Abstract 4625: ASIC1 regulates neuronal differentiation of neuroblastoma through Notch signaling pathway

Background: Neuroblastoma arises when neuroblasts of the sympathetic nervous system fails to undergo proper differentiation. The Notch signaling pathway is critical for cell differentiation, growth and proliferation. In neurons, it has been shown that up regulation of Notch activity either inhibited neurite extension or caused retraction of neurites. Conversely, inhibition of Notch1 signaling facilitated neurite extension. Acid-sensing ion channels (ASICs) are proton-gated cation channels. In addition to the role in synaptic plasticity, learning and memory, a good correlation has been found between ASIC1a expression and spine density suggesting that ASIC1a plays an important role in the processes of spine morphogenesis, maintenance and remodeling which are involved in the process of neuronal differentiation. The underlying mechanism however remains elusive. We hypothesize that ASIC1 regulates neurite growth through Notch signaling in neuroblastoma. Methods: 1) We employed proteomic approach and pathway analysis to examine changes in Notch signaling by ASIC gene deletion. By comparison adult ASIC1a-/- and WT mice, quantitative mass spectrometric techniques of spectral counting followed by analysis with SEQUEST were used to identify reliable proteins. 2) We determined the effects of ASIC1a on neurite growth in a mouse neuroblastoma cell line, NS20Y cells by down regulating ASIC1a expression with shRNA or inhibiting ASIC1a function with PcTX, a specific pharmacological inhibitor. 3) We determined the effects of DAPT, a Notch signal inhibitor on neuronal differentiation in NS20Y cells. Results: 1) The enrichment analyses by the bioinformatics system Metacore showed that development Notch signaling pathway (p = 1.526E-05), development Notch-induced epithelial-mesenchymal transition (EMT, p = 3.902E-05) and development Notch-mediated pathway for NF-κB modulation (p = 2.323E-04) were changed in WT mice compared to ASIC1a-/-. 2) Down regulation of ASIC1a in NS20Y cells with shRNA inhibits CPT-cAMP induced neurite growth, while over expression of ASIC1a promotes its growth. 3) Down regulation of ASIC1 by shRNA increased Notch1 and its target gene Survivin in NS20Y cells. 4) DAPT, an inhibitor of Notch significantly protects inhibition of CPT-cAMP induced neurite extension by down-regulation of ASIC1 in NS20Y cells. Conclusion: These data indicate that Notch1 signaling may be required for ASIC1 mediated neurite growth and neuronal differentiation of neuroblastoma. Promoting neural differentiation of neuroblastoma in vitro provides insight into potential clinical applications in the treatment of patients with neuroblastoma (This work is supported by R01NS066027, NIMHD S21MD000101, and U54NS08932) Citation Format: Mingli Liu, Koichi Inoue, Zhi-gang Xiong. ASIC1 regulates neuronal differentiation of neuroblastoma through Notch signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4625.