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

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

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.

Technical Summary

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.
 
Description BBSRC Impact Acceleration Account (IAA)
Amount £5,000 (GBP)
Funding ID BH152503 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2016 
End 06/2016
 
Description MRC PhD Studentship
Amount £35,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2008 
End 09/2013
 
Description PhD studentship/Randerson Foundation
Amount £80,000 (GBP)
Organisation Newcastle University 
Department Randerson Foundation
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 09/2015
 
Description Wellcome Trust Equipment Grant
Amount £444,300 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2009 
End 06/2014
 
Description RNA seq 
Organisation Newcastle University
Department Institute for Cell and Molecular Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of cells (differentiated and undifferentiated SH-SY5Y cells) for RNA seq analysis and fluorimetric data (Ca2+ signalling) from these cell types.
Collaborator Contribution RNA seq expertise and analysis
Impact Data included thesis of PhD student. A full paper is in preparation.
Start Year 2015
 
Description Wound healing 
Organisation Newcastle University
Department Institute of Cellular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Studies using single cell imaging techniques to determine the intracellular concentration and distribution of calcium ions
Collaborator Contribution The wound healing biological system (keratinocytes)
Impact Full paper currently under review in Journal of Cellular Physiology (March 2021)
Start Year 2019
 
Description amino acid transport 
Organisation Newcastle University
Department Institute for Cell and Molecular Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution expertise in intracellular calcium imaging techniques and neuronal cell lines
Collaborator Contribution expertise in amino acid transport mechanisms
Impact Conference abstract (FASEB J, 2013)
Start Year 2009
 
Description metal binding 
Organisation Newcastle University
Department Institute for Cell and Molecular Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental technique (confocal microscopy) and intellectual input.
Collaborator Contribution Expertise in metal chelation and bacterial biochemistry
Impact PMID: 18948958
Start Year 2007
 
Description patch-clamp 
Organisation University of Washington
Department Department of Pharmacology
Country United States 
Sector Academic/University 
PI Contribution Expertise in calcium signalling in electrically excitable cells and training of staff
Collaborator Contribution provision of technical expertise and equipment
Impact PMID: 16806464
 
Description retinoids 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in calcium signalling mechanisms in neuronal cells and confocal microscopy
Collaborator Contribution Expertise in retinoid-induced differentiation of cells
Impact Several publications, notably PMID 16914200. See also entry for G0000190 PhD studentship funded
 
Description retinoids 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in calcium signalling in neuronal cells
Collaborator Contribution Expertise in retinoid-induced differentiation of cells
Impact PMID:15673285 PMID:16095688 PMID:16914200 PhD studentship funded
 
Description ryanodine 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in mammalian ryanodine receptor signalling mechanisms
Collaborator Contribution MRC Functional Genomics Unit: Expertise in insect signalling mechanisms
Impact PMID: 18696132
Start Year 2007
 
Description urology 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution measurements of changes in concentration of intracellular Ca2+
Collaborator Contribution increased understanding of mechanisms of smooth muscle cell differentiation
Impact PMID: 20546875 multi-disciplinary: molecular and cellular techniques
Start Year 2006
 
Description Bristish Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Members of the general public attended the workshop which was run each hour throughout the day
Year(s) Of Engagement Activity 2013
 
Description Institute booklet 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Institute research booklet, aimed at the public

Unknown
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description School Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public Open Days - c5 per year. for prospective undergraduate and postgraduate students, and their parents.

Increase in number of 'Unconditional/Conditional Firm' UCAS applicants to School (Department). Info: Board of Studies, School of Biomedical Sciences
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019,2020
 
Description School children - Sunderland 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Field questions on email from primary school children in Sunderland who wanted to talk to a 'real scientist'.

The children were very pleased.
Year(s) Of Engagement Activity 2007,2008,2009
 
Description School pupils - work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact School pupils work in the lab during vacation for work experience (ongoing dates)

increase in requests for information on career pathways to become a researcher
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016
 
Description Website - public 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact General University website

Unkown
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012,2013,2014,2015,2016
 
Description Website - specialised 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Personal and Institute websites

Contact from prospective PhD students and post-doctoral researchers
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015,2016