p53 family members and apoptosis in malignant melanoma: a role in chemoresistance?

Lead Research Organisation: Queen Mary, University of London
Department Name: Blizard Institute of Cell and Molecular

Abstract

We are investigating why melanoma, an aggressive form of skin cancer, is so resistant to chemotherapy, and how new treatments might be able to overcome this resistance. In the UK 8,100 new cases of melanoma are diagnosed annually with the incidence predicted to treble in the next 30 years. Melanoma arises from the pigment-producing cells (melanocytes) in skin and both its development and resistance to standard cancer treatments is thought in part due to the resistance of cells to apoptosis - a form of cell death. Essential to apoptosis is the p53 gene and loss of p53 activity is frequently implicated in other cancers. p53 also plays a crucial role in protecting against skin cancer induced by ultraviolet radiation (UVR). The aim of this project is to study the p53 family of proteins and their interacting roles in UVR-induced cell death in melanocytes and melanoma. Melanoma represents a major burden on public health and healthcare resources. This project will determine the mechanism of p53 inactivation in melanoma. Information obtained will further our understanding of changes in melanocytes as they undergo malignant progression and provide new ways to develop treatments that are effective.

Technical Summary

Malignant melanoma is the most aggressive form of skin cancer. Its incidence has increased more rapidly than that of any other cancer in the last 30 years. Advanced disease has poor prognosis and is notoriously resistant to all current therapeutic modalities. Unlike many other malignancies, p53 mutations are infrequent in melanoma implicating aberrant upstream or downstream components of the p53 signalling pathway. In the proposed study the hypothesis that the p53 family members are determinants of chemosensitivity and chemoresistance in melanoma will be tested.

The specific aims of the study are to assess the integrity of the p53 signalling pathway including downstream target genes and co-activators of p53 family members in normal melanocytes and melanoma cell lines and to relate this to cellular response to genotoxins including ultraviolet radiation (UV) and cytotoxic drugs. Experiments will first establish the existence of post-translational modifications (specifically phosphorylation and acetylation) of p53 and p73 following genotoxic stress in melanocytes and melanoma cell lines. Using p53 target gene micro-arrays the p53 transcriptosome will be analysed following genotoxic stress in primary melanocytes and melanoma cell lines of varying sensitivity to DNA damage. Expression patterns of pro-apoptotic co-activators will be monitored (in collaboration) to seek correlations between patterns of target gene expression, co-activator expression and cellular response to DNA damage.

Expression patterns of each p53 family member and their splice variants will be established in control cultures and in tissue samples obtained prospectively. The role of individual p53 family members in UV-induced apoptosis will be examined by FACS analysis by exposing p53 wild-type and p53 null melanoma cell lines to different doses of UVB, UVA and DNA-damaging agents. Experiments to upregulate (by transfection or lentiviral infection) and/or deplete p63, p73 and their splice variants (by siRNAi and micro-siRNAi) will determine the chemosensitivity of melanoma cells to relevant genotoxic stimuli. Melanoma cell lines and a spectrum of melanocytic lesions from the patient cohort will also be screened for p53 polymorphic variants to investigate their synergistic roles with p53 family members in UV-induced apoptotic cell death and correlation with clinical outcomes respectively.

The proposed work combines both functional/mechanistic studies and the molecular pathological analysis of tumour samples to assess the clinical significance of in vitro results. Determining the molecular basis of the intrinsic resistance of melanocytes to apoptosis during malignant progression may provide insight into potential therapeutic targets for overcoming their acquired chemoresistance.

Publications

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Matin RN (2013) Synchronous melanoma and renal carcinoma: a clinicopathological study of five cases. in Clinical and experimental dermatology

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Matin RN (2011) Cutaneous mucinous carcinoma arising in extramammary Paget disease of the perineum. in The American Journal of dermatopathology

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Matin RN (2008) Melanoma in organ transplant recipients: clinicopathological features and outcome in 100 cases. in American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

 
Description BSF PhD Studentship
Amount £81,000 (GBP)
Funding ID Title: Dissecting the role of iASPP, a novel crucial regulator of epidermal homeostasis, in keratinocyte skin carcinogenesis. 
Organisation British Skin Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 10/2015
 
