ABSTRACT
Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with significant remodeling, and although this remodeling is reversed by current treatment regimens, symptoms of EoE and associated remodeling reappear upon cessation of therapies. We hypothesized that structural remodeling of cell-cell adhesion is a key factor in the pathogenesis of EoE and that epithelial to mesenchymal transition (EMT) was a viable molecular process to lead to this remodeling. Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were evaluated by transmission electron microscopy to measure intercellular spaces (IS) between cells. Biopsy samples from all groups were analyzed for cellular levels of cell-cell adhesion proteins: E-cadherin, zonula occludens associated protein-1 (ZO-1), and N-cadherin. We also analyzed for cellular levels and localization two of transcription factors, Twist1 and ß-catenin, that are associated with promoting EMT. The IS was significantly increased in the EoE group compared to the control. We observed a significant decrease in E-cadherin and ZO-1 levels and a concomitant increase in N-cadherin levels in EoE samples compared to control. Further, while there was no significant change in cellular levels of ß-catenin, we observed an altered localization of the protein from the cell membrane in control tissue to a nuclear/perinuclear localization in EoE. We observed higher levels of the transcription factor Twist1 in the EoE group compared to normal which was localized mainly at the nucleus. Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. Our data also suggest that EMT, potentially regulated by transcription factors ß-catenin and Twist1, may be responsible for the molecular alteration which leads to the remodeling of esophageal epithelia in EoE.
Subject(s)
Eosinophilic Esophagitis , Epithelial-Mesenchymal Transition , Nuclear Proteins , Twist-Related Protein 1 , beta Catenin , Cadherins/physiology , Child , Eosinophilic Esophagitis/pathology , Humans , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , beta Catenin/physiologyABSTRACT
Purpose E-cadherin is a calcium-dependent glycoprotein whose main role is cellcell adhesion. Its transcriptional repressor TWIST1 is a basic helixloophelix (bHLH) protein that participates in gastrulation and formation of mesodermal tissues during embryogenesis. In adult tissues, the high expression of TWIST1 induces the epithelialmesenchymal transition (EMT)a process in which cells become motile and able to metastasize. In this paper, we investigated the involvement of E-cadherin and TWIST1 in the carcinogenesis of brain metastases originating from two different primary sitesbreast and lung. Methods The localization and expression of E-cadherin and its transcriptional repressor TWIST1 were investigated using a DAB-labeled streptavidinhorseradish peroxidase immunohistochemical reaction and specific monoclonal antibodies against TWIST1 and E-cadherin. Image J software was used for semi-quantitative analysis while H-score served for statistical evaluations. Results Immunohistochemistry showed that the expression of E-cadherin was downregulated in 85.7% of brain metastases, while at the same time, 82.2% of them showed upregulated TWIST1. Statistical analysis confirmed a significant negative correlation between expressions of TWIST1 and E-cadherin (p = 0.001). When the brain metastases expression levels were compared to primary breast tumors in corresponding patients, E-cadherin showed higher expression in primary pairs compared to corresponding metastases. Consistent to its role, TWIST1 was downregulated in all primary tumor samples in comparison to corresponding metastases pairs (p = 0.034). Conclusion This research provides valuable data regarding molecular events involving two EMT key components that could give directions for new possibilities for brain metastases diagnosis and treatment (AU)
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Brain Neoplasms , Cadherins/metabolism , Lung Neoplasms/pathology , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , Up-Regulation , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Brain Neoplasms/secondaryABSTRACT
Mitochondria of hematopoietic stem cells (HSCs) play crucial roles in regulating cell fate and preserving HSC functionality and survival. However, the mechanism underlying HSC regulation remains poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Twist1 deletion results in significantly decreased lymphoid-biased HSC frequency, markedly reduced HSC dormancy and self-renewal capacity, and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil-induced stresses and exhibit typical phenotypes of senescence. Mechanistically, Twist1 deletion induces transactivation of voltage-gated calcium channel (VGCC) Cacna1b, which exhausts lymphoid-biased HSCs, impairs genotoxic hematopoietic recovery, and enhances mitochondrial calcium levels, metabolic activity, and reactive oxygen species production. Suppression of VGCC by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through the CACNA1B/Ca2+/mitochondria axis and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate.
