Abstract A127: Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across neuroblastoma cells lines: new prognostic marker and therapeutic target

Abstract

Abstract Butrylcholinesterase (BChE) is a glycoprotein found in the peripheral and central nervous system including in neuroblastoma, a tumor of the peripheral sympathetic nervous system. The use of BChE as a tumor biomarker and potential therapeutic target has been proposed. The purpose of this study was to assess the association between BChE and tumorigenicity in two NB cell lines and their respective stem cell populations. Two MYCN amplified neuroblastoma cell lines, BE-2 (highly MYCN amplified) and IMR-32 (intermediately MYCN amplified) were studied along with neuroblastoma stem cells identified by side population (SP) in flow cytometry. MYCN protein, which is a poor prognostic proliferative marker in neuroblastoma, was measured by western blot in each of the two cell lines to characterize and confirm the amplification phenotype. Cell cycle characteristics of each cell line was evaluated using a propidium iodide based analysis by flow cytometry. BChE protein was measured using western blot in the two cell lines as well as in their SP and non-SP (NSP) populations after sorting. An MYCN non-amplified neuroblastoma cell line (SH-SY5Y) was tested for both MYCN and BChE protein as a potential low MYCN and BChE expressing tumor. The metabolic profile (glycolysis and OXPHOS) was determined for both unsorted, SP and NSP cells using Seahorse® metabolic analysis. BE-2 and IMR-32 tumorigenicity was characterized by growing cells subcutaneously in the flanks of NSG immunodeficient mice. BE-2c tumors developed earlier and grew more rapidly than IMR-32 tumors after injection of a similar number of cells. MYCN protein was similarly expressed in BE-2c and IMR-32 despite higher in-vivo tumorigenicity of BE-2c. In contrast, BChE was higher in BE-2 compared to IMR-32 corresponding to the order of tumorigenicity in-vivo. BE-2 and IMR-32 SP stem cells were relatively quiescent as measured by lower BChE protein compared to NSP cells and lower metabolic rates as measured by extracellular acidification (glycolysis) and oxygen consumption (OXPHOS metabolism). Cell cycle analysis and cell growth curves support BE-2 being a more proliferative cell line than IMR-32. MYCN has been the traditional marker to identify aggressive poor prognostic NB but data presented suggests BChE may also identify high risk disease generally correlating with growth of tumor in NSG mice and cellular growth characteristics. The use of small molecule inhibitors of BChE are currently under pre-clinical evaluation and given the relationship between BChE expression and tumorigenicity, BChE may be a promising new target. Ongoing work in our laboratories is investigating mechanisms of association between BChE and high-risk features of NB. Citation Format: Katie Greenwood, Timothy R. McGuire, Donald Coulter, Erin McIntyre, Nagendra K. Chaturvedi, Paul Kortylewicz, Shantaram S. Joshi, Xiaoyu Chen, John G. Sharp, Janina Baranowska-Kortylewicz. Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across neuroblastoma cells lines: new prognostic marker and therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A127.