Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis.
ABSTRACT: To metastasize, carcinoma cells must attenuate cell-cell adhesion to disseminate into distant organs. A group of transcription factors, including Twist1, Snail1, Snail2, ZEB1, and ZEB2, have been shown to induce epithelial mesenchymal transition (EMT), thus promoting tumor dissemination. However, it is unknown whether these transcription factors function independently or coordinately to activate the EMT program. Here we report that direct induction of Snail2 is essential for Twist1 to induce EMT. Snail2 knockdown completely blocks the ability of Twist1 to suppress E-cadherin transcription. Twist1 binds to an evolutionarily conserved E-box on the proximate Snail2 promoter to induce its transcription. Snail2 induction is essential for Twist1-induced cell invasion and distant metastasis in mice. In human breast tumors, the expression of Twist1 and Snail2 is highly correlated. Together, our results show that Twist1 needs to induce Snail2 to suppress the epithelial branch of the EMT program and that Twist1 and Snail2 act together to promote EMT and tumor metastasis.
Project description:Snail2 has an important role in the epithelial-mesenchymal transition (EMT) and tumor metastasis. Here, we report that Snail2 is highly expressed during TGF-? induced EMT in HL-7702 cells. Additionally, overexpression of Snail2 successfully promotes the migration and invasion of these cells, both in vitro and in a mouse model. Furthermore, our results show that HDAC1 and HDAC3 could suppress the Snail2 gene promoter. Moreover, we find that the acetylation of H3K4 and H3K56 are significantly reduced during the EMT process of liver HL-7702 cells. Thus, our results indicate that HDAC1 and HDAC3 epigenetically suppress the expression of Snail2 during the EMT of liver cells, revealing an opposing function of HDACs during the migration of malignant tumors.
Project description:The Twist1 transcription factor is known to promote tumor metastasis and induce Epithelial-Mesenchymal Transition (EMT). Here, we report that Twist1 is capable of promoting the formation of invadopodia, specialized membrane protrusions for extracellular matrix degradation. Twist1 induces PDGFR? expression, which in turn activates Src, to promote invadopodia formation. We show that Twist1 and PDGFR? are central mediators of invadopodia formation in response to various EMT-inducing signals. Induction of PDGFR? and invadopodia is essential for Twist1 to promote tumor metastasis. Consistent with PDGFR? being a direct transcriptional target of Twist1, coexpression of Twist1 and PDGFR? predicts poor survival in breast tumor patients. Therefore, invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis.
Project description:Several E-box-binding transcription factors regulate individual and collective cell migration and enhance the motility of epithelial cells by promoting epithelial-mesenchymal transition (EMT). Here, we characterized the role of a subset of these transcription factors and the EMT proteome in branching morphogenesis of mammary epithelial tissues using a three-dimensional organotypic culture model of the mammary duct. We found that the transcription factors Snail1, Snail2, and E47 were transiently upregulated at branch sites; decreasing the expression of these transcription factors inhibited branching. Conversely, ectopic expression of Snail1, Snail2, and E47 induced branching in the absence of exogenous stimuli. These changes correlated with the expression of mesenchymal markers and repression of E-cadherin, which was essential for branching. Snail1 and Snail2 also promoted cell survival at branch sites, but this was not sufficient to induce branching. These findings indicate that Snail1, Snail2, and E47 can promote collective migration during branching morphogenesis of mammary epithelial tissues through key regulators of EMT.
Project description:Neural crest cells form within the neural tube and then undergo an epithelial to mesenchymal transition (EMT) to initiate migration to distant locations. The transcriptional repressor Snail2 has been implicated in neural crest EMT via an as of yet unknown mechanism. We report that the adaptor protein PHD12 is highly expressed before neural crest EMT. At cranial levels, loss of PHD12 phenocopies Snail2 knockdown, preventing transcriptional shutdown of the adhesion molecule Cad6b (Cadherin6b), thereby inhibiting neural crest emigration. Although not directly binding to each other, PHD12 and Snail2 both directly interact with Sin3A in vivo, which in turn complexes with histone deacetylase (HDAC). Chromatin immunoprecipitation revealed that PHD12 is recruited to the Cad6b promoter during neural crest EMT. Consistent with this, lysines on histone 3 at the Cad6b promoter are hyperacetylated before neural crest emigration, correlating with active transcription, but deacetylated during EMT, reflecting the repressive state. Knockdown of either PHD12 or Snail2 prevents Cad6b promoter deacetylation. Collectively, the results show that PHD12 interacts directly with Sin3A/HDAC, which in turn interacts with Snail2, forming a complex at the Cad6b promoter and thus revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.
Project description:The neural crest is an induced tissue that is unique to vertebrates. In the clawed frog Xenopus laevis, neural crest induction depends on signals secreted from the prospective dorsolateral mesodermal zone during gastrulation. The transcription factors Snail2 (Slug), Snail1 and Twist1 are expressed in this region. It is known that Snail2 and Twist1 are required for both mesoderm formation and neural crest induction. Using targeted blastomere injection, morpholino-based loss of function and explant studies, we show that: (1) Snail1 is also required for mesoderm and neural crest formation; (2) loss of snail1, snail2 or twist1 function in the C2/C3 lineage of 32-cell embryos blocks mesoderm formation, but neural crest is lost only in the case of snail2 loss of function; (3) snail2 mutant loss of neural crest involves mesoderm-derived secreted factors and can be rescued synergistically by bmp4 and wnt8 RNAs; and (4) loss of snail2 activity leads to changes in the RNA levels of a number of BMP and Wnt agonists and antagonists. Taken together, these results identify Snail2 as a key regulator of the signals involved in mesodermal induction of neural crest.
