Abstract 3744: G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations.

Abstract

Abstract Neuroblastoma is a neural crest derived malignancy arising form peripheral sympathetic nervous system of infants and young children. We have recently defined a highly tumorigenic sub-population of neuroblastoma cells based on surface expression of CD114 (the G-CSF receptor, CSF3R). These cells meet the operational criteria for cancer stem cells based on their self-renewal, differentiation and lineage restricted tumorigenicity, and can be isolated directly from primary tumor biopsy specimens. As STAT3 activation is a canonical downstream event of G-CSF receptor ligation, is critical for neuronal specification, and is implicated in cancer stem cell maintenance, we hypothesized that inhibition of the STAT3 pathway may functionally inhibit the cancer stem cell-like neuroblastoma subpopulation. We used pathway-specific qPCR arrays to evaluate differential gene expression in CD114+ and CD114- tumor subpopulations. As predicted, STAT3 target genes were up-regulated in the CD114+ stem cell-like population. In addition, critical genes controlling early neural crest specification, cell cycle progression, self-renewal, and maintenance of pluripotency were up-regulated in CD114+ population while markers of mature mesenchymal neural crest were increased in CD114- population. Treatment of Neuroblastoma cell lines with G-CSF increased both CD114 expression and STAT3 transcriptional activation in a dose and time dependent manner. Phospho-flow analysis confirmed STAT3 phosphorylation (Y705) in response to exogenous G-CSF. The addition of a STAT3 inhibitor which prevents phosphorylation and dimerization of STAT3 (Stattic), specifically abrogated the effect of exogenous G-CSF treatment. Finally, we identified putative STAT3 binding sites within the 5’UTR of the CSF3R gene and confirmed binding of STAT3 and pSTAT3 using chromatin immunoprecipitation analysis (ChIP-qPCR). We demonstrate significant enrichment of binding in response to exogenous G-CSF within CD114 receptor positive subpopulation in contrast to CD114- population. These data support a model for a positive-feedback loop where G-CSF triggers STAT3 dependent up-regulation of the G-CSF receptor, further sensitizing these cells to exogenous ligand, promoting STAT3 transcriptional activation, and contributing to stem cell maintenance. Our data support further evaluation of the potential clinical role for specific STAT3 inhibitors as anti-cancer stem cell therapy in neuroblastoma. Citation Format: Saurabh Agarwal, Zaowen Chen, Danielle Hsu, Eugene Kim, Jason M. Shohet. G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3744. doi:10.1158/1538-7445.AM2013-3744