Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulationReportar como inadecuado




Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Cancer

, 9:235

First Online: 15 July 2009Received: 17 December 2008Accepted: 15 July 2009DOI: 10.1186-1471-2407-9-235

Cite this article as: Hugo, H.J., Wafai, R., Blick, T. et al. BMC Cancer 2009 9: 235. doi:10.1186-1471-2407-9-235

Abstract

BackgroundA 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-δEF1. 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.

MethodsPMC42-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-δEF1 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.

ResultsWhen 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-δEF1 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-δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.

ConclusionST 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-δEF1 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.

AbbreviationsEMTepithelial to mesenchymal transition

STstaurosporine

aPKCatypical protein kinase C

EGFepidermal growth factor

MT-PCRmultiplex tandem PCR

ECMextracellular matrix

EGFRepidermal growth factor receptor

CAV1caveolin 1

CTGFconnective tissue growth factor

CYR61cysteine-rich, angiogenic inducer, 61

CD44CD44 antigen Indian blood group

S100A4S100 calcium binding protein A4

SNAI2Snail2

NLFnuclear localized factor

EPHB4EPH receptor B4

FGFfibroblast growth factor

Wntwingless-type

BMPbone morphogenic protein

HGFhepatocyte growth factor

IGFinsulin growth factor

TNFαtumor necrosis factor alpha

PI3Kphosphoinositide 3 kinase

TGFβtransforming growth factor beta

MAPKmitogen activated protein kinase

AKTv-akt murine thymoma viral oncogene homolog 1

CLDNclaudin

CD24CD24 molecule

CDHcadherin

LGALS1lectin, galactoside-binding, soluble, 1

DCNdecorin

SPARCsecreted protein, acidic, cysteine-rich osteonectin

WT1Wilms tumor 1

PAXpaired box gene

KRTkeratin

MLCKmyosin light chain kinase

PKAprotein kinase A

PKGprotein kinase G

ILKintegrin linked kinase

NFkBnuclear factor-kappa B.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-9-235 contains supplementary material, which is available to authorized users.

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Autor: Honor J Hugo - Razan Wafai - Tony Blick - Erik W Thompson - Donald F Newgreen

Fuente: https://link.springer.com/







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