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1.  Structure-Function Studies of the bHLH Phosphorylation Domain of TWIST1 in Prostate Cancer Cells12 
Neoplasia (New York, N.Y.)  2015;17(1):16-31.
The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice.
doi:10.1016/j.neo.2014.10.009
PMCID: PMC4309734  PMID: 25622896
EMT, epithelial-mesenchymal transition; bHLH, basic helix-loop-helix; T-T, TWIST1-TWIST1; E12/E47, E2A proteins; T-E, TWIST1-E12; IHC, immunohistochemistry; PI, propidium iodide; PKA, protein kinase A
2.  Twist1 Controls a Cell-Specification Switch Governing Cell Fate Decisions within the Cardiac Neural Crest 
PLoS Genetics  2013;9(3):e1003405.
Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.
Author Summary
During vertebrate development, a unique population of cells, termed neural crest cells, migrates throughout the developing embryo, generating various cell types, for example, the smooth muscle that divides the aorta and pulmonary artery where they connect to the heart, and the autonomic neurons, which coordinate organ function. The distinctions between neural crest cells that will form smooth muscle and those that will become neurons are thought to occur prior to migration. Here, we show that, in mice with mutations of the transcription factor Twist1, a subpopulation of presumptive smooth muscle cells, following migration to the heart, instead mis-specify to resemble autonomic neurons. Twist1 represses transcription of the pro-neural factor Phox2b both through antagonism of its upstream effector, Sox10, and through direct binding to its promoter. Phox2b is absolutely required for autonomic neuron development, and indeed, the aberrant neurons in Twist1 mutants disappear when Phox2b is also mutated. Ectopic Twist1 expression within all neural crest cells disrupts the specification of normal autonomic neurons. Collectively, these data reveal that neural crest cells can alter their cell fate from mesoderm to ectoderm after they have migrated and that Twist1 functions to maintain neural crest cell potency during embryonic development.
doi:10.1371/journal.pgen.1003405
PMCID: PMC3605159  PMID: 23555309
3.  Phosphorylation of Serine 68 of Twist1 by MAPKs Stabilizes Twist1 Protein and Promotes Breast Cancer Cell Invasiveness 
Cancer research  2011;71(11):3980-3990.
Twist1, a bHLH transcription factor, promotes breast tumor cell epithelial-mesenchymal transition (EMT), invasiveness and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (S68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This S68 is phosphorylated by p38, JNK and ERK1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in HEK293 and breast cancer cells. Prevention of S68 phosphorylation by an alanine (A) mutation (S68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of MAPKs by an active Ras protein or TGF-β treatment significantly increases S68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, while blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and un-induced S68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness upon stimulation with TGF-β or the active Ras as well as taxol resistance compared to same cells expressing the S68A-Twist1 mutant. Importantly, the levels of S68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor-negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1.
doi:10.1158/0008-5472.CAN-10-2914
PMCID: PMC3107354  PMID: 21502402
4.  Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome 
Journal of Clinical Investigation  2001;107(9):1153-1161.
The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased cell growth in mutant osteoblasts compared with control cells. The mutation also caused increased alkaline phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associated with increased type I collagen expression. Mutant cells also showed increased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Twist mutant osteoblasts displayed a cell-autonomous reduction of osteocalcin mRNA expression in basal conditions and during osteogenesis. The data show that genetic deletion of Twist causing reduced Twist dosage increases cell growth, collagen expression, and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossification induced by deletion of the bHLH Twist domain in Saethre-Chotzen syndrome.
PMCID: PMC209280  PMID: 11342579
5.  The Twist Box Domain is Required for Twist1-induced Prostate Cancer Metastasis 
Molecular cancer research : MCR  2013;11(11):10.1158/1541-7786.MCR-13-0218-T.
Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial-mesenchymal transition (EMT) that promotes cancer metastasis. Structure-function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer (PCa). Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in PCa cells using in vitro assays which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extra-thoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for PCa cells to colonize metastatic lung lesions and extra-thoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in PCa cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and PCa metastasis.
doi:10.1158/1541-7786.MCR-13-0218-T
PMCID: PMC3833995  PMID: 23982216
Twist1; Twist box; epithelial-mesenchymal transition; prostate cancer; metastasis; Hoxa9
6.  Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms 
Cell Research  2011;22(1):90-106.