Subject(s)
Calcium Channels, N-Type/physiology , Hematopoietic Stem Cells/cytology , Twist-Related Protein 1/physiology , Adenosine Triphosphate/metabolism , Animals , Apoptosis , Calcium Channel Blockers/pharmacology , Calcium Signaling , Cell Cycle , Cell Self Renewal , DNA Damage , Fluorouracil/pharmacology , Fluorouracil/toxicity , Gene Expression Regulation , Gene Ontology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/radiation effects , Mice , Mice, Congenic , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Myelopoiesis , RNA, Messenger/biosynthesis , Radiation Injuries, Experimental/prevention & control , Reactive Oxygen Species/metabolism , Twist-Related Protein 1/deficiency , Twist-Related Protein 1/geneticsABSTRACT
The nuclear transcription factor twist-related protein 1 (Twist1) is associated with tumor malignant transformation and metastasis in various types of carcinomas. We found that Twist1 was highly expressed in clinical multiple myeloma (MM) cells, and explored its roles in proliferation and apoptosis in human MM cell lines U266 and RPMI-8226. In these cells, Twist1 transcriptionally regulated the miRNA hsa-miR138-5p, which targeted caspase-3 to control apoptosis. Silencing of Twist1 significantly suppressed cell proliferation and increased apoptosis, which was reversed by overexpression of hsa-miR138-5p or simultaneous silencing of caspase-3. This reversion was further substantiated by attenuated apoptotic signaling, including downregulated expression of the cleaved forms of caspase-3 and peroxisome proliferator-activated receptor 1 (PPAR1). We demonstrate here for the first time that the novel Twist1/hsa-miR138-5p/caspase-3 pathway contributes significantly to the proliferation and survival of human MM cells. Our study provides new insight for novel MM treatments by developing Twist1-targeted therapeutics.
Subject(s)
Cell Proliferation/genetics , Multiple Myeloma/pathology , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , Aged , Aged, 80 and over , Apoptosis/genetics , Caspase 3/genetics , Caspase 3/physiology , Cell Line, Tumor , Cell Movement/genetics , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , MicroRNAs/genetics , MicroRNAs/physiology , Middle Aged , Multiple Myeloma/genetics , Nuclear Proteins/genetics , Signal Transduction/genetics , Twist-Related Protein 1/geneticsABSTRACT
PURPOSE: E-cadherin is a calcium-dependent glycoprotein whose main role is cell-cell adhesion. Its transcriptional repressor TWIST1 is a basic helix-loop-helix (bHLH) protein that participates in gastrulation and formation of mesodermal tissues during embryogenesis. In adult tissues, the high expression of TWIST1 induces the epithelial-mesenchymal transition (EMT)-a process in which cells become motile and able to metastasize. In this paper, we investigated the involvement of E-cadherin and TWIST1 in the carcinogenesis of brain metastases originating from two different primary sites-breast and lung. METHODS: The localization and expression of E-cadherin and its transcriptional repressor TWIST1 were investigated using a DAB-labeled streptavidin-horseradish peroxidase immunohistochemical reaction and specific monoclonal antibodies against TWIST1 and E-cadherin. Image J software was used for semi-quantitative analysis while H-score served for statistical evaluations. RESULTS: Immunohistochemistry showed that the expression of E-cadherin was downregulated in 85.7% of brain metastases, while at the same time, 82.2% of them showed upregulated TWIST1. Statistical analysis confirmed a significant negative correlation between expressions of TWIST1 and E-cadherin (p = 0.001). When the brain metastases expression levels were compared to primary breast tumors in corresponding patients, E-cadherin showed higher expression in primary pairs compared to corresponding metastases. Consistent to its role, TWIST1 was downregulated in all primary tumor samples in comparison to corresponding metastases pairs (p = 0.034). CONCLUSION: This research provides valuable data regarding molecular events involving two EMT key components that could give directions for new possibilities for brain metastases diagnosis and treatment.
Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/secondary , Breast Neoplasms/pathology , Cadherins/biosynthesis , Lung Neoplasms/pathology , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , Up-Regulation , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle AgedABSTRACT
Twist-related protein 1 (TWIST1), a highly conserved basic helix-loop-helix transcription factor, stimulates epithelial-mesenchymal transition (EMT) and plays a crucial role in the regulation of the extracellular matrix (ECM) and cell-cell adhesion. Our aim in this study was to evaluate the functional correlation between TWIST1 and MMP genes in human ESCC cell lines, KYSE-30 and YM-1. To generate recombinant retroviral particles, the Pruf-IRES-GFP-hTWIST1 was co-transfected into HEK293T along with pGP and pMD2. G as well as Pruf-IRES-GFP control plasmid. Stably transduced high-expressing GFP-hTWIST1 and GFP-control KYSE-30 cells were generated. The produced retroviral particles were transduced into the KYSE-30 and YM-1 ESCC cells. Ectopic expression of TWIST1 mRNA and expression of the MMP genes (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10) were examined by relative comparative real-time PCR. In silico analysis of the MMP markers and their promoter elements was explored. Moreover, the scratch wound assay was used to evaluate the migration of TWIST1-induced cells. TWIST1 level was up-regulated by nearly 5-fold and 7.4-fold in GFP-hTWIST1 KYSE-30 and YM-1 cells compared to GFP control cells, respectively. Interestingly, this enforced expression of TWIST1 subsequently caused significant overexpression of transcripts for selected MMP genes in GFP-hTWIST1 in comparison with GFP control cells in both ESCC cell lines. Also, the scratch assay indicated that TWIST1 expression effectively increased the migration of GFP-TWIST1 KYSE-30 cells against GFP KYSE-30 control cells in vitro. The present findings illuminate that TWIST1 may contribute broadly to ESCC development in concert with up-regulation of MMPs expression and further suggest the potential advantage of exerting TWIST1/MMPs signaling axis as a framework from which to expand our understanding about the mechanisms of ESCC tumorigenesis.
Subject(s)
Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , Matrix Metalloproteinases/genetics , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , Up-Regulation/physiology , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Esophageal Neoplasms/enzymology , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/enzymology , Esophageal Squamous Cell Carcinoma/pathology , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Promoter Regions, GeneticABSTRACT
BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is an oral precancerous disorder associated with the habit of areca nut chewing. MiR-10b has been shown to be upregulated in the oral cancer cells and induced by Twist. Our previous work has revealed that Twist participated in the pathogenesis of OSF and therefore we aimed to investigate whether Twist/miR-10b axis was involved in the activation of myofibroblast in the oral cavity. METHODS: The expression levels of miR-10b in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined. Besides, the expression of miR-10b was determined in fBMFs following knockdown of Twist or in BMFs after arecoline stimulation. Myofibroblast activities, including collagen gel contraction, migration and wound healing abilities, as well as the expression of α-SMA were measured in fBMFs treated with miR-10b inhibitor. Last, we investigated whether the effect of Twist overexpression could be reversed by suppression of miR-10b. RESULTS: MiR-10b expression was overexpressed in both OSF tissues and fBMFs. The silence of Twist resulted in the downregulation of miR-10b in fBMFs and arecoline treatment led to an increase of miR-10b in a dose-dependent manner. Inhibition of miR-10b ameliorated the activation of myofibroblasts and the expression of α-SMA. Moreover, we demonstrated that suppression of miR-10b hindered the increased collagen gel contraction caused by Twist overexpression. CONCLUSION: MiR-10b upregulation in OSF may be due to the stimulation of areca nut, leading to elevated myofibroblast activation. Our findings showed that the areca nut-induced expression of miR-10b was under the regulation of Twist and inhibition of miR-10b may provide a direction for treatment of OSF.
