Epithelial-to-mesenchymal Transition In Mammary Epithelial Cells Research Articles

Spatial Gradients of E-Cadherin and Fibronectin in TGF-β1-Treated Epithelial Colonies Are Independent of Fibronectin Fibril Assembly.

Epithelial to Mesenchymal Transition (EMT) is a dynamic, morphogenetic process characterized by a phenotypic shift in epithelial cells towards a motile and often invasive mesenchymal phenotype. We have previously demonstrated that EMT is associated with an increase in assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils. We have also demonstrated that Transforming Growth Factor-β1 (TGF-β1) localizes to FN fibrils, and disruption of FN assembly or disruption of TGF-β1 localization to FN fibrils attenuates EMT. Previous studies have shown that TGF-β1 induces spatial gradients of EMT in mammary epithelial cells cultured on FN islands, with cells at free edges of the island preferentially undergoing EMT. In the current work, we sought to investigate: (a) whether FN fibril assembly is also spatially patterned in response to TGF-β1, and (b) what effects FN fibril inhibition has on spatial gradients of E-Cadherin and FN fibrillogenesis. We demonstrate that mammary epithelial cells cultured on square micropatterns have fewer E-Cadherin-containing adherens junctions and assemble more FN fibrils at the periphery of the micropattern in response to increasing TGF-β1 concentration, indicating that TGF-β1 induces a spatial gradient of both E-Cadherin and FN fibrils. Inhibition of FN fibril assembly globally diminished E-Cadherin-containing adherens junctions and FN fibrillogenesis, but did not eliminate the spatial gradient of either. This suggests that global inhibition of FN reduces the degree of both FN fibrillogenesis and E-Cadherin-containing adherens junctions, but does not eliminate the spatial gradient of either, suggesting that spatial gradients of EMT and FN fibrillogenesis are influenced by additional factors.

Mnt Represses Epithelial Identity To Promote Epithelial-to-Mesenchymal Transition.

The multistep process of epithelial-to-mesenchymal transition (EMT), whereby static epithelial cells become migratory mesenchymal cells, plays a critical role during various developmental contexts, wound healing, and pathological conditions such as cancer metastasis. Despite the established function of basic helix-loop-helix (bHLH) transcription factors (TFs) in cell fate determination, only a few have been examined for their role in EMT. Here, using transcriptome analysis of distinct stages during stepwise progression of transforming growth factor beta (TGFβ)-induced EMT in mammary epithelial cells, we revealed distinct categories of bHLH TFs that show differential expression kinetics during EMT. Using a short interfering RNA-mediated functional screen for bHLH TFs during EMT, we found Max network transcription repressor (MNT) to be essential for EMT in mammary epithelial cells. We show that the depletion of MNT blocks TGFβ-induced morphological changes during EMT, and this is accompanied by derepression of a large number of epithelial genes. We show that MNT mediates the repression of epithelial identity genes during EMT by recruiting HDAC1 and mediating the loss of H3K27ac and chromatin accessibility. Lastly, we show that MNT is expressed at higher levels in EMT-High breast cancer cells and is required for their migration. Taken together, these findings establish MNT as a critical regulator of cell fate changes during mammary EMT. IMPORTANCE The bHLH TF Mnt promotes epithelial to mesenchymal transition through epigenetic repression of the epithelial gene expression program.

News and views on EMT Fra-1 controls EMT in mammary epithelial cells

Epithelial-to-mesenchymal transition (EMT), a process critical for breast cancer cell dissemination, engages the Fos-related antigen 1 (Fra-1) transcription factor. Molecular and cellular analyses revealed an evolutionary conservation of Fra-1 functions in EMT of mouse and human mammary cells, establishing a solid basis for preclinical studies in genetically engineered mouse models.

Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFβ expression.

Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models.

CPEB1 promotes differentiation and suppresses EMT in mammary epithelial cells

Downregulation of CPEB1, a sequence-specific RNA-binding protein, in a mouse mammary epithelial cell line (CID-9) causes epithelial-to-mesenchymal transition (EMT), based on several criteria. First, CPEB1 knockdown decreases protein levels of E-cadherin and β-catenin but increases those of vimentin and Twist1. Second, the motility of CPEB1-depleted cells is increased. Third, CID-9 cells normally form growth-arrested, polarized and three-dimensional acini upon culture in extracellular matrix, but CPEB1-deficient CID-9 cells form nonpolarized proliferating colonies lacking a central cavity. CPEB1 downregulates Twist1 expression by binding to its mRNA, shortening its poly(A) tract and repressing its translation. CID-9 cultures contain both myoepithelial and luminal epithelial cells. CPEB1 increases during CID-9 cell differentiation, is predominantly expressed in myoepithelial cells, and its knockdown prevents expression of the myoepithelial marker p63. CPEB1 is present in proliferating subpopulations of pure luminal epithelial cells (SCp2) and myoepithelial cells (SCg6), but its depletion increases Twist1 only in SCg6 cells and fails to downregulate E-cadherin in SCp2 cells. We propose that myoepithelial cells prevent EMT by influencing the polarity and proliferation of luminal epithelial cells in a mechanism that requires translational silencing of myoepithelial Twist1 by CPEB1.

Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells

Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β (TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-β-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β-signaling pathways.

Abstract 324: The integrin-linked kinase (ILK) / Rictor complex is required for TGFβ-1-induced epithelial to mesenchymal transition (EMT)

Abstract Epithelial to mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase is a focal adhesion adaptor and serine/threonine protein kinase that regulates cell proliferation, survival, and EMT. Elucidating the molecular mechanisms necessary for the development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1 mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mTORC2 component Rictor as well as its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK-Rictor complex using siRNA-mediated knockdown. Furthermore, we identified formation of the ILK/Rictor complex formation in cancer but not normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells following TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 324. doi:1538-7445.AM2012-324

Abstract 1494: The role of microenvironment in mammary epithelial cell plasticity

Abstract Understanding breast cancer heterogeneity is crucial for the development of targeted therapeutic interventions. One source of this intratumoral diversity comes from changes induced in epithelial cells by signals they receive from the stromal microenvironment. These changes include the epithelial-to-mesenchymal transition (EMT), a process of cellular plasticity by which epithelial cells disengage cell-cell and cell-extracellular matrix interactions, alter cytoskeletal structure, and implement a transcriptional program that facilitates the acquisition of aggressive traits, such as increased motility, invasiveness and resistance to apoptosis. Bissell lab has demonstrated that an extracellular protease, matrix metalloproteinase-3 (MMP3), is able to initiate the formation of mammary tumors in mice, as well as trigger EMT in mammary epithelial cells when activated. Furthermore, the observed EMT in MMP3-overexpressing cells was found to be dependent upon reactive oxygen species (ROS) production. However, it is not clear how this extracellular secreted protein functions with an epithelial cell to affect its intracellular events. To understand the mechanism by which MMP3 drives transformation of mammary cells, we asked which domain of MMP3 is required for ROS production or EMT in mammary epithelial cells, and we are investigating cell surface proteins that may be target of degradation or binding partner of MMP3 during tumorigenesis. This work will unravel new candidates for specific inhibition, providing attractive options for treatment of this and other diseases in which MMP3 plays a pathological role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1494. doi:10.1158/1538-7445.AM2011-1494