genes that demonstrated robust increases in gene expression across treatment conditions in vitro in both OVCAR 3 and Hey cells: vascular endothelial growth factor (VEGF-A), solute carrier family 2 (SLC2A1; Glut-1), phosphoglycerate mutase 1 (PGAM1),lactate dehydrogenase A (LDHA), prolyl 4-hydroxylase, alpha polypeptide (P4HA1), adrenomedullin (ADM), v-yc myelocytomatosis viral related oncogene ( NDRG1; N-myc), aldolase A (ALDOA), carbonic anhydrase 9 (CA9). Xenograft tissue showed high overexpression of all 9 of these hypoxia regulated genes when normalized to the Hey normoxia cell line control grown in vitro, with CA9 levels being maximally elevated. On average, primary tumors showed highest expression levels of VEGF and CA9 when normalized to the in vitro Hey cell normoxia control. When the primary tumor samples were compared to matched metastatic tissue there was a strong correlation between expression patterns of VEGF-A and SLC2A1. Two of five matched samples showed over 3 fold elevation in VEGF and SL2A1 in the metastatic tissue relative to primary tumor. Our data suggests that there may be significant heterogeneity between the primary and metastatic tumors which could lead to sampling bias when investigating hypoxia regulated genes or gene products such as VEGF-A and SL2A1 as prognostic or therapeutic biomarkers.

Citation Format: Amanda F. Baker, Scott Malm, Cindy Laughren, Setsuko K. Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A6.