Subject(s)
MicroRNAs , Nuclear Proteins , Oral Submucous Fibrosis , Twist-Related Protein 1 , Areca , Arecoline/pharmacology , Cell Transdifferentiation , Fibroblasts , Humans , MicroRNAs/genetics , Mouth Mucosa , Myofibroblasts , Nuclear Proteins/physiology , Twist-Related Protein 1/physiologyABSTRACT
Dendritic cells (DCs) play a central role in autoimmunity, immune homeostasis, and presentation of tumor antigens to T cells in order to prime antitumor responses. The number of tumor-infiltrating DCs is associated with survival and prognosis in cancer. Twist1 is a well-known regulator of tumor initiation and promotion, but whether and how DC-derived Twist1 regulates antitumor responses remains poorly understood. Here, we generated a mouse line with Twist1 conditionally depleted in DCs and found that Twist1-deficiency in DCs did not affect the DCs and T cell homeostasis under steady-state conditions; however, in melanoma models, the proportion of conventional DCs (cDCs) in draining lymph nodes (DLNs) was significantly decreased. Accordingly, a decreased ratio and number of tumor-infiltrating cDCs were observed, which reduced the recruitment of tumor-infiltrating T cells. Furthermore, production of IFN-γ, a crucial antitumor factor, by T cells, was dramatically decreased, which can further dampen the T cell antitumor functions. Collectively, our data indicate that Twist1 in DCs regulates antitumor functions by maintain the number of tumor-infiltrating DCs and T cells, and their antitumor activity.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Immunity, Cellular/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma, Experimental/immunology , Twist-Related Protein 1/physiology , Animals , Antigens, Neoplasm/immunology , Dendritic Cells/metabolism , Dendritic Cells/pathology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, KnockoutABSTRACT
AIMS: To investigate the effects and mechanisms of DLL3 in inflammation-mediated A2058 melanoma cell invasion and metastasis. MATERIALS AND METHODS: Melanoma A2058 cells was stimulated with lipopolysaccharide (LPS), with or without transfection of DLL3 siRNA, or DLL3 overexpression vector, or Twist1 siRNA. Cell migration and invasion were detected by wound healing and transwell invasion assay. The production of inflammatory factors TNF-α and IL-6 was measured by ELISA. The expression of Notch signaling-related molecules was detected by PCR and western blot. The protein expression of MMP1, MMP9, VEGF, DLL3, and EMT-related molecules was tested by western blot. KEY FINDINGS: LPS treatment increased migration and invasion of A2058 cells, accompanied by increased expression of TNF-α and IL-6. DLL3 was both upregulated in the LPS- or TNF-α-stimulated A2058 cells, and DLL3 knockdown inhibited LPS-induced inflammation, migration and invasion of A2058 cells, accompanied by down-regulation of MMP1, MMP9 and VEGF. Besides, DLL3 knockdown inhibits the expression of Twist1, a key EMT regulating factor, as well as the EMT hallmarks slug, N-cadherin and vimentin. Moreover, Twist1 silence inhibited EMT, and limited LPS-induced migration and invasion of A2058 cells, with decreased expression of MMP1, MMP9 and VEGF and reduced production of TNF-α and IL-6 in LPS-stimulated A2058 cells. SIGNIFICANCE: Knockdown of DLL3 restricts LPS-induced inflammation, migration and invasion of A2058 melanoma cells via blocking Twist1-mediated EMT. Therefore, targeting DLL3 may be a promising therapeutic strategy against inflammation-aggravated melanoma progression.
Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Melanoma/metabolism , Membrane Proteins/genetics , Membrane Proteins/physiology , Cadherins , Cell Line, Tumor , Cell Movement/genetics , Down-Regulation , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Knockdown Techniques , Humans , Inflammation/genetics , Interleukin-6 , Lipopolysaccharides/pharmacology , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Melanoma/genetics , Neoplasm Invasiveness/genetics , Nuclear Proteins/physiology , Signal Transduction , Transcriptional Activation , Tumor Necrosis Factor-alpha/drug effects , Twist-Related Protein 1/physiology , Up-Regulation , Vascular Endothelial Growth Factor A , VimentinABSTRACT
Metastasis is the main cause of cancer mortality. However, the triggering mechanisms and regulation of epithelial-mesenchymal transition (EMT) factors in the commitment of metastasis have not been well characterized. Spermatogenic Zip 1 (SPZ1) acts as a proto-oncogene and an upstream regulator of EMT during tumorigenesis. Here we report that the HIV-1 Tat-interacting protein 60 kDa (Tip60) acetyltransferase mediates acetylation at lysine residues of SPZ1 at positions 369 and 374, and of TWIST1 at positions 73 and 76, which are required for SPZ1-TWIST1 complex formation and cancer cell migration in vitro and in vivo. Ectopic SPZ1 and TWIST1 expression, but not that of TWIST1 alone, enhanced vascular endothelial growth factor (VEGF) expression via the recruitment of bromodomain-containing protein 4 (BRD4), thus enhancing RNA-Pol II-dependent transcription and inducing metastasis. Neutralization of VEGF using humanized monoclonal antibodies such as Avastin, effectively abrogated the EMT and oncogenesis induced by the acetylated SPZ1-TWIST1 complex. Our findings highlight the importance of acetylation signaling in the SPZ1-TWIST1-BRD4 axis in the mediation of EMT and its regulation during tumor initiation and metastasis.