Description Barts and the London Charitable Foundation/CRUK project grant
Amount £162,862 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Barts and the London Charitable Foundation/CRUK project grant
Amount £99,858 (GBP)
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Barts and the London Charity Research Fund
Amount £18,400 (GBP)
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Barts and the London Charity Strategic Research Grant
Amount £138,800 (GBP)
Funding ID Title: Exploring novel molecular targets in non-BRAF mutated metastatic melanoma. 
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 11/2013
 
Description Melanoma Focus Patient Impact Project
Amount £10,000 (GBP)
Organisation Melanoma Focus 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description PhD studentship
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description PhD studentship 2
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Research Grant Award
Amount £15,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Title Tissue Bank 
Description Melanoma tissue samples and cell lines of varying stage of disease. 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2008 
Provided To Others? Yes  
Impact Investigation of aberrations in other molecular pathways e.g. Wnt signalling pathway 
 
Description ASPP Proteins 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Involvement of the ASPP proteins in Autophagy
Collaborator Contribution Involvement of the ASPP proteins in Autophagy
Impact Publication listed
Start Year 2010
 
Description Characterisation of melanoma 
Organisation Fondazione IRCCS Istituto Nazionale dei Tumori
Country Italy 
Sector Charity/Non Profit 
PI Contribution We have provided the melanoma cell lines in which to determine the mutation status
Collaborator Contribution Established melanoma cell lines with characterized status of BRAF/NRAS mutationsGenome wide microarray analysis and microRNA profiling of melanoma cell lines silenced for p63Dissecting the role of YAP in melanomaApoptosis and DNA repair in melanoma
Impact Multi-disciplinary - resulted in characterisation of mutation status of melanoma lines which has led to further funding and included in publication (pending)
Start Year 2006
 
Description Characterisation of melanoma 
Organisation Fondo Edo Tempia Biella
Country Italy 
Sector Charity/Non Profit 
PI Contribution We have provided the melanoma cell lines in which to determine the mutation status
Collaborator Contribution Established melanoma cell lines with characterized status of BRAF/NRAS mutationsGenome wide microarray analysis and microRNA profiling of melanoma cell lines silenced for p63Dissecting the role of YAP in melanomaApoptosis and DNA repair in melanoma
Impact Multi-disciplinary - resulted in characterisation of mutation status of melanoma lines which has led to further funding and included in publication (pending)
Start Year 2006
 
Description Characterisation of melanoma 
Organisation National Cancer Institute (CRO)
Country Italy 
Sector Academic/University 
PI Contribution We have provided the melanoma cell lines in which to determine the mutation status
Collaborator Contribution Established melanoma cell lines with characterized status of BRAF/NRAS mutationsGenome wide microarray analysis and microRNA profiling of melanoma cell lines silenced for p63Dissecting the role of YAP in melanomaApoptosis and DNA repair in melanoma
Impact Multi-disciplinary - resulted in characterisation of mutation status of melanoma lines which has led to further funding and included in publication (pending)
Start Year 2006
 
Description Characterisation of melanoma 
Organisation University of Calabria
Country Italy 
Sector Academic/University 
PI Contribution We have provided the melanoma cell lines in which to determine the mutation status
Collaborator Contribution Established melanoma cell lines with characterized status of BRAF/NRAS mutationsGenome wide microarray analysis and microRNA profiling of melanoma cell lines silenced for p63Dissecting the role of YAP in melanomaApoptosis and DNA repair in melanoma
Impact Multi-disciplinary - resulted in characterisation of mutation status of melanoma lines which has led to further funding and included in publication (pending)
Start Year 2006
 
Description Establishing a correlation between ?Np63 and IGFBP7 in melanoma cells 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Melanoma cell lines provision and data sharing
Impact Pending
Start Year 2010
 
Description Serum Epigenetic Biomarkers for Metastatic Melanoma 
Organisation University of Dundee
Department Centre for Oncology and Molecular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative study investigating biomarkers in melanoma relapse - retrospective and prospective collection of samples.
Collaborator Contribution Collaborative study investigating biomarkers in melanoma relapse - lab research and statistical analyses undertaken at this site.
Impact Pending
Start Year 2013
 
Description Targeting p63 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell lines, reagents and data sharing
Collaborator Contribution Publication listedThe role of TINT in melanoma metastasisEpigenetic regulation of novel target genes in melanomaEpigenetic regulation of novel target genes in melanoma
Impact Publication listed
Start Year 2007
 
