Calcium signalling and differentiation in neuroblastoma cells

Abstract

Neuroblastoma is a cancer of the sympathetic nervous system derived from neural crest cells that fail to differentiate during development. Neuroblastoma tumours and cell lines are heterogeneous, comprised of ‘neuroblastic’ N-type cells, precursors to a neuronal neural crest cell lineage and ‘substrate-adherent’ S-type cells, precursors to a non-neuronal neural crest cell lineage. Retinoids, such as retinoic acid (RA), cause both N- and S-type cells to switch from proliferation to differentiation. This underlies the use of RA in the treatment of neuroblastoma disease. The aim of this study was to investigate the role of Ca2+ signalling in the process of differentiation. N- and S-type cell populations were enriched from the SH-SY5Y neuroblastoma cell line to allow characterisation of Ca2+ signalling and differentiation within the two cell phenotypes. The RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in store-operated Ca2+ entry (SOCE) in N-type cells but not in S-type cells. In N-type cells expression of the ER Ca2+ sensor protein STIM1 and the channel protein Orai1 also became down-regulated, whilst expression of the channel protein TRPC1 became up-regulated. Knockdown of STIM1 and Orai1 in proliferating N-type cells down-regulated SOCE. Knockdown of Orai1, but not STIM1, induced differentiation and also enhanced differentiation induced by RA. Overexpression of STIM1 and Orai1 in RA-differentiated cells restored SOCE and reduced the extent of differentiation. Knockdown of TRPC1 had no effect on SOCE or differentiation in proliferating N-type cells but reduced the extent of SOCE down-regulation and differentiation induced by RA. These observations suggest that Orai1 may be a negative regulator of differentiation in N-type cells whereas STIM1 down-regulation may be required to maintain the differentiated state. TRPC1 expression may be required for a fully functional differentiated phenotype. These proteins could represent putative drug targets in the multi-modal treatment of neuroblastoma disease.