Spatial, temporal and metabolic regulation of PI 3-kinase derived lipid signals in health and disease

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

The correct functions of our cells and tissues are controlled by a wide range of signalling molecules such as hormones and growth factors. These usually bind to receptors on the outer surface of cells and trigger the production of intracellular signals called second messengers. We study a special class of second messengers called inositol phospholipids and one in particular which is phosphatidylinositol trisphosphate or PIP3. PIP3 is the second messenger produced by insulin action and it plays an important role in controlling the amount of sugar in the blood. Failure to produce PIP3 in insulin responsive tissues after a meal is a major cause of diabetes. Cells control PIP3 levels through enzymes that make it, called PI 3-kinases, and other enzymes, called phosphatases, which destroy it. Mutations in one of the PI 3-kinases which cause its activation at the wrong time can cause cancer and occur commonly in breast and colon tumours whilst inactivating mutations in one of the phosphatases, called PTEN, cause many other tumours. We study the regulation of PI 3-kinase signalling especially focussing on the phosphatases that destroy PIP3. We collaborate with several large pharmaceutical companies who are targeting PI 3-kinase signalling to develop drugs for the treatment of cancer, type 2 diabetes and inflammatory diseases.

Technical Summary

Inositol glycerophospholipids are amongst the most versatile intracellular signal molecules whose functions include acting as second messengers in response to hormones and growth factors and the regulation of membrane trafficking events. There are currently eight known species which differ with respect to the number and distribution of phosphate groups around the inositol ring and they function by binding to effector proteins which possess lipid binding domains many of which are highly specific for a particular headgroup configuration. This programme focuses on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), the product of type I phosphoinositide 3-kinases (PI 3-kinases) and PtdIns(3,4P)P2, produced mainly by the action of 5-phosphatases on the former lipid signal. PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumour suppressor that inactivates PtdIns(3,4,5)P3 by hydrolysing the 3-phosphate. Fundamental studies of PTEN mechanism and membrane targeting will be continued. We discovered that PI 3-kinase signalling can be stimulated by reactive oxygen species (ROS) which inactivate PTEN through reversible oxidation of the active site cysteine and we have preliminary evidence that PtdIns(3,4)P2 is also metabolised by a redox-sensitive phosphatase. The roles of endogenously generated ROS in regulating these enzymes and hence PI 3-kinase lipid products will therefore be studied in cell lines and extended to xenograft models of solid tumours. We have also exploited a range of lipid binding protein domains to develop a suite of methods that allow quantitative and spatial analysis of lipid signals in enzyme assays, live cells and subcellular structures, the latter using immuno-electron microscopy. These approaches will be used to study localised generation and metabolism of signalling lipids in polarised cellular responses such as the formation of macropinosomes in dendritic cells and on the dendritic cell side of the ?immune synapse? between these cells and T-cells, as well as in models of disease. The ultrastuctural studies aim to determine the molecular basis for the occurrence of PI 3-kinase lipid products in many cellular compartments including identifiying the isoforms of enzymes involved in their synthesis and metabolism and the identification of novel PtdIns(3,4,5)P3 binding proteins. Further work will lead to the development of improved methods for measuring protein/lipid interactions and for the detection of PtdIns(3,4,5)P3 in small cell numbers sorted by FACS analysis. Lastly we will study novel mechanisms of feedback regulation of insulin simulated PI 3-kinase responses that may contribute to an insulin resistant state.

Publications

10 25 50
 
Description BBSRC CASE Studentship (Bohringer Ingelheim)
Amount £80,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2006 
End 09/2010
 
Description DTI: Succeeding Through Innovation
Amount £250,746 (GBP)
Organisation Department for Business, Energy & Industrial Strategy 
Sector Public
Country United Kingdom
Start  
 
Title Antibodies 
Description Purified polyclonal antibodies produced during research programme 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact Research publications and progress 
 
Title Expression construct collection 
Description An extensive range of expression constructs (plasmids and viral vectors) for use in several systems. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact We frequently supply other academic research groups with expression constructs (approx 10-15 times per year). Some of these interactions take the forms of collaborations, others simply acknowledged reagent supply. These reagents have been used in many subsequent published projects. 
 
Title Lipid Kinase selectivity screen 
Description This is a service provided to our pharmaceutical collaborators and the broader academic community supporting drug discovery efforts in the area of PI3K. It is the first of its kind developed within an academic setting. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Supports intensive research in 5 global pharmaceutical companies developing novel cancer treatments targeting the PI 3-kinase pathway. Will develop a large data matrix relating compound structures to efficacy against a large number of lipid kinase enzymes. 
URL http://www.kinase-screen.mrc.ac.uk/kinase-lipid-kinase-panel
 
Description Acadamic collaboration - Rick Randall 
Organisation University of St Andrews
Department Centre for Biomolecular Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Sharing data and analysing samples
Collaborator Contribution Sharing unpublished data
Impact Publications: PM ID: 18029356 and 20133840
Start Year 2006
 
Description Academic collaboration - Britta Eickholt 
Organisation Charité - University of Medicine Berlin
Country Germany 
Sector Academic/University 
PI Contribution Extensive academic collaboration, involving reagent exchange, sharing ideas and data. One postdoctoral researcher worked in the Eickholt lab in Berlin for 2 months in 2013.
Collaborator Contribution The Eickholt lab have provided reagents and shared unpublished data to allow collaborative projects. They also hosted an NRL lab postdoctoral researcher working in the Eickholt lab in Berlin for 2 months in 2013
Impact 3 research Publications: PubMed IDs: 19767745, 23940795, 23085752
 
