Abstract B32: The role of Exon 0 in mediating Mxi0 activity in neuroblastoma

Background: Neuroblastoma is the most common extracranial malignancy of childhood. The Myc family of proteins regulates cell growth and proliferation and has been implicated in the etiology of many cancers. MYCN amplified neuroblastoma is associated with a poor prognosis. Investigating specific tumor pathways will further our understanding of neuroblastoma pathogenesis and lead to future therapeutic options. Mxi1 is a member of the MAD family that inhibits N-Myc activity. Mxi0 is an alternatively spliced variant of Mxi1 with a different first exon (Exon 0) whose function has not been determined. These proteins appear to have differential functions in neuroblastoma pathogenesis. Objective: Elucidate the impact of Mxi1 and Mxi0 expression on neuroblastoma physiology and determine the role of Exon 0 in the function of Mxi0. Design: We created neuroblastoma cell lines with inducible expression of Mxi1 and Mxi0 to tightly control expression. Cell proliferation and survival were quantified using BrdU and MTT assays. Chemosensitivity was assessed by treating cells with doxorubicin or etoposide after induction of Mxi1 or Mxi0 expression. Cell viability was then measured by by MTT assay. To help examine the role of Exon 0, we utilized GFP tagged constructs of Mxi1, Mxi0, and Exon 0. These proteins were expressed in 293T cells and subcellular localization of Mxi1, Mxi0, and Exon 0 proteins was then detected by immunofluorescence. To assess the role of nuclear export in cell localization, cells were treated with leptomycin B. Results: Overexpression of Mxi1 inhibits N-Myc mediated cell proliferation. Conversely, overexpression of Mxi0 in neuroblastoma cell lines leads to enhanced proliferation, suggesting that Mxi0 has a counter-regulatory role to that of Mxi1. Compared with Mxi1, expression of Mxi0 results in cells becoming more chemoresistant. Examination of Mxi1 and Mxi0 subcellular location reveals that Mxi1 resides in the nucleus while Mxi0 is found primarily in the cytoplasm. Exon 0 alone also is found in the cytoplasm, indicating that this differential localization is determined by the presence of Exon 0. Finally, treatment with leptomycin B resulted in accumulation of Mxi0 in the nucleus, suggesting that it may cycle in and out of the nucleus in response to appropriate signals. 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 enhances chemosensitivity of neuroblastoma cells, while Mxi0 has the opposite effect. Exon 0 directs the cytoplasmic localization of Mxi0 and may play an important role in its differential function. A better understanding of how the interaction between Mxi1 and Mxi0 affects neuroblastoma physiology and how Exon 0 imparts the differential function of Mxi0 may aid in developing more effective targeted therapies to improve outcomes in children with neuroblastoma. Citation Format: Stephen J. Kirchner, James T. Bartram, D. Christian Ellis, Daniel S. Wechsler, Michael B. Armstrong. The role of Exon 0 in mediating Mxi0 activity in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B32.