This article reviews the molecular structure, expression pattern, physiological function, pathological roles and molecular mechanisms of Twist1 in development, genetic disease and cancer. Twist1 is a basic helix-loop-helix domain-containing transcription factor. It forms homo- or hetero-dimers in order to bind the Nde1 E-box element and activate or repress its target genes. During development, Twist1 is essential for mesoderm specification and differentiation. Heterozygous loss-of-function mutations of the human Twist1 gene cause several diseases including the Saethre-Chotzen syndrome. The Twist1-null mouse embryos die with unclosed cranial neural tubes and defective head mesenchyme, somites and limb buds. Twist1 is expressed in breast, liver, prostate, gastric and other types of cancers, and its expression is usually associated with invasive and metastatic cancer phenotypes. In cancer cells, Twist1 is upregulated by multiple factors including SRC-1, STAT3, MSX2, HIF-1α, integrin-linked kinase and NF-κB. Twist1 significantly enhances epithelial-mesenchymal transition (EMT) and cancer cell migration and invasion, hence promoting cancer metastasis. Twist1 promotes EMT in part by directly repressing E-cadherin expression by recruiting the nucleosome remodeling and deacetylase complex for gene repression and by upregulating Bmi1, AKT2, YB-1, etc. Emerging evidence also suggests that Twist1 plays a role in expansion and chemotherapeutic resistance of cancer stem cells. Further understanding of the mechanisms by which Twist1 promotes metastasis and identification of Twist1 functional modulators may hold promise for developing new strategies to inhibit EMT and cancer metastasis.
doi:10.1038/cr.2011.144
PMCID: PMC3351934  PMID: 21876555
Twist1; development; differentiation; cancer; epithelial-mesenchymal transition; metastasis
7.  TWIST1 promotes invasion through mesenchymal change in human glioblastoma 
Molecular Cancer  2010;9:194.
Background
Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro.
Results
To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells.
Conclusions
Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.
doi:10.1186/1476-4598-9-194
PMCID: PMC2920263  PMID: 20646316
8.  Phosphoregulation of Twist1 Provides a Mechanism of Cell Fate Control 
Current medicinal chemistry  2008;15(25):2641-2647.
Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist’s biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.
PMCID: PMC2744367  PMID: 18855684
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation
9.  Phosphoregulation of Twist1 Provides a Mechanism of Cell Fate Control 
Current Medicinal Chemistry  2008;15(25):2641-2647.
Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically, Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist’s biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus, the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.
doi:10.2174/092986708785908987
PMCID: PMC2744367  PMID: 18855684
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation.
10.  Nonsense mutations of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients cause dysregulation of periostin 
Setleis Syndrome (OMIM ID: 227260) is a rare autosomal recessive disease characterized by abnormal facial development. Recently, we have reported that two nonsense mutations (c.486C>T [Q119X] and c.324C>T [Q65X]) of the basic helix-loop-helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Here we show that periostin, a cell adhesion protein involved in connective tissue development and maintenance, is down-regulated in Setleis Syndrome patient fibroblast cells and that periostin positively responds to manipulations in TWIST2 levels, suggesting that TWIST2 is a transactivator of periostin. Functional analysis of the TWIST2 mutant form (Q119X) revealed that it maintains the ability to localize to the nucleus, forms homo and heterodimers with the ubiquitous bHLH protein E12, and binds to dsDNA. Reporter gene assays using deletion constructs of the human periostin promoter also reveal that TWIST2 can activate this gene more specifically than Twist1, while the Q119X mutant results in no significant transactivation. Chromatin immunoprecipitation assays show that both wild-type TWIST2 and the Q119X mutant bind the periostin promoter, however only wild-type TWIST2 is associated with higher levels of histone acetylation across the 5′-regulatory region of periostin. Taken together, these data suggest that the C-terminal domain of TWIST2, which is missing in the Q119X mutant form of TWIST2, is responsible for proper transactivation of the periostin gene. Improper regulation of periostin by the mutant form of TWIST2 could help explain some of the soft tissue abnormalities seen in these patients therefore providing a genotype-phenotype relationship for Setleis Syndrome.
doi:10.1016/j.biocel.2011.07.003
PMCID: PMC3163740  PMID: 21801849
TWIST1; Dermo-1; Focal Facial Dermal Dysplasia; skin development
11.  TWIST is Expressed in Human Gliomas and Promotes Invasion1 
Neoplasia (New York, N.Y.)  2005;7(9):824-837.
Abstract
TWIST is a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, promotes tumor cell metastasis, and, in response to cytotoxic stress, enhances cell survival. Our screen for bHLH gene expression in rat C6 glioma revealed TWIST. To delineate a possible oncogenic role for TWIST in the human central nervous system (CNS), we analyzed TWIST message and protein expression in gliomas and normal brain. TWIST was detected in the large majority of human glioma-derived cell lines and human gliomas examined. Increased TWIST mRNA levels were associated with the highest grade gliomas, and increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis and subsequent malignant progression. TWIST was also expressed in embryonic and fetal human brain, and in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.