Description Academic collaboration - Britta Eickholt 
Organisation King's College London
Department MRC Centre for Developmental Neurobiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Extensive academic collaboration, involving reagent exchange, sharing ideas and data. One postdoctoral researcher worked in the Eickholt lab in Berlin for 2 months in 2013.
Collaborator Contribution The Eickholt lab have provided reagents and shared unpublished data to allow collaborative projects. They also hosted an NRL lab postdoctoral researcher working in the Eickholt lab in Berlin for 2 months in 2013
Impact 3 research Publications: PubMed IDs: 19767745, 23940795, 23085752
 
Description Academic collaboration - Carola Neumann 
Organisation Medical University of South Carolina
Country United States 
Sector Academic/University 
PI Contribution Sharing reagents and data
Collaborator Contribution Provided reagents and shared data to pursue collaborative work
Impact Publications: PM ID 19369943 and unpublished data
Start Year 2008
 
Description Academic collaboration - Joao Barata 
Organisation University of Lisbon
Department Institute for Molecular Medicine
Country Portugal 
Sector Academic/University 
PI Contribution Sharing data and reagents and hosting a visiting student to analyse samples.
Collaborator Contribution Sharing reagents and unpublished data
Impact Publication. PM ID: 18830414
Start Year 2006
 
Description Academic collaboration - Kees Weijer 
Organisation University of Dundee
Department Division of Cell and Developmental Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Sharing data and reagents within a shared project
Collaborator Contribution Sharing data and providing novel experimental capabilities within a shared project
Impact Publications: PM IDs: 17240336, 19915616, 16246156 and unpublished data
 
Description Academic collaboration - Seth Schorr 
Organisation University of Dundee
Department College of Medicine, Dentistry & Nursing
Country United Kingdom 
Sector Academic/University 
PI Contribution Project instigation and direction. Some experiments. Writing and over-seeing publication
Collaborator Contribution Extensive experimnetal collaboration, significant scientific dialog, and experiments mostly performed by rsesearchers funded from collaborators lab.
Impact Publication: Ellis et al, PMID: 20600851
Start Year 2007
 
Description Commercial service collaboration - Novartis 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution We tested several compounds for their ability to inhibit PTEN in vitro
Collaborator Contribution It motivated us to test several control compounds as inhibitors of the tumour suppressor phosphatase PTEN
Impact We obtained scientifically valuable data from several control compounds and supplied data to Novartis regarding their test compounds.
Start Year 2011
 
Description DSTT Pharameutical Consortium 
Organisation Dundee Signal Transduction Therapy (DSTT) Consortium
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration is funded by a pharmaceutical consortium, supporting several activities including a research Division sited in the University of Dundee College of Life Sciences. From 1998-2012 this was overseen by the co-directors, Peter Downes, the original PI on this grant and Professors Philip Cohen and Dario Alessi. Dr Leslie and Prof Downes and their research groups also provided advice and expertise to the consortium companies. Since the last renewal of the collaboration in 2012, the grant holders have not been formally involved but occasionally act as informal consultants to the consortium companies, the last instance being in October 2013.
Collaborator Contribution We have had occasional access to novel compounds from these pharmaceutical collaborators for use as experimental enzyme inhibitors in our research. Extensive interactions with the pharmaceutical collaborators has also been effectiove in broadening the translational training of the research team.
Impact The collaboration has supported the drug discovery efforts of 6 major global pharmaceutical companies in the field of PI 3-Kinase signalling, an extrememly active area, especially for cancer. The collaboration has supported 2 or 3 research posts in the core CPD/NRL labs since its inception in 1998 contributing to many of the CPD lab publications over this time. It has also funded a level of around ten active laboratory positions since 1998, with these trained staff going into further academic and commercial positions.
 
Title Lipid kinase selectivity screen 
Description The development of a lipid kinase selectivity screen is not strictly a discovery, but it requires a high level of expertise and reputation in the lipid signalling field as well as the resources required to generate large quantities of many enzymes grouped together under a single assay platform. We also have 'first mover' advantage in terms of the marketability of the screen and existing collaborators in the form of the DSTT member pharmaceutical companies. 
IP Reference  
Protection Protection not required
Year Protection Granted 2008
Licensed No
Impact A screen which supports and drives PI 3-kinase pathway drug discovery within 5 major pharmaceutical companies.
 
Description College Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Several group members presenting science at College Open Day

High attendence (around 300)
Year(s) Of Engagement Activity 2010,2011,2012
 
Description Press and media coverage 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Peter Downes and Nick Leslie have been interviewed by press and television to discuss individual research publications.

MRC funded work in the CPD/NRL laboratory is also of great interest to the pharmaceutical industry and underpined the ground breaking collaboration between Dundee scientists and five leading pharmaceutical companies known as the Division of Signal Transduction Therapy (DSTT). Peter Downes was a founding co-director of DSTT which has attracted much external interest 1998-2012. During the period of support we have hosted at least eight visits from members of the international press and other publishing activities, Scottish and international development agencies and industry.

Elevated profile of MRC funded research and its translation to drug discovery through collaboration with global pharmaceutical companies. The PI3K pathway which is the subject of this funding and which was co-discovered by Peter Downes, is one of the most fertile areas of mechanism based drug dicovery, especially relating to human cancer.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description School and early undergraduate project placements 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact We regularly host laboratory placements for school and early undergraduate students to experience research

To my knowledge, all of these students have entered University scientific degree programmes or in the case of relevant undregraduates continued a research/scientific career
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Visit from School pupils - Madras Academy St Andrews 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact A seminar regarding our research and careers in science

Good positive feedback
Year(s) Of Engagement Activity 2006