FOSTER, CLARE,RUTH (2012) A proteomic and genomic investigation into the role of lamin A in colorectal cancer cell motility. Doctoral thesis, Durham University.
|PDF - Accepted Version|
Lamins are type V intermediate filament proteins found at the nuclear envelope. Expression of lamin A in colorectal cancer (CRC) tumours is correlated with poor prognosis and expression of lamin A in CRC cell lines promotes greatly increased cell motility. The aim of this study was to identify proteins that promote cell motility in response to lamin A expression and to investigate lamin A regulated changes in gene/protein expression and cytoskeletal organisation that might underpin the increased cell motility.
The effects of lamin A expression were studied using quantitative proteomic and genomic methods using cells from the colorectal cancer cell line SW480 which had been transfected with GFP-lamin A (SW480/lamA) or GFP as a control (SW480/cntl). A biochemical fractionation technique was optimised for the preparation of cytoskeletal fractions which were analysed by 2D DIGE (2D difference in-gel electrophoresis) to reveal accurate and reproducible changes in the representation of proteins within the cytoskeleton in SW480/lamA cells compared to controls. The majority of proteins identified were either components of the actin/intermediate filament cytoskeleton, protein chaperones or translation initiation/elongation factors. Interestingly, tissue transglutaminase 2, a protein which modifies elements of the cytoskeleton and is associated with cancer progression, was highly over-represented in the cytoskeleton fraction of SW480/lamA cells.
Ingenuity Pathway Analysis was used to analyse genome-wide Affymetrix microarray analysis of SW480/cntl and SW480/lamA cell lines. A highly significant interaction network was identified which clustered together genes linked to cancer, cellular movement and cellular growth and proliferation. Epithelial markers such as CDH1 were down-regulated and mesenchymal markers such as FN1 were up-regulated in cells expressing GFP-lamin A, which suggested that lamin A over-expression may lead to an epithelial-mesenchymal transition (EMT). As A-type lamins are known to modulate downstream effects of TGFβ signalling, and TGFβ is an inducer of EMT, changes in genes involved in TGFβ signalling were investigated. Knockdown of lamin A using siRNA led to decreased expression of TGFBI and SNAI2 followed by reduced cell motility.
The data suggest that expression of lamin A in CRC cells causes changes in the organisation of the actin cytoskeleton and in TGFβ signalling, potentially involving an epithelial to mesenchymal transition, leading to increased cell motility and an increased risk of death from cancer.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Lamins, Cancer, Cell, Biology, Colorectal Cancer|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||16 Mar 2012 13:12|