Investigating the role of P-Rex1 in mouse models of melanoma

Lead Research Organisation: Beatson Institute for Cancer Research
Department Name: Research Group 18 (Owen Sansom)

Abstract

The skin cancer malignant melanoma (MM) is increasing in rate more than any other common cancer in the UK, and is doubling in rate every 10-20 years in Western countries. Early spread of MM to other organs is nearly always the cause of death from this cancer. Identifying and studying new proteins which are both responsible for this spread and treatable is crucial for this cancer which is known for being unresponsive to traditional therapies.

We have genetically removed a protein called P-Rex1 from black mice, and discovered that their bellies and feet remain white. These white areas suggest that melanocytes (the skin cells responsible for MM) aren‘t reaching the furthest regions of migration during normal mouse development. We also know that the genetic changes required for melanocyte movement during development are very similar to the genetic changes required during MM tumour spread. Therefore we have obtained mice which develop human-like melanomas and I plan to assess if removing P-Rex1 can stop the lethal migration of melanocytes (known as metastasis) to other organs. I will also analyse resected human melanoma specimens to evaluate P-Rex1 in a comprehensive manner which is correlated with how well the patient did with their melanoma.

Technical Summary

Aims: The incidence of malignant melanoma (MM) is increasing more than any other common cancer in the UK. Worldwide its incidence is doubling in rate every 10-20 years in countries with white populations. Metastasis is almost invariably the ultimate cause of death in MM. My aim is to study the impact of P-Rex1 on metastasis in humanised mouse models of melanoma.

Hypothesis: Several humanised mouse models of human melanoma have been characterised, reflecting both common gain-of-function mutations (BrafV600E, NrasQ61K) as well as loss-of-function in known MM tumour-suppressor genes (PTEN, CDKN2A). We have crossed a P-Rex1 knockout mouse to a pure C57BL6 background, revealing a previously uncharacterised ‘white belly‘ phenotype with matching white feet and tail. These areas are the extremities of melanoblast migration during embryogenesis, suggesting a migratory deficit of the cells. Previous work done in our laboratory has also shown P-Rex1 to be consistently up-regulated in a host of melanoma cell lines. I therefore hypothesised that P-Rex1 up-regulation is a central component of the melanocyte migration involved in melanoma metastasis.

Objectives:
1. To study incidence, latency, and localisation of MM primary tumours and metastasis between P-Rex1 knockout and WT mice crossed to both BrafV600E and NrasQ61K melanoma mouse models.
2. To apply both GFP and DCT-lacZ reporter constructs to the above genotypes in order to study melanoblast migration as a paradigm for MM metastasis.
3. To perform a comprehensive survey of P-Rex1 expression in a host of human melanoma samples (including tissue microarrays) and correlate with outcome data.

Design & Methodology:
We already have access to characterised BrafV600E and NrasQ61K mouse models of MM, as well as P-Rex1 knockout mice. With these mice I plan to perform a comprehensive cohort comparison study to answer my hypothesis with regard to objective 1. To fully characterise the MM model(s) I will use histologicnd immunohistochemistry (IHC) techniques. The transgenic expression of GFP or ?-galactosidase under the control of melanocyte-specific promoters will allow me to assess morphology, speed, and localisation of melanoblasts in various forms of MM genetically modified mice. I will further consolidate the importance of these signalling pathways using clinical MM tissue microarrays (TMA) to explore the role of P-Rex1 in primary and metastatic human samples.

Medical and Scientific Opportunities: Identifying and characterising novel proteins which are responsible for metastasis and amenable to therapeutic intervention is a priority for MM, which is characterised by its resistance to traditional chemotherapeutics and ability to metastasise early.

Publications

10 25 50
 
Description Murine melanoma allograft development
Geographic Reach Local/Municipal/Regional - UK Only 
Policy Influence Type Influenced training of practitioners or researchers
Impact I have led development and characterisation of murine melanoma allografts following subcutaneous and tail-vein injection of immuno-competent mice. I have disseminated my experience to other groups within the institute, primarily to advise how they can best use in vivo models of melanoma to characterise their tissue culture results.
 
Title Melanoblast migration and cell number assays 
Description Melanoblast migration and cell number assays using DCT-lacZ mice 
Type Of Material Data analysis technique 
Year Produced 2009 
Provided To Others? Yes  
Impact results in recent Nature Comms paper 
 
Title Melanoblast speed and distance assays 
Description Assays developed to study dynamic characteristics of melanoblast movement using live imaging 
Type Of Material Data analysis technique 
Year Produced 2011 
Provided To Others? Yes  
Impact results in recent Nature Comms paper 
 
Title Nras Prex melanocyte cell lines 
Description Cell lines derived from pup skin of various Nras and Prex genotypes 
Type Of Material Cell line 
Year Produced 2011 
Provided To Others? Yes  
Impact None as yet 
 
Title murine melanoma allograft model 
Description Cell lines derived from murine melanocytes (described previously) re-injected into immuno-competent C57Bl6 mice by both subcutaneous and tail-vein routes. Served as nice models of primary and metastatic melanomagenesis for with which we could apply genetic manipulations. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact Results in recent Nature Comms paper. 
 
Description ICR, London 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution Braf mice at an advanced stage of breeding with Prex mice central to grant application. Various comparisons of cancer characteristics between Prex wild-type and knockout mice are underway.
Collaborator Contribution Provided oncogenic Braf mice for breeding to Prex mice
Impact one paper do far, due for submission within 2 wks.
Start Year 2008
 
Description Institut Curie, Paris 
Organisation Marie Curie
Department Marie Curie Research Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Tyr Cre mice essential to cancer cohort and reporter studies described in grant application. Allows melanocyte-specific expression of various mutations of interest.
Collaborator Contribution Provided Tyr Cre mice, offered advice and expertise.
Impact one paper due for submission within 2 weeks
Start Year 2006
 
Description MRC, Edinburgh 
Organisation University of Edinburgh
Department MRC Centre for Inflammation Research
Country United Kingdom 
Sector Public 
PI Contribution DCT-lacZ mice now bred to Prex mice and embryological studies at an advanced stage.
Collaborator Contribution Provided DCT-lacZ mice, offered expertise and advice
Impact Similar interests between collaborators, so I wouldnt describe as multi-disciplinary. One paper so far is due for submission in next 2 weeks.
Start Year 2007
 
Description University of North Carolina 
Organisation University of North Carolina at Chapel Hill
Country United States 
Sector Academic/University 
PI Contribution Collaborative immunohistochemistry, co-ordinated between 2 centres
Collaborator Contribution Examining human tissue immunohistochemically, offering expertise and advice
Impact paper due for submission within 2 wks.
Start Year 2006
 
Description Beatson Institute Open Evening 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Approximately 10 people attended a tour of the institute with myself. These consisted of school pupils, as well as patients and relatives affected by cancer. I used my MRC-funded research as an example of how translational cancer is performed here.

I have been asked to participate in such events again in the future, and will likely be presenting to larger public audiences at some point.
Year(s) Of Engagement Activity 2011