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/physiology , Epithelial-Mesenchymal Transition/physiology , Liver Neoplasms/metabolism , Lysine Acetyltransferase 5/physiology , Neoplasm Proteins/physiology , Nuclear Proteins/physiology , Protein Processing, Post-Translational , Transcription Factors/physiology , Twist-Related Protein 1/physiology , Acetylation , Animals , Bevacizumab/pharmacology , Bevacizumab/therapeutic use , Carcinogenesis , Cell Cycle Proteins , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/blood supply , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/genetics , Mice , Mice, Nude , Mice, Transgenic , Neoplasm Invasiveness , Neoplasm Metastasis , Neovascularization, Pathologic/metabolism , Protein Interaction Mapping , Proto-Oncogene Mas , Signal Transduction , Sorafenib/pharmacology , Sorafenib/therapeutic use , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , Xenograft Model Antitumor AssaysABSTRACT
Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. The poor survival may be due to a high proportions of tumor recurrence and metastasis. Kinesin family member C1 (KIFC1) is highly expressed in a variety of neoplasms and is a potential marker for non-small cell lung cancer or ovarian adenocarcinoma metastasis. Nevertheless, the role of KIFC1 in HCC metastasis remains obscure. We investigated this in the present study using HCC cell lines and clinical specimens. Our results indicated that increased levels of KIFC1 were associated with poor prognosis and metastasis in HCC. In addition, KIFC1 induced epithelial-to-mesenchymal transition (EMT) and HCC metastasis both in vitro and in vivo. This tumorigenic effect depended on gankyrin; inhibiting gankyrin activity reversed EMT via activation of protein kinase B (AKT)/Twist family BHLH transcription factor 1 (AKT/TWIST1). We also found that KIFC1 was directly regulated by the microRNA miR-532-3p, whose downregulation was associated with metastatic progression in HCC. These results denote that a decrease in miR-532-3p levels results in increased KIFC1 expression in HCC, leading to metastasis via activation of the gankyrin/AKT/TWIST1 signaling pathway.
Subject(s)
Carcinoma, Hepatocellular/secondary , Epithelial-Mesenchymal Transition/physiology , Kinesins/physiology , Liver Neoplasms/pathology , Lung Neoplasms/secondary , MicroRNAs/physiology , Neoplasm Proteins/physiology , RNA, Neoplasm/physiology , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/mortality , Cell Line, Tumor , Down-Regulation , Heterografts , Humans , Kaplan-Meier Estimate , Kinesins/antagonists & inhibitors , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/mortality , Lung Neoplasms/physiopathology , Male , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/biosynthesis , MicroRNAs/genetics , Neoplasm Proteins/antagonists & inhibitors , Nuclear Proteins/physiology , Prognosis , Proteasome Endopeptidase Complex/physiology , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt/physiology , RNA Interference , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Signal Transduction , Twist-Related Protein 1/physiologyABSTRACT
The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.
Subject(s)
Limb Deformities, Congenital/genetics , Twist-Related Protein 1/genetics , Twist-Related Protein 1/physiology , Animals , Branchial Region/metabolism , Enhancer Elements, Genetic/genetics , Extremities/embryology , Gene Expression Regulation, Developmental/genetics , Genes, Homeobox , Histone Deacetylases/genetics , Homeodomain Proteins/genetics , Limb Buds/metabolism , Limb Deformities, Congenital/embryology , Mice , Mice, Inbred C57BL , Organogenesis , Repressor Proteins/genetics , Transcription Factor AP-2 , Transcription Factors/genetics , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially, in eastern Asia, and its prognosis is poor once metastasis occurs. Niclosamide, a US Food and Drug Administration-approved antihelmintic drug, was shown to inhibit the growth of various cancers including HCC, but the effect of niclosamide on cell motility and the underlying mechanism have not yet been completely defined. The present study demonstrated that niclosamide, at 0-40 nM, concentration-dependently inhibited wound closure and the migratory/invasive capacities of human Huh7 and SK-Hep-1 HCC cells without exhibiting cytotoxicity. A protease array analysis showed that CD10 was dramatically downregulated in Huh7 cells after niclosamide treatment. Western blot and flow cytometric assays further demonstrated that CD10 expression was concentration-dependently downregulated in Huh7 and SK-Hep-1 cells after niclosamide treatment. Mechanistic investigations found that niclosamide suppressed Twist-mediated CD10 transactivation. Moreover, knockdown of CD10 expression by CD10 small interfering RNA in HCC cells suppressed cell migratory/invasive abilities and overexpression of CD10 relieved the migration inhibition induced by niclosamide. Taken together, our results indicated that niclosamide could be a potential agent for inhibiting metastasis of HCC, and CD10 is an important target of niclosamide for suppressing the motility of HCC cells.
