Project description:In this study we studied the presence of tumor cells that underwent epithelial-to-mesenchymal transition within polyoma middle T antigen (PyMT) breast tumors. For this we dissociated tumors and isolated Ecad positive tumor cells by FACS sorting. We confirmed that PyMT tumors contain a small set of tumor cells that have undergone EMT in the primary tumor and that E-cadherin can be used as a marker on single cell level for mesenchymal status in this model.
Project description:Forced overexpression and/or downregulation of proteins regulating epithelial-to-mesenchymal transition (EMT) has been reported to alter metastasis by changing migration and stem cell capacity of tumor cells. However, these manipulations artificially keep cells in fixed states, while in vivo cells may adapt transient and reversible states. Here, we have tested the existence and role of epithelial-mesenchymal plasticity in metastasis of mammary tumors without artificially modifying EMT regulators. In these tumors, we found by intravital microscopy that the motile tumor cells have undergone EMT, while their epithelial counterparts were not migratory. Moreover, we found that epithelial-mesenchymal plasticity renders any EMT-induced stemness differences, as reported previously, irrelevant for metastatic outgrowth, because mesenchymal cells that arrive at secondary sites convert to the epithelial state within one or two divisions, thereby obtaining the same stem cell potential as their arrived epithelial counterparts. We conclude that epithelial-mesenchymal plasticity supports migration but additionally eliminates stemness-enhanced metastatic outgrowth differences.
Project description:Epithelial-mesenchymal transition (EMT) has been linked to cancer progression and metastatic propensity. The 4T1 tumor is a clinically relevant model of spontaneous breast cancer metastasis. Here we characterize 4T1-derived cell lines for EMT, in vitro invasiveness and in vivo metastatic ability. Contrary to expectations, the 67NR cells, which form primary tumors but fail to metastasize, express vimentin and N-cadherin, but not E-cadherin. 4T1 cells, however, express E-cadherin, are highly migratory and invasive, and metastasize to multiple sites. The 66cl4 metastatic cells display mixed epithelial and mesenchymal markers, but are less migratory and invasive than 67NR cells. These findings demonstrate that the metastatic ability of breast cancer cells does not correlate with genotypic and phenotypic properties of EMT per se, and suggest that other processes may govern metastatic capability. Gene expression analysis also has not identified differences in EMT markers, but has identified several candidate genes that may influence metastatic ability. Keywords: cell type comparison
Project description:In this study we want to compare the expression profiles of E-cadherin high and low tumor cells from 4 different mice with breast cancer brain metastasis. We used a breast cancer brain metastasis model derived from the PyMT E-cadherin-mCFP breast cancer model (Beerling et al. Cell Reports 2016) and FACS sorted samples based on endogenous E-cadherin-mCFP expression.
Project description:BackgroundEribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) in human breast cancer cells were investigated.MethodsTriple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER-)/progesterone receptor (PR-)/human epithelial growth receptor 2 (HER2-) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting.ResultsTreatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model.ConclusionsEribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.
Project description:Experimental evidence accumulated over decades has implicated epithelial-mesenchymal plasticity (EMP), which collectively encompasses epithelial-mesenchymal transition and the reverse process of mesenchymal-epithelial transition, in tumour metastasis, cancer stem cell generation and maintenance, and therapeutic resistance. However, the dynamic nature of EMP processes, the apparent need to reverse mesenchymal changes for the development of macrometastases and the likelihood that only minor cancer cell subpopulations exhibit EMP at any one time have made such evidence difficult to accrue in the clinical setting. In this Perspectives article, we outline the existing preclinical and clinical evidence for EMP and reflect on recent controversies, including the failure of initial lineage-tracing experiments to confirm a major role for EMP in dissemination, and discuss accumulating data suggesting that epithelial features and/or a hybrid epithelial-mesenchymal phenotype are important in metastasis. We also highlight strategies to address the complexities of therapeutically targeting the EMP process that give consideration to its spatially and temporally divergent roles in metastasis, with the view that this will yield a potent and broad class of therapeutic agents.
