Phenotype-specific store-operated calcium entry and the differentiation response in neuroblastoma cells /

Abstract

Understanding the fundamental molecular mechanisms that control the proliferationdifferentiation cellular switch and maintenance of the differentiated state is needed to fully harness the therapeutic potential for highly detrimental diseases such as cancer and neurodegenerative disorders. Intracellular free Ca2+ plays an essential role in the differentiation process and, more specifically, the ubiquitous Ca2+ signalling pathway; storeoperated Ca2+ entry (SOCE) is altered with differentiation. The SH-SY5Y neuroblastoma cancer cell line was utilised in this study to investigate the role of SOCE in phenotype-specific differentiation responses using morphological, biochemical and functional single cell, Ca2+ imaging techniques. Neuroblastoma is a paediatric malignancy of the sympathetic nervous system that is comprised of immature neural crest cells. Retinoic acid is used to treat neuroblastoma patients however many respond poorly, leading to aggressive disease progression. The SHSY5Y neuroblastoma-derived cell line consists of three morphologically distinct phenotypes; immature neuroblastic N-type cells, non-neuronal S-type cells and putative intermediate Itype cells, which exhibit variable tumourgenicity and can be induced to differentiate using 9- cis-retinoic acid (9cRA). The 9cRA-induced differentiation response of N-type and S-type populations involved morphological changes accompanied by an uncoupling of SOCE from Ca2+ store release that could be observed from the first day of 9cRA treatment. SOCE down-regulation was attributed to changes in expression and localisation of the CRAC channel protein Orai1 and the Ca2+ sensing protein STIM1. The extent of SOCE uncoupling was influenced in N-type and I-type cells but not S-type cells by the predominant background cell environment. Conditioned media from proliferating and differentiating N-type and S-type populations was also able to influence cell phenotype and the associated SOCE responses. This study describes how the 9cRA-induced differentiation response occurred in a multi-step manner in N-type populations and in gradual manner in S-type populations and raises the possibility that SOCE proteins could potentially be utilised as drug targets in neuroblastoma treatment or neurodegenerative disease therapy.