Abstract 1442: Side population analysis and gene expression profiling identify Notch pathway genes in neuroblastoma

Abstract

Abstract High-risk neuroblastoma (NB) has a very poor prognosis. Upon relapse, NB is chemotherapy-resistant and almost universally fatal. Cancer stem-like cells have been identified in both primary tumors and in cell lines and may have inherent resistance to traditional chemotherapeutic agents. We previously investigated NB cancer stem-like cells using side population (SP) analysis of three pairs of NB cell lines derived from single patients at the time of diagnosis and at relapse after therapy. We reported that the proportion of SP cells in the relapsed cell lines was significantly increased compared with its paired pretreatment cell line. Based on these findings, we hypothesized that specific signaling pathways would be differentially expressed in the pre- versus post-relapse SP cells. Therefore, we performed gene expression profiling using Affymetrix whole-genome arrays on the SP fraction of the cell lines SMS-KCN (pre-treatment) and SMS-KCNR (relapse). Microarray analysis revealed 126 genes at least 2-fold up-regulated in the post-relapse SP cells compared to the pre-relapse SP fraction. Specifically, we identified several members of the Notch signaling pathway including Notch 2. To confirm the finding of increased notch expression, we measured mRNA expression in 84 genes involved in Notch signaling in SMS-KCN SP and SMS-KCNR SP cells using an RT*2 Profiler™ PCR Array. Multiple notch genes were confirmed to be upregulated from ∼1.5-fold (NOTCH1) to 15-fold (HES1) in the post-relapse compared to the pre-treatment cell line SP cells. Subsequently, mRNA expression levels of nine Notch pathway genes, NOTCH1-4, HES1, HEY1, HEYL, and JAG1 and 2, were measured in six paired cell lines (SMS-KCN and KCNR, SMS-KAN and KANR, and CHLA-122 and 136) by qRT-PCR and were increased in each of the unsorted post-relapse cell lines. To assess the functional role of Notch signaling, we treated four of the cell lines with a γ-secretase inhibitor (DAPT, 5µM) every 72 hours for 7 days which resulted in a >50% decrease in viability in all cell lines. Evaluation of the publicly available NCI NB gene expression profiling database (http://home.ccr.cancer.gov/oncology/oncogenomics/) showed that increased expression of NOTCH1, 2, and 3 correlated with a worse clinical outcome compared to those with low Notch expression levels. These results suggest that side population analysis coupled with gene expression profiling is a useful method for identifying novel pathways in NB and that upregulation of the Notch pathway may be important in the biology of NB cancer stem-like cells. Inhibition of the Notch pathway using γ-secretase inhibitors may provide new therapeutic options in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1442. doi:1538-7445.AM2012-1442