PMCID: PMC1501937  PMID: 16229805
cancer; brain tumor; neuron; oncogene; invasion
12.  Twist-2 Controls Myeloid Lineage Development and Function 
PLoS Biology  2008;6(12):e316.
Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2–deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPα. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-γ (IFNγ) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.
Author Summary
Hematopoiesis is coordinated by transcription factors that regulate proliferation, differentiation, and cell fate determinations. Myelopoiesis refers to the development of all white blood cells, excluding lymphocytes (B and T cells); however, the molecular regulation of this developmental process is still incompletely understood. In this study using mice that lack expression of Twist-2, we establish a novel role for this basic helix-loop-helix transcription factor as regulator of myeloid progenitors and fully differentiated myeloid cells. Specifically, Twist-2 acts to inhibit proliferation as well as differentiation of progenitors that give rise to macrophages, neutrophils, and basophils by inhibiting the important transcription factors Runx1 and C/EBPα. In mature myeloid cells, Twist-2 negatively regulates the production of proinflammatory cytokines while positively promoting the production of regulatory cytokine IL-10 by these cells. These findings provide significant insight into regulation of myeloid lineage development and function.
The transcription factor Twist-2 is a new regulator that inhibits the proliferation and differentiation of granulocyte macrophage progenitors. Twist-2 also inhibits proinflammatory cytokine production, while stimulating IL-10 by myeloid cells.
doi:10.1371/journal.pbio.0060316
PMCID: PMC2602725  PMID: 19090621
13.  Significance of Twist expression and its association with E-cadherin in esophageal squamous cell carcinoma 
Background
Twist is a basic helix-loop-helix (bHLH) transcriptional factor that has been identified to play an important role in epithelial-mesenchymal transition (EMT)-mediated metastasis through the regulation of E-cadherin expression. However, few authors have examined the expression of Twist and E-cadherin and their prognostic value in patients with esophageal squamous cell carcinoma (ESCC). The purpose of this study is to evaluate the clinical significance of Twist and E-cadherin expression in ESCC.
Methods
We immunohistochemically investigated the relationship between their expression and clinicopathological factors including prognosis in surgical specimens of primary tumors in 166 patients with ESCC.
Results
The expression rate of high Twist was 42.0% and that of preserved E-cadherin was 40.4%. The expression of high Twist and reduced E-cadherin was significantly associated with depth of tumor invasion, lymph node metastasis, distant nodal metastasis, stage and lymphatic invasion, and poor prognosis. High Twist expression significantly correlated with reduced E-cadherin expression. In the preserved E-cadherin group, the 5-year survival rate was better for patients who were low for Twist expression than for those who were high for Twist expression. Multivariate analysis indicated that the combination of low Twist and preserved E-cadherin expression was an independent prognostic factor along with tumor depth, distant nodal metastasis and E-cadherin expression.
Conclusions
Evaluation of Twist and E-cadherin expressions should be useful for determining tumor properties, including prognosis, in patients with ESCC.
doi:10.1186/1756-9966-28-158
PMCID: PMC2808309  PMID: 20025748
14.  The Role of Nuclear β-Catenin Accumulation in the Twist2-Induced Ovarian Cancer EMT 
PLoS ONE  2013;8(11):e78200.
Background
Twist2 has been shown to promote human tumor invasion as in breast cancer and cervical cancer. However, whether Twist2 promotes human ovarian cancer progression remains to be elucidated. Here, we investigate the role of Twist2 in ovarian cancer invasion and metastasis as well as the underlying molecular mechanisms.
Methods
Twist2 expression was detected by Immunohistochemistry (IHC) on tissue microarray of human ovarian cancers with scoring procedure according to the staining intensity and pattern. Twist2 gene was stably introduced into SKOV-3 ovarian cancer cells to examine the changes of cellular morphology, motility, invasiveness, and EMT molecular markers.
Results
Twist2 expression is significantly increased in ovarian cancers along with the FIGO disease stage, indicating that Twist2 may be associated with ovarian cancer metastasis. Overexpression of Twist2 induced the EMT phenotype including downregulation of E-cadherin, and upregulation of N-cadherin and β-catenin in human ovarian cancer cells, suggesting that Twist2 might promote β-catenin release from the E-cadherin/β-catenin complex through inhibition of E-cadherin. Thus, β-catenin degradation was inhibited due to inhibition of APC, and the Wnt/β-catenin pathway was then activated by nuclear β-catenin accumulation, which may activate transcription of downstream target genes to promote tumor invasion and metastasis. Collectively, these data indicated that β-catenin is involved in Twist2-induced EMT in ovarian cancer.