Description Targeting p63 
Organisation Queen Mary University of London
Department Barts and The London School of Medicine and Dentistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell lines, reagents and data sharing
Collaborator Contribution Publication listedThe role of TINT in melanoma metastasisEpigenetic regulation of novel target genes in melanomaEpigenetic regulation of novel target genes in melanoma
Impact Publication listed
Start Year 2007
 
Description Targeting p63 
Organisation University of Cologne
Country Germany 
Sector Academic/University 
PI Contribution Cell lines, reagents and data sharing
Collaborator Contribution Publication listedThe role of TINT in melanoma metastasisEpigenetic regulation of novel target genes in melanomaEpigenetic regulation of novel target genes in melanoma
Impact Publication listed
Start Year 2007
 
Description Targeting p63 
Organisation University of Dundee
Department Dundee Cancer Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell lines, reagents and data sharing
Collaborator Contribution Publication listedThe role of TINT in melanoma metastasisEpigenetic regulation of novel target genes in melanomaEpigenetic regulation of novel target genes in melanoma
Impact Publication listed
Start Year 2007
 
Description World melanoma study 
Organisation Fondazione IRCCS Istituto Nazionale dei Tumori
Country Italy 
Sector Charity/Non Profit 
PI Contribution World melanoma study to compare multiple versus sporadic melanoma
Impact Pending
Start Year 2010
 
Description p63 expression 
Organisation Medical Research Council (MRC)
Department MRC Toxicology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Tools reagents and antibodies for detecting p63 expression in melanoma cell lines
Collaborator Contribution Publication pending and funding approvalTools reagents and antibodies for detecting p63 expression in melanoma cell linesCD133 expression in p63 positive melanoma cells
Impact Publication reported and one pending plus funding
Start Year 2007
 
Description p63 expression 
Organisation Technical University of Dresden
Country Germany 
Sector Academic/University 
PI Contribution Tools reagents and antibodies for detecting p63 expression in melanoma cell lines
Collaborator Contribution Publication pending and funding approvalTools reagents and antibodies for detecting p63 expression in melanoma cell linesCD133 expression in p63 positive melanoma cells
Impact Publication reported and one pending plus funding
Start Year 2007
 
Description p63 expression 
Organisation University of Rome Tor Vergata
Country Italy 
Sector Academic/University 
PI Contribution Tools reagents and antibodies for detecting p63 expression in melanoma cell lines
Collaborator Contribution Publication pending and funding approvalTools reagents and antibodies for detecting p63 expression in melanoma cell linesCD133 expression in p63 positive melanoma cells
Impact Publication reported and one pending plus funding
Start Year 2007
 
Description p63/p53 signalling in melanoma 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of melanoma cell lines, reagents and sharing of data
Collaborator Contribution Signalling pathwaysSignallng pathwaysp53 short isoforms and p53 Arg72Pro SNP in melanomaBak & p53 mitochondrial relocation in melanoma
Impact Publications listed and other outputs pending
Start Year 2006
 
Description p63/p53 signalling in melanoma 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution Provision of melanoma cell lines, reagents and sharing of data
Collaborator Contribution Signalling pathwaysSignallng pathwaysp53 short isoforms and p53 Arg72Pro SNP in melanomaBak & p53 mitochondrial relocation in melanoma
Impact Publications listed and other outputs pending
Start Year 2006
 
Description p63/p53 signalling in melanoma 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of melanoma cell lines, reagents and sharing of data
Collaborator Contribution Signalling pathwaysSignallng pathwaysp53 short isoforms and p53 Arg72Pro SNP in melanomaBak & p53 mitochondrial relocation in melanoma
Impact Publications listed and other outputs pending
Start Year 2006
 
Description p63/p53 signalling in melanoma 
Organisation University of Rome Tor Vergata
Country Italy 
Sector Academic/University 
PI Contribution Provision of melanoma cell lines, reagents and sharing of data
Collaborator Contribution Signalling pathwaysSignallng pathwaysp53 short isoforms and p53 Arg72Pro SNP in melanomaBak & p53 mitochondrial relocation in melanoma
Impact Publications listed and other outputs pending
Start Year 2006