Stopped in translation: EMT control meets eukaryotic elongation

Recently reporting in Molecular Cell, Hussey et?al. (2011) find that hnRNP E1 inhibits the translation of several genes essential for the epithelial-to-mesenchymal transition (EMT) by blocking eEF1A1 release during translation elongation. Phosphorylation of hnRNP E1 in response to TGF-β signaling disrupts the hnRNP E1-eEF1A1 interaction, triggering EMT.     

aV integrins and TGF-β-induced EMT: a circle of regulation

Transforming growth factor-β (TGF-β) has roles in embryonic development, the prevention of inappropriate inflammation and tumour suppression. However, TGF-β signalling also regulates pathological epithelial-to-mesenchymal transition (EMT), inducing or progressing a number of diseases ranging from inflammatory disorders, to fibrosis and cancer. However, TGF-β signalling does not proceed linearly but rather induces a complex network of cascades that mutually influence each other and cross-talk with other pathways to successfully induce EMT. Particularly, there is substantial evidence for cross-talk between αV integrins and TGF-β during EMT, and anti-integrin therapeutics are under development as treatments for TGF-β-related disorders. However, TGF-β's complex signalling network makes the development of therapeutics to block TGF-β-mediated pathology challenging. Moreover, despite our current understanding of integrins and TGF-β function during EMT, the precise mechanism of their role during physiological versus pathological EMT is not fully understood. This review focuses on the circle of regulation between αV integrin and TGF-β signalling during TGF-β induced EMT, which pose as a significant driver to many known TGF-β-mediated disorders.     

Regulation of the TGF-β pathway by deubiquitinases in cancer

The transforming growth factor-β (TGF-β) pathway regulates diverse cellular processes. It signals via serine/threonine kinase receptors and intracellular Smad and non-Smad effector proteins. In cancer cells, aberrant TGF-β signalling can lead to loss of growth inhibition and an increase in invasion, epithelial-to-mesenchymal transition (EMT) and metastasis. Therapeutic targeting of the pro-oncogenic TGF-β responses is currently being explored as a potential therapy against certain invasive and metastatic cancer types. The ubiquitin post-translational regulation system is emerging as a key regulatory mechanism for the control of TGF-β pathway components. In this review, we focus on the role of deubiquitinases (DUBs), which counteract the activity of E3 ubiquitin ligases. We will discuss the mechanisms by which specific DUBs control Smad and non-Smad TGF-β signalling routes, and how perturbation of the expression and function of DUBs contributes to misregulation of TGF-β signalling in cancer.     

Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth.     

Role of Dlg5/lp-dlg, a membrane-associated guanylate kinase family protein, in epithelial-mesenchymal transition in LLc-PK1 renal epithelial cells

Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT.     

The iron chelators Dp44mT and DFO inhibit TGF-β-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1)

The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.     

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity

Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.     

Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-β (TGF-β). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-β-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-β. A positive role for Notch1 was revealed in three experiments: (1) TGF-β induced Notch1 mRNA in valve ECs, (2) a γ-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.     

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells

Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-β (TGF-β) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-β-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-β-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1β. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-β, TNF-α, and IL-1β, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-β-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-β-induced EMT in A549 cells, which is transduced in part by NFκB signalling.     

In vitro and ex vivo vanadium antitumor activity in (TGF-β)-induced EMT. Synergistic activity with carboplatin and correlation with tumor metastasis in cancer patients

Epithelial to mesenchymal transition (EMT) plays a key role in tumor progression and metastasis as a crucial event for cancer cells to trigger the metastatic niche. Transforming growth factor-β (TGF-β) has been shown to play an important role as an EMT inducer in various stages of carcinogenesis. Previous reports had shown that antitumor vanadium inhibits the metastatic potential of tumor cells by reducing MMP-2 expression and inducing ROS-dependent apoptosis. However, the role of vanadium in (TGF-β)-induced EMT remains unclear. In the present study, we report for the first time on the inhibitory effects of vanadium on (TGF-β)-mediated EMT followed by down-regulation of ex vivo cancer stem cell markers. The results demonstrate blockage of (TGF-β)-mediated EMT by vanadium and reduction in the mitochondrial potential of tumor cells linked to EMT and cancer metabolism. Furthermore, combination of vanadium and carboplatin (a) resulted in synergistic antitumor activity in ex vivo cell cultures, and (b) prompted G₀/G₁ cell cycle arrest and sensitization of tumor cells to carboplatin-induced apoptosis. Overall, the findings highlight the multifaceted antitumor action of vanadium and its synergistic antitumor efficacy with current chemotherapy drugs, knowledge that could be valuable for targeting cancer cell metabolism and cancer stem cell-mediated metastasis in aggressive chemoresistant tumors.     

MIR-99a and MIR-99b modulate TGF-β induced epithelial to mesenchymal plasticity in normal murine mammary gland cells

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs.Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells.Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated.In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.