Abstract
Epithelial-mesenchymal transition (EMT) is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D) reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.
Highlights
Epithelial–mesenchymal transition (EMT) is a cellular program required during normal developmental processes such as embryogenesis and tissue remodeling and in the progression of diseases such as cancer [1]
We showed previously that reduced expression of pseudopod-enriched proteins resulted in reduced actin cytoskeleton dynamics and cell size that were associated with a reversal of EMT in six metastatic cancer cell lines [18]
We show that depolymerization of the actin cytoskeleton of cancer cells with cytochalasin D (Cyt D) induces nuclear-cytoplasmic translocation of EMT-associated transcription factors, increased E-cadherin expression, reduced cell shape and size and reduced activation of RhoA
Summary
Epithelial–mesenchymal transition (EMT) is a cellular program required during normal developmental processes such as embryogenesis and tissue remodeling and in the progression of diseases such as cancer [1]. During this process, disruption of cell-cell and cell-extracellular matrix (ECM) adhesions releases epithelial cells from the surrounding tissue. Actin cytoskeleton remodeling is mediated by the Rho GTPases and represents a basic mechanism critical to cell migration during processes such as cancer metastasis. Expression of actin regulatory proteins such as Arp2/3 and WAVE2 correlates with poor prognosis in breast and liver carcinomas supporting a role for actin cytoskeleton dynamics and organization as critical regulators of cancer progression [16]. A recent study has implicated increased myosin IIB expression and myosin IIA heavy chain phosphorylation in enhancing mammary epithelial cell migration and invasion in TGF-β–induced EMT [17]
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