BACKGROUND: A feature of epithelial to mesenchymal transition (EMT) relevant to 
tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, 
influencing cellular ECM adherence and motility. This is coupled with the 
transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and 
Zeb1/deltaEF1. These genes, overexpressed in breast carcinomas, are known 
targets of growth factor-initiated pathways, however it is less clear how 
alterations in ECM attachment cross-modulate to regulate these pathways. EGF 
induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor 
staurosporine (ST) induces EMT in embryonic neural epithelial cells, with 
F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of 
aPKC.
METHODS: PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or 
both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, 
Zeb1/deltaEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) 
and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) 
were visualized by confocal microscopy. A public database of human breast 
cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.
RESULTS: When PMC42-LA were treated with EGF, Snail2 was the principal 
E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with 
more extreme EMT and Zeb1/deltaEF1 induction seen with ST+EGF. ST reduced stress 
fibres and focal contact size rapidly and independently of gene transcription. 
Gene expression analysis by MT-PCR indicated that ST repressed many genes which 
were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes 
which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the 
public database of breast cancers revealed tumours exhibiting higher Snail1 
expression have an increased risk of disease-recurrence. This was not seen for 
Snail2, and Zeb1/deltaEF1 showed a reverse correlation with lower expression 
values being predictive of increased risk.
CONCLUSION: ST in combination with EGF directed a greater EMT via actin 
depolymerisation and focal contact size reduction, resulting in a loosening of 
cell-ECM attachment along with Snail1-Zeb1/deltaEF1 induction. This appeared 
fundamentally different to the EGF-induced EMT, highlighting the multiple 
pathways which can regulate EMT. Our findings add support for a functional role 
for Snail1 in invasive breast cancer.