Abstract
Liver fibrosis and cancer metastasis remain significant health problems, with a mortality rate of nine million each year worldwide. Effective therapies for these problems are very limited due to lack of a clear understanding of the underlying mechanisms. Epithelial-mesenchymal-transition (EMT) is a naturally occurring and strictly regulated trans-differentiation process essential for tissue morphogenesis during early embryonic development. Recent studies suggest that aberrant reactivation of EMT in adults may be a key event in both tissue fibrosis and cancer metastasis. Nevertheless, the pathological significance of EMT in the progression of fibrosis and tumor metastasis remains unclear. Despite the apparently different contributions of EMT in these processes, EMT seems to be induced by several common key regulators, including snail homolog 1 (Snail1). Understanding the role of this regulator in disease-associated processes (e.g. production of extracellular matrix proteins, apoptosis resistance and cell migration) is necessary to decipher the underlying mechanisms of tissue fibrosis and cancer metastasis. In this study, we utilized transforming growth factor-beta 1 (TGF-β1) to establish EMT in non-malignant murine biliary epithelial cells (cholangiocytes, 603B cells) and human lung epithelial carcinoma cells (A495 cells). We examined collagen production and cell survival in 603B cells and cell migration in A549 cells during TGF-β1-induced EMT. The involvement of Snail1 in mediating these disease-associated alterations was further evaluated to investigate the potential direct links between EMT and liver fibrosis as well as cancer metastasis. |We showed that TGF-β1 stimulation induced EMT-like alterations in 603B and A549 cells. TGF-β1-treated cells displayed EMT-associated morphological changes, and lost expression of epithelial marker E-cadherin (E-cad) and gained expression of mesenchymal marker N-cadherin (N-cad). In addition, TGF-β1 treatment induced a transient up-regulation of Snail1 in 603B cells and a persistent up-regulation of Snail1 in A549 cells. |We found that TGF-β1 transiently increased Collagen 1 alpha 1(Col1A1) production in 603B cells. Up-regulation of Snail1 following TGF-1 stimulation was required for EMT-associated changes in 603B cells. Interestingly, Snail1 induction was dispensable for TGF-β1 to enhance Col1A1 expression, arguing against the concept that cholangiocyte EMT is one of the major mechanisms of generating collagen-producing cells during liver fibrosis. TGF-β1 treatment protected 603B cells from tumor necrosis factor-alpha (TNF-α)-induced apoptosis. We demonstrated that Snail1 knockdown inhibited TGF-β1-induced apoptosis resistance in 603B cells. Moreover, Snail1 repressed the expression of multiple pro-apoptotic genes in 603B cells, suggesting the direct contributions of EMT to enhancing cell survival. Since the accumulation of cholangiocytes (ductular reactions) is a common feature of liver fibrosis, our data indicate a potential new mechanism of EMT to promote the development of liver fibrosis by enhancing cholangicocyte survival thus favoring their accumulation during fibrosis. |Our results demonstrated that Snail1 is a key mediator in the TGF-β1 signaling pathway that induces EMT-associated cell migration in A549 cells. TGF-β1 treatment dramatically enhanced cell migration as determined by the transwell assay. Moreover, Snail1 negatively regulated the expression of SCARA5, a metastasis suppressor inhibiting focal adhesion kinase (FAK) activation. Snail1 binds to promoter of SCARA5, implying that it is a Snail1 target gene. Consistent with this concept, we found that non-coding RNA-a7 (ncRNA-a7), a positive snail1 regulator, inhibited SCARA5 expression in A549 cells. Enforced expression of SCARA5 abolished TGF-β1-induced A549 cell migration, implying that SCARA5 may be a key Snail1 target involved in modulating cell mobility during EMT. However, down-regulation of SCARA5 alone was not sufficient to induce A549 migration, indicating the involvement of other EMT-associated genes. |It has been speculated that Snail1 may recruit chromatin modifying proteins to epigenetically repress gene transcription. Our data showed that DNA-methyltransferase 1 (DNMT1) interacts with a long intergenic ncRNA (lincRNA-STXPB5) and Snail1 in A549 cells. DNMT1 activity was required to repress SCARA5 expression during TGF-β1-induced EMT in A549 cells. However, differing from a previous study suggesting that Snail1 recruits DNMT1 to methylate E-cad promoter in cells with chronic TGF-β1 exposure, we found that TGF-β1 induced a rapid SCARA5 down-regulation in a DNA methylation-independent manner, highlighting the complexity of Snail1-mediated epigenetic regulation. Collectively, these data suggest the possibility that EMT regulator Snail1 may promote cancer progression via epigenetically silencing the expression of metastasis suppressors. |In summary, our data indicate that Snail1 is an essential mediator of TGF-β1-induced EMT-like alterations in the murine non-tumorigenic cholangiocyte line 603B and human lung cancer line A549. Snail1-mediated gene repression contributes to EMT-associated pathological alterations in both 603B cells and A549 cells. During TGF-β1-induced EMT, the expression of multiple pro-apoptotic genes is down-regulated in 603B cells in a Snail1-dependent manner thereby protecting 603B cells from apoptosis, implying the involvement of EMT in ductular reactions. In the A549 cells, Snail1 interacts with DNMT1 to repress the expression of metastasis suppressor SCARA5 and promote cell migration, which might be a key event for cancer progression. Taken together, our research provides new evidence to support that EMT-regulator snail1 might directly contribute to the development of disease-associated pathological alterations. This suggests a novel mechanism of EMT promoting disease progression and highlighting the therapeutic potential of targeting EMT for liver fibrosis and cancer metastasis.