Project description:p27 restrains normal cell growth, but PI3K-dependent C-terminal phosphorylation of p27 at threonine 157 (T157) and T198 promotes cancer cell invasion. Here, we describe an oncogenic feedforward loop in which p27pT157pT198 binds Janus kinase 2 (JAK2) promoting STAT3 (signal transducer and activator of transcription 3) recruitment and activation. STAT3 induces TWIST1 to drive a p27-dependent epithelial-mesenchymal transition (EMT) and further activates AKT contributing to acquisition and maintenance of metastatic potential. p27 knockdown in highly metastatic PI3K-activated cells reduces STAT3 binding to the TWIST1 promoter, TWIST1 promoter activity and TWIST1 expression, reverts EMT and impairs metastasis, whereas activated STAT3 rescues p27 knockdown. Cell cycle-defective phosphomimetic p27T157DT198D (p27CK-DD) activates STAT3 to induce a TWIST1-dependent EMT in human mammary epithelial cells and increases breast and bladder cancer invasion and metastasis. Data support a mechanism in which PI3K-deregulated p27 binds JAK2, to drive STAT3 activation and EMT through STAT3-mediated TWIST1 induction. Furthermore, STAT3, once activated, feeds forward to further activate AKT.
Project description:Twist1 is an epithelial-mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) <i>Twist1</i> or tumor cell-specific <i>Twist1</i> knockout (Twist1<sup>TKO</sup>). <i>Twist1</i> knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in ?6% of the advanced WT tumor cells. Most of these Twist1<sup>+</sup> cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ER? and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin<sup>+</sup>/vimentin<sup>+</sup>). In advanced Twist1<sup>TKO</sup> tumor cells, <i>Twist1</i> knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1<sup>TKO</sup> tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1<sup>TKO</sup> tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.
Project description:Epithelial-mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype.Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.
Project description:The heterogeneous breast cancers can be classified into different subtypes according to their histopathological characteristics and molecular signatures. Foxa1 expression is linked with luminal breast cancer (LBC) with good prognosis, whereas Twist1 expression is associated with basal-like breast cancer (BLBC) with poor prognosis owing to its role in promoting epithelial-to-mesenchymal transition (EMT), invasiveness and metastasis. However, the regulatory and functional relationships between Twist1 and Foxa1 in breast cancer progression are unknown. In this study, we demonstrate that in the estrogen receptor (ER?)-positive LBC cells Twist1 silences Foxa1 expression, which has an essential role in relieving Foxa1-arrested migration, invasion and metastasis of breast cancer cells. Mechanistically, Twist1 binds to Foxa1 proximal promoter and recruits the NuRD transcriptional repressor complex to de-acetylate H3K9 and repress RNA polymerase II recruitment. Twist1 also silences Foxa1 promoter by inhibiting AP-1 recruitment. Twist1 expression in MCF7 cells silenced Foxa1 expression, which was concurrent with the induction of EMT, migration, invasion and metastasis of these cells. Importantly, restored Foxa1 expression in these cells largely inhibited Twist1-promoted migration, invasion and metastasis. Restored Foxa1 expression did not change the Twist1-induced mesenchymal cellular morphology and the expression of Twist1-regulated E-cadherin, ?-catenin, vimentin and Slug, but it partially rescued Twist1-silenced ER? and cytokeratin 8 expression and reduced Twist1-induced integrin ?5, integrin ?1 and MMP9 expression. In a xenografted mouse model, restored Foxa1 also increased Twist1-repressed LBC markers and decreased Twist1-induced BLBC markers. Furthermore, Twist1 expression is negatively correlated with Foxa1 in the human breast tumors. The tumors with high Twist1 and low Foxa1 expressions are associated with poor distant metastasis-free survival. These results demonstrate that Twist1's silencing effect on Foxa1 expression is largely responsible for Twist1-induced migration, invasion and metastasis, but less responsible for Twist1-induced mesenchymal morphogenesis and expression of certain EMT markers.
Project description:We have demonstrated that the oncogenic activation of B-RAF (using a truncated delta-BRAF-ER version inducible with tamoxifen) in the melan-a melanocyte cell line triggers the activation of Zeb1 and Twist1 at the expanse of Zeb2 and Snail2. Enforced maintenance of Zeb2 or Snail2 expression reduces the B-RAF oncogenic potential while ectopic expression of Zeb1 or Twist1 cooperates with B-RAF in melan-a cell transformation. To get an insight into the properties of these embryonic transcription factors, gene expression profiles of melan-a-derived cell lines either expressing a non-activated B-RAF (- tamoxifen) or an activated BRAF (+ tamoxifen) alone or in combination with Snail2, Zeb2, Twist1 or Zeb1 have been established. Melan-a cells were transduced with an activated version of BRAF(delta-BRAF-ER, inducible with tamoxifen) alone or in combination with SNAIL2, ZEB2, ZEB1 or TWIST1. Gene expression profiles before or following BRAF activation (alone or in combination with the embryonic transcription factors) were determined. Ectopic expression of SNAIL2, ZEB2, ZEB1 or TWIST1 on BRAF-target genes in the murine melanocytic melan-a cell line.