Regulation of cell morphology, migration and adhesion by class II phosphoinositide 3-kinase enzymes

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

In this project we are trying to better understand what makes human cells move. This is a very important question because large numbers of cancer patients die each day worldwide because tumour cells leave the original site of disease to spread throughout their body. This spreading makes it difficult to provide a cure by surgery alone if the cancer is discovered late.
Our work focuses on breast cancer since these tumour cells often move faster when they meet a protein called epidermal growth factor or EGF. Once this happens, proteins inside the tumour, act in a series of steps to make the cells move. We have previously shown that one of these steps involves a protein called PI3K-C2b. At the moment we know little about how PI3K-C2b acts inside cells to increase their speed.
We will grow human breast cancer cells in the lab and record how they move after EGF treatment over many hours using a microscope and video camera. We will then see what happens to cancer cells that make more PI3K-C2b protein or we block its production. This will show us if PI3K-C2b can be used as a target for new medicines against cancer.

Technical Summary

Phosphatidylinositol (PtdIns)(3,4,5)P3 has been shown to play a pivotal regulatory role in the re-organisation of actin filaments in response to integrin, growth factor and chemokine receptor stimulation. In recent years, several downstream targets of PtdIns(3,4,5)P3 have been identified and the mechanisms responsible for its effects on actin filaments elucidated. However, the full spectrum of phosphoinositide 3-kinase (PI3K) enzymes responsible for the production of PtdIns(3,4,5)P3 remains unclear as is the contribution made by other 3-phophoinositides to actin filament reorganisation. I have demonstrated that stable expression of the class II enzyme PI3K-C2beta in human embryonic kidney (HEK) cells decreased their adhesion and increased their migration. Consistent with these effects, PI3K-C2beta decreased expression of both the alpha4 and beta1 integrin subunits that together form VLA4 that binds ICAM1 and fibronectin. PI3K-C2beta expression also increases actin filaments at the cell cortex and actin rich lamellipodia and filopodia in HEK293 cells plated onto fibronectin. Each of these PI3K-C2beta mediated effects were dependent upon its catalytic activity. Critically, co-expression of 2xFYVE domains that selectively bind PtdIns(3)P inhibited the PI3K-C2beta mediated effects on filopodia and lamellipodia formation. These data domonstrate that not only might the class II PI3K enzymes form novel mediators of cell migration, the molecular mechanism responsible require elucidation. Consequently, in this proposal we will:

1. Compare and contrast the effects of acute versus chronic expression of PI3K-C2beta on actin filament reorganisation, integrin expression, migration and vesicle transport.
2. Establish the effect of topically administered or microinjection PtdIns(3)P on actin filaments
3. Identify the molecular mechanisms downstream of 3-phoshoinositide production that mediate the effects of PI3K-C2beta on actin assembly, cell migration and morphology.
4. Evaluate the effect of chronic PI3K-C2beta expression in growth factor dependent chemotaxis in vitro and xenograft metastasis in vivo

Publications

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Leibiger B (2010) Insulin-feedback via PI3K-C2alpha activated PKBalpha/Akt1 is required for glucose-stimulated insulin secretion. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description Diabetes UK, Project Grant
Amount £240,000 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2008 
End 04/2011
 
Title Scatter assay 
Description Validated breast cancer cell line and conditions to study growth factor induced scatter of breast cancer cells in vitro 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? Yes  
Impact Manuscript 
 
Title siRNA 
Description validating oligonuleotides to silence PI3K gene expression 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Publication 
 
Description FM 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution Intellectual input, reagents, biochemical analysis
Collaborator Contribution Intellectual input, microscopy
Impact 18843041
 
Description HB 
Organisation University of Zagreb
Country Croatia 
Sector Academic/University 
PI Contribution Intellectual input, providing laboratory access, reagents
Collaborator Contribution Expertise in the analysis of proteins in nuclei
Impact 19496756
Start Year 2007
 
Description JO 
Organisation University of Illinois
Country United States 
Sector Academic/University 
PI Contribution Intellectual input, cDNA and antibodies, biochemical analysis
Collaborator Contribution Intellectual input, cDNA and antibodies
Impact 17875942
Start Year 2006
 
Description KS 
Organisation University of Ioannina
Country Greece 
Sector Academic/University 
PI Contribution Intellectual input, biochemical analysis
Collaborator Contribution Providing intellectual input, chemical libraries and lead compounds
Impact 19950162
Start Year 2007
 
Description RM 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input, biochemical analysis, reagents
Collaborator Contribution Intellectual input, technical support
Impact Project grant
Start Year 2008
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Providing an overview of cancer research and career options in Biomedical science

Raising public awareness and improving career advice
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact The post doc researcher returned to her secondary school to promote careers in science

Raised significant interest in reading a biochemical science/medicine at degree level
Year(s) Of Engagement Activity 2006,2007,2008,2009