Subject(s)
Anthelmintics/pharmacology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Neprilysin/genetics , Niclosamide/pharmacology , Administration, Oral , Anthelmintics/administration & dosage , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Humans , Liver Neoplasms/genetics , Neoplasm Invasiveness , Neoplasm Metastasis , Niclosamide/administration & dosage , RNA, Small Interfering/genetics , Twist-Related Protein 1/physiologyABSTRACT
The mesenchymal gene program has been shown to promote the metastatic progression of ovarian cancer; however, specific proteins induced by this program that lead to these metastatic behaviors have not been identified. Using patient derived tumor cells and established human ovarian tumor cell lines, we find that the Epithelial-to-Mesenchymal Transition inducing factor TWIST1 drives expression of discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase (RTK) that recognizes fibrillar collagen as ligand. The expression and action of DDR2 was critical for mesothelial cell clearance, invasion and migration in ovarian tumor cells. It does so, in part, by upregulating expression and activity of matrix remodeling enzymes that lead to increased cleavage of fibronectin and spreading of tumor cells. Additionally, DDR2 stabilizes SNAIL1, allowing for sustained mesenchymal phenotype. In patient derived ovarian cancer specimens, DDR2 expression correlated with enhanced invasiveness. DDR2 expression was associated with advanced stage ovarian tumors and metastases. In vivo studies demonstrated that the presence of DDR2 is critical for ovarian cancer metastasis. These findings indicate that the collagen receptor DDR2 is critical for multiple steps of ovarian cancer progression to metastasis, and thus, identifies DDR2 as a potential new target for the treatment of metastatic ovarian cancer.
Subject(s)
Discoidin Domain Receptor 2/genetics , Nuclear Proteins/physiology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Twist-Related Protein 1/physiology , Animals , Biomarkers, Tumor/physiology , Cell Movement/genetics , Cells, Cultured , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Ovarian Neoplasms/mortality , Up-Regulation/geneticsABSTRACT
Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.
Subject(s)
Carotid Arteries/pathology , Endothelium, Vascular/pathology , Epithelial-Mesenchymal Transition , Gene Expression Regulation , Plaque, Atherosclerotic/pathology , Snail Family Transcription Factors/metabolism , Stress, Mechanical , Animals , Aorta/metabolism , Aorta/pathology , Carotid Arteries/metabolism , Cell Proliferation , Cells, Cultured , Endothelium, Vascular/metabolism , Humans , Mice , Mice, Knockout , Nuclear Proteins/physiology , Plaque, Atherosclerotic/metabolism , Receptor, TIE-1/physiology , Snail Family Transcription Factors/genetics , Swine , Twist-Related Protein 1/physiologyABSTRACT
The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. Expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/physiology , Carcinoma, Hepatocellular/pathology , Epithelial-Mesenchymal Transition , Liver Neoplasms/pathology , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , Adult , Aged , Aged, 80 and over , Carcinogenesis , Carcinoma, Hepatocellular/etiology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Liver Neoplasms/etiology , Male , Middle Aged , Nuclear Proteins/genetics , Twist-Related Protein 1/geneticsABSTRACT
Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/physiology , Lung Neoplasms/drug therapy , Nuclear Proteins/physiology , Small Cell Lung Carcinoma/drug therapy , Animals , Drug Resistance, Neoplasm , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Humans , Mice , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Twist-Related Protein 1/physiologyABSTRACT
