Genetic Analysis of the Putative Oncogene INT6 in Zebrafish

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Molecular. Genetics & Pop Health

Abstract

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Technical Summary

Significant insight into cancer development and treatment is found, in part, by studying cancer genes in model systems, such as the zebrafish. Many genes are highly conserved between zebrafish and humans, and mutations in zebrafish cancer genes can induce tumours that strongly resemble human cancers. Here, I propose to use the zebrafish system to rigorously probe the function of the putative cancer gene, INT6, in multi-drug resistance and cancer development.
INT6 is highly conserved with a clear role in cancer development, although the mechanisms behind this involvement remain mysterious. In fission yeast, Int6 maintains genome integrity, and regulates the multi-drug exposure response. In mice, integration of a virus into the INT6 gene results in truncated alleles of INT6 that appear to act in a dominant manner to promote breast cancer formation. Interestingly, in human cancers, loss of INT6 gene expression is frequently detected suggesting Int6 may also participate in suppressing tumour formation.
For my first Aim, I will use gene knockdown and overexpression methods in zebrafish embryos to rapidly establish Int6 function in genome stability and multi-drug resistance. Genomic instability is a hallmark of cancer, and these studies will be greatly enhanced by collaborating with Dr. Charles Lee, Assistant Director, Harvard Cancer Center Cytogenetic Core.
For my second Aim, I will create transgenic and mutant zebrafish lines that mimic Int6 mutations found in mammalian cancers, and yeast multi-drug resistance strains. In collaboration with Dr. Leonard Zon (Children‘s Hospital Boston) I will identify Int6 loss-of-function alleles using a new method called TILLING that involves screening over 1500 F1 mutagenized fish for INT6 mutations. With an array of Int6 lines, I will ultimately determine the contribution of Int6 to tumourigenesis. As well, I will identify Int6 genetic interacting partners, focusing on those that reveal novel insights into Int6 mechanisms thatead to multi-drug resistance and increased rates of tumour development. Zebrafish tumour pathology will be in consultation with Dr. James Amatruda (Children‘s Hospital), who has established a carcinogenesis assay in zebrafish. Finally, I will correlate Int6 tumour promoting mutations found in the zebrafish to those found in human cancers.
These Aims will be the first to establish INT6 gene networks in vertebrates, as well as address the effects of multiple Int6 mutations in tumourigenesis. Many tumours acquire resistance to chemotherapy drugs, and pursuing the function of Int6 in multi-drug resistance has direct implications for a clinically important question in cancer treatment.

Publications

10 25 50
 
Description AICR funding (Gene-environment interactions in cancer)
Amount £180,000 (GBP)
Organisation Association for International Cancer Research (AICR) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2007 
End 08/2010
 
Description FP7 Framework
Amount £200,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 04/2008 
End 04/2011
 
Description Medical Research Scotland funding
Amount £150,000 (GBP)
Organisation Medical Research Scotland 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2008 
End 09/2011
 
Description Wellcome Trust Project Grant (A Critical Requirement for MAPK-signaling in Development and Disease)
Amount £222,258 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2007 
End 07/2010
 
Title CFC-zebrafish 
Description We have developed an assay to test the activity of cardio-facio-cutaneous alleles, and to test the effects of specific clinically active drugs on restoring their "normal" activity 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Publication -- Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors. Anastasaki C, Estep AL, Marais R, Rauen KA, Patton EE. Hum Mol Genet. 2009 Jul 15;18(14):2543-54. Epub 2009 Apr 17. PMID: 19376813 [PubMed - indexed for MEDLINE] Anastasaki C, Rauen KA, Patton EE. Continual low-level MEK inhibition ameliorates cardio-facio-cutaneous phenotypes in zebrafish. Dis Model Mech. 2012 Jul;5(4):546-52. 
URL http://europepmc.org/abstract/MED/19376813
 
Title Small molecule 
Description a small molecule that causes a specific developmental phenotype (kills developing and differentiated melanocytes) 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact n/a considering for patent 
 