Conclusion
Our data indicates that upregulation of Twist2 is correlated with the FIGO stage in human ovarian cancers. In this report, we demonstrated that nuclear β-catenin is accumulated in Twist2-induced EMT cells to facilitates ovarian cancer invasion and metastasis.
doi:10.1371/journal.pone.0078200
PMCID: PMC3823872  PMID: 24244294
15.  Zoledronic Acid Reverses the Epithelial-Mesenchymal Transition and Inhibits Self-Renewal of Breast Cancer Cells through Inactivation of Nuclear Factor kappa B 
Molecular cancer therapeutics  2013;12(7):1356-1366.
Zoledronic acid (ZA), a third generation bisphosphonate, has been shown to reduce cell migration, invasion, and metastasis. However, the effects of ZA on the epithelial-mesenchymal transition (EMT), a cellular process essential to the metastatic cascade, remain unclear. Therefore, the effects of ZA on EMT, using triple negative breast cancer cells (TNBC) as a model system, were examined in greater detail. ZA treatment decreased expression of mesenchymal markers N-cadherin, Twist and Snail, and subsequently upregulated expression of E-cadherin. ZA also inhibited cell viability, induced cell cycle arrest and decreased the proliferative capacity of TNBC, suggesting that ZA inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype, the effects of ZA on self-renewal in TNBC were also studied. Treatment with ZA decreased expression of self-renewal proteins BMI-1 and Oct-4, and both prevented and eliminated mammosphere formation. To understand the mechanism of these results, the effect of ZA on established EMT regulator NFκB was investigated. ZA inhibited phosphorylation of RelA, the active subunit of NFκB, at serine 536 and modulated RelA subcellular localization. Treatment with ZA reduced RelA binding to the Twist promoter, providing a direct link between inactivation of NFκB signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased, correlating modulation of EMT to decreased self-renewal. Based on these results, it is proposed that, through inactivation of NFκB, ZA reverses EMT, which leads to a decrease in self-renewal.
doi:10.1158/1535-7163.MCT-12-0304
PMCID: PMC3711266  PMID: 23619300
zoledronic acid; epithelial mesenchymal transition; self-renewal; triple negativebreast cancer; nuclear factor kappa B
16.  Twist expression promotes migration and invasion in hepatocellular carcinoma 
BMC Cancer  2009;9:240.
Background
Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC).
Methods
We examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion.
Results
We detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively.
Conclusion
Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.
doi:10.1186/1471-2407-9-240
PMCID: PMC2720986  PMID: 19615090
17.  Phosphorylation of the Twist1-Family Basic Helix-Loop-Helix Transcription Factors Is Involved in Pathological Cardiac Remodeling 
PLoS ONE  2011;6(4):e19251.
Background
The Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.
Principal Findings
Here, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability.
Conclusions
This study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.
doi:10.1371/journal.pone.0019251
PMCID: PMC3084786  PMID: 21559426
18.  The basic domain of myogenic basic helix-loop-helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist. 
Molecular and Cellular Biology  1997;17(11):6563-6573.
In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (i) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 failed to rescue the inhibitory effects of M-Twist on MyoD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain failed to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain was sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retained other M-Twist functions such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD.
PMCID: PMC232510  PMID: 9343420
19.  Mutations within Helix I of Twist1 Result in Distinct Limb Defects and Variation of DNA-Binding Affinities 
The Journal of biological chemistry  2007;282(37):27536-27546.
Twist1 is a basic helix-loop-helix (bHLH) factor that plays an important role in limb development. Haploinsufficiency of Twist1 results in polydactyly via the inability of Twist1 to antagonistically regulate the related factor Hand2. The mechanism modulating Twist1-Hand2 antagonism is via phosphoregulation of conserved threonine and serine residues in helix I of the bHLH domain. Phosphoregulation alters the dimerization affinities for both proteins. Here we show that the expression of Twist1 and Twist1 phosphoregulation mutants result in distinct limb phenotypes in mice. In addition to dimer regulation, Twist1 phosphoregulation affects the DNA-binding affinities of Twist1 in a partner dependent and cis-element dependent manner. In order to gain a better understanding of the specific Twist1 transcriptional complexes that function during limb morphogensis, we employ a series of Twist1-tethered dimers that include the known Twist1 partners, E12 and Hand2, as well as a tethered Twist1 homodimer. We show that these dimers behave in a manner similar to monomerically expressed bHLH factors and result in distinct limb phenotypes that correlate well with those observed from the limb expression of Twist1 and Twist1 phosphoregulation mutants. Taken together, this study shows that the Twist1 dimer affinity for a given partner can modulate the DNA binding affinity and that Twist1 dimer choice determines phenotypic outcome during limb development.
doi:10.1074/jbc.M702613200
PMCID: PMC2556885  PMID: 17652084
bHLH factors; Twist1; Hand2; limb development; transcription; and dimerization
20.  The basic helix loop helix transcription factor Twist1 is a novel regulator of ATF4 in osteoblasts 
Journal of cellular biochemistry  2012;113(1):70-79.
