Actin Cytoskeleton Regulation of Epithelial Mesenchymal Transition in Metastatic Cancer Cells

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.

Fluid shear stress induces epithelial-mesenchymal transition (EMT) in Hep-2 cells

“…During EMT, polymerization of the actin cytoskeleton is clearly observed. Shankar et al [46] indicated that Cytochalasin D (Cyt D) reduced cell size and F-actin levels and induced an up-regulation of E-cad at both protein and mRNA level. We found in addition that the actin cytoskeleton was rearranged with FSS stimulation.…”

Nonmuscle myosin II-B regulates epicardial integrity and epicardial derived mesenchymal cell maturation

“…Shankar et al were among the first to propose that actin dynamics controls EMT in metastatic cancer cells. This hypothesis was based on their studies with cultured metastatic cancer cell lines showing that knockdown of pseudopodia-enriched proteins (Shankar et al, 2010) or cytochalasin D-induced disruption of actin filaments (Shankar and Nabi, 2015) decreased pseudopod formation and tumor cell invasion. This was associated with reduced actin filament stability, the loss of N-cadherin and vimentin expression, and the regaining of E-cadherin expression.…”

The cytoskeleton as a novel target for treatment of renal fibrosis

“…Parrish 6 cytoskeleton are linked with EMT (Liu, 2004;Shankar and Nabi, 2015), this review does not address the numerous studies addressing the pathways that regulate SMA expression.…”