Abstract
BackgroundDuring cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance.MethodsThe expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay.ResultsInduction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2.ConclusionTaken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.
Highlights
During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state
Four Anterior gradient 2 protein (AGR2)-positive cancer cell lines showing different epithelial mesenchymal transition traits were selected to investigate the effect of Transforming growth factor beta (TGF-β) on EMT induction with respect to AGR2 expression
We found that AGR2 expression positively correlates with the expression of the epithelial marker E-cadherin, while TGF-β-induced reduction of AGR2 was concomitant with the classical features of mesenchymal cells such as the loss of E-cadherin, induction of N-cadherin and morphological changes arising from cytoskeleton reorganisation including diffuse cytoplasmic distribution of vimentin and re-localization of actin
Summary
Epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. Epithelial-mesenchymal transition (EMT) is a reversible process of cellular reprogramming that plays an important role in normal development and injury response. Phenotypic changes occur during EMT, such as the loss of epithelial proteins including E-cadherin, cytokeratins and claudins, and the acquisition of mesenchymal proteins such as N-cadherin and vimentin [1]. Following these changes at the level of gene expression, polarized epithelial cells undergo morphological changes into spindle-shaped migratory mesenchymal cells [2]. AGR2 participates in the attenuation of degradation processes and prevents the induction of apoptosis, leading to increased cellular survival [12, 14, 18]
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