Abstract B37: The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity

Background: Neuroblastoma is the most common extracranial malignancy of childhood. Myc family proteins regulate cell growth and proliferation in response to mitogenic stimulation, and Myc proteins are implicated in the etiology of many cancers. MYCN amplified neuroblastoma carries a poor overall survival with limited therapeutic options. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options for children with this disease. Mxi1 is a member of the MAD family of proteins that inhibit N-Myc function. Mxi0 is an alternatively-spliced variant of Mxi1 whose function has not been determined. We hypothesize that Mxi1 and Mxi0 expression levels affect N-Myc-dependent neuroblastoma cell growth. Objective: To determine the impact of modulating Mxi1 and Mxi0 expression on N-Myc-dependent neuroblastoma cell proliferation and survival. Design: We expressed Mxi1 and Mxi0 in SHEP neuroblastoma cells and SHEP cells stably transfected to express high levels of MYCN (SHEP/ MYCN ). We also utilized native neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0. Cell proliferation and survival were quantified using BrdU and MTT assays, respectively. Apoptosis was measured by propidium iodide staining and caspase-3 immunohistochemistry. Cellular localization of Mxi1 and Mxi0 proteins was detected by immunofluorescence. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Additionally, in the absence of N-Myc, Mxi1 overexpression independently inhibits cell proliferation and induces cell apoptosis. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Expression of Mxi1 increased sensitivity of the neuroblastoma cells to doxorubicin, while higher levels of Mxi0 made the cells more chemoresistant. Finally, examination of Mxi1 and Mxi0 cellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Conclusions: Overexpression of Mxi1 in neuroblastoma cell lines leads to inhibition of N-Myc-mediated cell proliferation while Mxi0 appears to promote cell growth. Mxi1 expression enhanced chemosensitivity of neuroblastoma cells, while Mxi0 had the converse effect. A better understanding of the interaction between Mxi1 and Mxi0 and how the balance of these proteins affect neuroblastoma physiology may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: D. Christian Ellis, R. Scott Widemon, Daniel S. Wechsler, Michael B. Armstrong. The impact of modulating Mxi1 and Mxi0 expression on N-Myc-mediated neuroblastoma tumor pathogenesis and chemosensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B37.