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.
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.
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.
People |
ORCID iD |
Tim Cheek (Principal Investigator) |
Publications

Bell N
(2013)
Store-operated Ca(2+) entry in proliferating and retinoic acid-differentiated N- and S-type neuroblastoma cells.
in Biochimica et biophysica acta

Clarke N.
(2007)
Changing the phenotype of urethral stroma by treatment with transforming growth factor beta-3
in EUROPEAN UROLOGY SUPPLEMENTS

Conlon Nichola J.
(2013)
Rheogenic amino acid transport by Drosophila CG4991 of the Amino Acid Auxin Permease (AAAP) transporter family
in FASEB JOURNAL

Edwards N
(2018)
Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters.
in Cellular and molecular life sciences : CMLS


Hudson L
(2021)
Dominant effect of gap junction communication in wound-induced calcium-wave, NFAT activation and wound closure in keratinocytes.
in Journal of cellular physiology

Sattelle DB
(2008)
Insect ryanodine receptors: molecular targets for novel pest control chemicals.
in Invertebrate neuroscience : IN

Tottey S
(2008)
Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.
in Nature

Whitworth CL
(2019)
Differentiation-Induced Remodelling of Store-Operated Calcium Entry Is Independent of Neuronal or Glial Phenotype but Modulated by Cellular Context.
in Molecular neurobiology
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 | 08/2008 |
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 | 08/2011 |
End | 09/2015 |
Description | Wellcome Trust Equipment Grant |
Amount | £444,300 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2009 |
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 |