OBJECTIVES: TWIST1 is a member of the class B of basic helix-loop-helix transcription factors that regulates cell lineage determination and differentiation and has been implicated in epithelial-to-mesenchymal transition. Here, we aimed to investigate the role of TWIST1 for the activation of resident fibroblasts in systemic sclerosis (SSc). METHODS: The expression of Twist1 in fibroblasts was modulated by forced overexpression or siRNA-mediated knockdown. Interaction of Twist1, E12 and inhibitor Of differentiation (Id) was analysed by co-immunoprecipitation. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitous or fibroblast-specific depletion of Twist1. Mice were either challenged with bleomycin or overexpressing a constitutively active transforming growth factor (TGF)ß receptor I. RESULT: The expression of TWIST1 was increased in fibroblasts in fibrotic human and murine skin in a TGFß/SMAD3-dependent manner. TWIST1 in turn enhanced TGFß-induced fibroblast activation in a p38-dependent manner. The stimulatory effects of TWIST1 on resident fibroblasts were mediated by TWIST1 homodimers. TGFß promotes the formation of TWIST1 homodimers by upregulation of TWIST1 and by induction of inhibitor of DNA-binding proteins, which have high affinity for E12/E47 and compete against TWIST1 for E12/E47 binding. Mice with selective depletion of Twist1 in fibroblasts are protected from experimental skin fibrosis in different murine models to a comparable degree as mice with ubiquitous depletion of Twist1. CONCLUSIONS: Our data identify TWIST1 as a central pro-fibrotic factor in SSc, which facilitates fibroblast activation by amplifying TGFß signalling. Targeting of TWIST1 may thus be a novel approach to normalise aberrant TGFß signalling in SSc.
Subject(s)
Fibroblasts/metabolism , Nuclear Proteins/physiology , Scleroderma, Systemic/metabolism , Twist-Related Protein 1/physiology , Animals , Case-Control Studies , Female , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Gene Knockdown Techniques , Humans , Male , Mice, Knockout , Nuclear Proteins/biosynthesis , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Multimerization/physiology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Scleroderma, Systemic/pathology , Signal Transduction/physiology , Skin/pathology , Transforming Growth Factor beta/pharmacology , Twist-Related Protein 1/biosynthesis , Twist-Related Protein 1/deficiency , Twist-Related Protein 1/genetics , Twist-Related Protein 1/metabolismABSTRACT
TWIST1 is a highly conserved basic helix-loop-helix transcription factor that contributes to cancer metastasis by promoting an epithelial-mesenchymal transition and repressing E-cadherin gene expression in breast cancer. In this study, we explored the potential role of miR-151 in TWIST1 expression and cancer properties in human breast cancer cells. We found that the human TWIST1 3'UTR contains a potential binging site for miR-151-3p at the putative target sequence 5'-CAGUCUAG-3'. Using a TWIST1-3'UTR luciferase reporter assay, we demonstrated that the target sequence within the TWIST1 3'UTR is required for miR-151-3p regulation of TWIST1 expression. Moreover, we found that ectopic expression of miR-151-3p by infection with adenoviruses expressing miR-151 significantly decreased TWIST1 expression, migration and invasion, but did not affect cell growth and tumorsphere formation of human breast cancer cells. In addition, overexpression of the protein coding region without the 3'UTR of TWIST1 reversed the repression of cell migration by miR-151-3p. Furthermore, knockdown of miR-151-3p increased TWIST1 expression, reduced E-cadherin expression, and enhanced cell migration. In conclusion, these results suggest that miR-151-3p directly regulates TWIST1 expression by targeting the TWIST1 3'UTR and thus repressing the migration and invasion of human breast cancer cells by enhancing E-cadherin expression. Our findings add to accumulating evidence that microRNAs are involved in breast cancer progression by modulating TWIST1 expression.
Subject(s)
Breast Neoplasms/physiopathology , Cell Movement/genetics , MicroRNAs/physiology , Nuclear Proteins/physiology , Twist-Related Protein 1/physiology , 3' Untranslated Regions , Blotting, Western , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation , Female , Humans , Neoplasm Invasiveness/physiopathology , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear. METHODS: We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism. RESULTS: OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial-mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration. CONCLUSIONS: Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.