Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

Abstract Background 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. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. Methods The 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. Results Induction 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. Conclusion Taken 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

ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, EF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis

Additional file 4: Figure S3. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

The effect of AGR2 expression on vimentin cellular localization. A scale bars correspond to 20 μm. (PDF 319 kb

Additional file 2: Table S1. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

Sequences of the primers used in quantitative PCR analysis. (PDF 213 kb

Additional file 3: Figure S2. of Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

The effect of PD98059 inhibitor on TGF-β induced nuclear accumulation of Smad2/Smad3 complex. (PDF 352 kb