Description Human genetic disease of the MAPK pathway 
Organisation University of California, San Francisco
Country United States of America 
Sector Academic/University 
PI Contribution Sharing of data, and ideas on human disease. We wrote a grant that was successfully funded, and have published in an medium impact journal. We are currently exploring the effects of small molecules on behaviour.
Collaborator Contribution Sharing of reagents, grant writing and publishing
Impact Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors. Anastasaki C, Estep AL, Marais R, Rauen KA, Patton EE. Hum Mol Genet. 2009 Jul 15;18(14):2543-54. Epub 2009 Apr 17. PMID: 19376813 [PubMed - indexed for MEDLINE] Proceedings from the 2009 genetic syndromes of the Ras/MAPK pathway: From bedside to bench and back. Rauen KA, Schoyer L, McCormick F, Lin AE, Allanson JE, Stevenson DA, Gripp KW, Neri G, Carey JC, Legius E, Tartaglia M, Schubbert S, Roberts AE, Gelb BD, Shannon K, Gutmann DH, McMahon M, Guerra C, Fagin JA, Yu B, Aoki Y, Neel BG, Balmain A, Drake RR, Nolan GP, Zenker M, Bollag G, Sebolt-Leopold J, Gibbs JB, Silva AJ, Patton EE, Viskochil DH, Kieran MW, Korf BR, Hagerman RJ, Packer RJ, Melese T. Am J Med Genet A. 2010 Jan;152A(1):4-24.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation Royal Netherlands Academy of Arts and Sciences (KNAW)
Department Hubrecht Institute
Country Netherlands, Kingdom of the 
Sector Academic/University 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation University of Bath
Department Department of Biology and Biochemistry
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation University of Edinburgh
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation University of St Andrews
Department School of Chemistry St Andrews
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Small molecule screening and melanoma models 
Organisation Virginia Commonwealth University
Department Department of Human and Molecular Genetics
Country United States of America 
Sector Academic/University 
PI Contribution We have been kindly given many small molecule tools which have been the basis for a successful grant for myself and a postdoc in the lab to Medical Research Scotland. We also had a paper accepted in Disease Models and Mechanisms.
Collaborator Contribution Sharing of chemical reagents and successfully won Medical Research Scotland grant.We have had an excellent collaboration generating new small molecules and identifying how the compounds work in living animals and tissues. We will be submitting a paper together.We won a Medical Research Scotland Award, and have used this in a successful collaboration to carry out a small molecule screen. We will be submitting this to Pigment Cell and Melanoma Research for publication.We are on an FP7-EU grant together, and have developed new models of melanoma.We are developing new melanoma models together.We are sequencing zebrafish melanoma models.
Impact We have successfully won funding from Medical Research Scotland, and have has a paper accepted in Disease Models and Mechanisms. We have two other papers arising from this collaboration to be submitted within the next few months.
Start Year 2007
 
Description Interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact BBC interview on melanoma and cancer TV and radio

Interview with journalist from Scottish newspaper

Interview for the Scotsman

Surprised by how many people in my local community told me they had seen the interview on TV. Less impact with the radio interview.

Many people commented to me in the community that they had seen the Scotsman article
Year(s) Of Engagement Activity 2006,2007,2010,2013
 
Description Presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Lecture at the Edinburgh science festival -- on cancer and zebrafish approaches to understanding disease and development, and why model organisms and basic science are so importnant

Many one-to-one discussions with audience members
Year(s) Of Engagement Activity 2008
 
Description Tour to cancer research fund raisers (lay people) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Two groups of 5 lay people who are interested in helping to raise money for cancer research. They toured the facility and we spent 1.5 hours with each group - showed melanoma models, discussed skin color and sun exposure.

Tour of facilities and interaction day for children from a primary school in Fife.

The attendees were very grateful and sent a follow up e-mail to say how much they enjoyed it.
Year(s) Of Engagement Activity 2010,2013