Project description:Tumor metastasis leads to high mortality; therefore, understanding the mechanisms that underlie tumor metastasis is crucial. Generally seen as a secretory protein, osteopontin (OPN) is involved in multifarious pathophysiological events. Here, we present a novel pro-metastatic role of OPN during metastatic colonization. Unlike secretory OPN (sOPN), which triggers the epithelial-mesenchymal transition (EMT) to initiate cancer metastasis, intracellular/nuclear OPN (iOPN) induces the mesenchymal-epithelial transition (MET) to facilitate the formation of metastases. Nuclear OPN is found to interact with HIF2α and impact the subsequent AKT1/miR-429/ZEB cascade. In vivo assays confirm that the progression of metastatic colonization is accompanied by the nuclear accumulation of OPN and the MET process. Furthermore, evidence of nuclear OPN in the lung metastases is exhibited in clinical specimens. Finally, VEGF in the microenvironment was shown to induce the translocation of OPN into the nucleus through a KDR/PLCγ/PKC-dependent pathway. Taken together, our results describe the pleiotropic roles of OPN in the tumor metastasis cascade, which indicate its potential as an effective target for both early and advanced tumors.
Project description:Epithelial-mesenchymal transition (EMT) has been linked to cancer progression and metastatic propensity. The 4T1 tumor is a clinically relevant model of spontaneous breast cancer metastasis. Here we characterize 4T1-derived cell lines for EMT, in vitro invasiveness and in vivo metastatic ability. Contrary to expectations, the 67NR cells, which form primary tumors but fail to metastasize, express vimentin and N-cadherin, but not E-cadherin. 4T1 cells, however, express E-cadherin, are highly migratory and invasive, and metastasize to multiple sites. The 66cl4 metastatic cells display mixed epithelial and mesenchymal markers, but are less migratory and invasive than 67NR cells. These findings demonstrate that the metastatic ability of breast cancer cells does not correlate with genotypic and phenotypic properties of EMT per se, and suggest that other processes may govern metastatic capability. Gene expression analysis also has not identified differences in EMT markers, but has identified several candidate genes that may influence metastatic ability. Experiment Overall Design: Female BALB/c mice were injected with 1x10^6 viable cells (3 mice with 4T1/CMVLUC, 3 mice with 66cl4/CMVLUC, and 3 mice with 67NR/CMVLUC) into the right fourth mammary gland. 15 days after injection primary tumors were excised, and total RNA for microarray hybridization was isolated from the tumor part of laser capture microdissected sections.
Project description:Distant metastasis is the major cause of cancer-related death in patients with colorectal cancer (CRC). Although the microRNA-200 (miR-200) family is a crucial inhibitor of epithelial-to-mesenchymal transition (EMT) in human cancer, the role of miR-200 members in the pathogenesis of metastatic CRC has not been investigated.Fifty-four pairs of primary CRC and corresponding matched liver metastasis tissue specimens were analysed for expression and methylation status of the miR-200 family members. Functional analysis of miR-200c overexpression was investigated in CRC cell lines, and cells were analysed for proliferation, invasion and migration. Expression of several miR-200c target genes (ZEB1, ETS1 and FLT1) and EMT markers (E-cadherin and vimentin) in CRC cell lines and tissue specimens was validated.Liver metastasis tissues showed higher expression of miR-200c (primary CRC = 1.31 vs. liver metastasis = 1.59; p = 0.0014) and miR-141 (primary CRC = 0.14 vs. liver metastasis = 0.17; p = 0.0234) than did primary CRCs, which was significantly associated with hypomethylation of the promoter region of these miRNAs (primary CRC = 61.2% vs. liver metastasis = 46.7%; p < 0.0001). The invasive front in primary CRC tissues revealed low miR-200c expression by in situ hybridization analysis. Transfection of miR-200c precursors resulted in enhanced cell proliferation but reduced invasion and migration behaviours in CRC cell lines. Overexpression of miR-200c in CRC cell lines caused reduced expression of putative gene targets, and resulted in increased E-cadherin and reduced vimentin expression. The associations between miR-200c, target genes and EMT markers were validated in primary CRCs and matching liver metastasis tissues.miR-200c plays an important role in mediating EMT and metastatic behaviour in the colon. Its expression is epigenetically regulated, and miR-200c may serve as a potential diagnostic marker and therapeutic target for patients with CRC.
Project description:This experiment is designed to evaluate gene expression alterations following miR-374a transduction in breast cancer cells. We find a significant elevation of Wnt/β-catenin signaling transcriptional targets. Total RNA were extracted from MCF-7 breast cancer cells stably transduced with miR-374a precursor or vector control. Two biological replications for each treatment.