Voltage-independent calcium entry and its role in the differentiation of neuronal cancer cell phenotypes

We are identifying defects in cell signalling pathways that are related to neuronal diseases. This will help in designing novel or improved chemotherapy drug treatments for cancers such as neuroblastoma disease.
We already know that a change in the level of calcium ions within neurones controls their normal function, such as the process of differentiation whereby the cells mature and stop growing. We have also found that, once neuroblastoma cells have differentiated, there is a key alteration in one of the pathways that changes the level of calcium ions in the cells.
What we don’t know, however, is exactly what this key alteration is and what controls it. Nor do we know whether the alteration is required for differentiation to occur, or to keep the cells in a differentiated state. By answering these questions we will be able to identify specific proteins in the calcium pathway that could be potential drug targets in the treatment of the disease.
It is not only patients with neuroblastoma disease who could benefit from our research. For example, some of the neuroblastoma cells that we use are so-called neuronal stem cells (NSC’s). NSC’s are important in the treatment of neurodegenerative brain diseases such as Alzheimer’s. Understanding the pathways of how NSC’s differentiate is therefore extremely important in its own right. We will be able to identify whether NSC’s have specific or ‘fingerprint’ calcium ion responses that define how these particular cells differentiate.

Human neuroblastoma cells differentiate in response to retinoids such as retinoic acid (RA), but not all cells in the population respond. Understanding the mechanisms underlying RA-induced differentiation, and its heterogeneity, will be key for the development of new and improved retinoid-based chemotherapeutic regimes that are used in the treatment of neuroblastoma disease, which is the most common solid-tumour cancer occurring in children. A key intracellular regulator of neuronal differentiation is a change in the concentration of cytosolic Ca2+ ([Ca2+]i), which controls gene transcription. One main mechanism by which an elevation in [Ca2+]i occurs is via the entry of Ca2+ from the extracellular medium, through plasma membrane Ca2+ channels. Such Ca2+ entry pathways include store-operated Ca2+ entry (SOCE), where entry is activated by depletion of intracellular Ca2+ stores, and non-SOCE, which is independent of store depletion. We will investigate these Ca2+ entry pathways in neuroblastoma cells, and determine their role RA-induced differentiation.

We will use human SH-SY5Y neuroblastoma cells, which contain a sub-population of putative neuronal stem cells. We have shown, firstly that undifferentiated SH-SY5Y cells possess a SOCE pathway that becomes downregulated in RA-differentiated cells whilst a non-SOCE pathway is upregulated concurrently. Secondly we have shown that Ca2+ signals can be restricted to phenotypic sub-populations of SH-SY5Y cells, raising the possibility that neuronal cancer stem cells may possess different, ‘fingerprint‘, Ca2+ entry or differentiation responses.

Objectives: 1) To determine the relationship between downregulation of SOCE, upregulation of non-SOCE, and differentiation in SH-SY5Y cell phenotypes, including putative stem cells. 2) To define the molecular mechanisms controlling SOCE and non-SOCE, and their role in the regulation of differentiation. We will investigate the roles played by the proteins Bcl-2, calcineurin, STIM1 and TRPC in these processes.

We will use confocal and epi-fluorescence single cell imaging techniques to measure Ca2+ entry (using fluorescent indicator dyes), differentiation and to assess cell phenotype (using immunofluorescence with specific markers), in combination with molecular biology techniques to alter the expression levels of wild type and mutant proteins.

This project will identify Ca2+ signalling pathways and proteins involved in the regulation of differentiation of human neuroblastoma cells. Since the induction of differentiation forms the basis of chemotherapeutic regimes used for the treatment of neuroblastoma disease, then these pathways and proteins will be potential chemotherapeutic drug targets. Results from the present study will therefore inform the design of more translational clinical investigations.