Parathyroid hormone (PTH) is an essential regulator of endochondral bone formation and an important anabolic agent for the reversal of bone loss. PTH mediates its functions in part by regulating binding of the bone-related activating transcription factor 4 (ATF4) to the osteoblast-specific gene, osteocalcin. The basic helix-loop-helix (bHLH) factors Twist1 and Twist2 also regulate osteocalcin transcription in part through the interaction of the C-terminal “box” domain in these factors and Runx2. In this study, we discovered a novel function of PTH: its ability to dramatically decrease Twist1 transcription. Since ATF4 is a major regulator of the PTH response in osteoblasts, we assessed the mutual regulation between these factors and determined that Twist proteins and ATF4 physically interact in a manner that affects ATF4 DNA binding function. We mapped the interaction domain of Twist proteins to the C-terminal “box” domain and of ATF4, to the N-terminus. Furthermore, we demonstrate that Twist1 overexpression in osteoblasts attenuates ATF4 binding to the osteocalcin promoter in response to PTH. This study thus identifies Twist proteins as novel inhibitory binding partners of ATF4 and explores the functional significance of this interaction.
doi:10.1002/jcb.23329
PMCID: PMC3414260  PMID: 21866569
Twist1; ATF4; PTH; osteoblasts; osteocalcin
21.  Epithelial-mesenchyme transition induced by TNF-α requires NF-κB-mediated transcriptional upregulation of Twist1 
Cancer Research  2012;72(5):1290-1300.
Pro-inflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchyme transition (EMT), however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNFα̣ are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-β and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB binding site in the Twist1 promoter and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.
doi:10.1158/0008-5472.CAN-11-3123
PMCID: PMC3350107  PMID: 22253230
22.  Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves 
Developmental biology  2010;347(1):167-179.
During embryogenesis the heart valves develop from undifferentiated mesenchymal endocardial cushions (EC), and activated interstitial cells of adult diseased valves share characteristics of embryonic valve progenitors. Twist1, a class II basic-helix-loop-helix (bHLH) transcription factor, is expressed during early EC development and is downregulated later during valve remodeling. The requirements for Twist1 down-regulation in the remodeling valves and the consequences of prolonged Twist1 activity were examined in transgenic mice with persistent expression of Twist1 in developing and mature valves. Persistent Twist1 expression in the remodeling valves leads to increased valve cell proliferation, increased expression of Tbx20, and increased extracellular matrix (ECM) gene expression, characteristic of early valve progenitors. Among the ECM genes predominant in the EC, Col2a1 was identified as a direct transcriptional target of Twist1. Increased Twist1 expression also leads to dysregulation of fibrillar collagen and periostin expression, as well as enlarged hypercellular valve leaflets prior to birth. In human diseased aortic valves, increased Twist1 expression and cell proliferation are observed adjacent to nodules of calcification. Overall, these data implicate Twist1 as a critical regulator of valve development and suggest that Twist1 influences ECM production and cell proliferation during disease.
doi:10.1016/j.ydbio.2010.08.021
PMCID: PMC2950232  PMID: 20804746
heart valve development; aortic valve disease; Twist1; bHLH transcription factor; extracellular matrix; cell proliferation
23.  Twist-1 Up-Regulation in Carcinoma Correlates to Poor Survival 
Epithelial-to-mesenchymal transition (EMT) facilitates tumor metastasis. Twist is a basic helix-loop-helix protein that modulates many target genes through E-box-responsive elements. There are two twist-like proteins, Twist-1 and Twist-2, sharing high structural homology in mammals. Twist-1 was found to be a key factor in the promotion of metastasis of cancer cells, and is known to induce EMT. Twist-1 participation in carcinoma progression and metastasis has been reported in a variety of tumors. However, controversy exists concerning the correlation between Twist-1 and prognostic value with respect to carcinoma. A systematic review and meta-analysis were performed to determine whether the expression of Twist-1 was associated with the prognosis of carcinoma patients. This analysis included 17 studies: four studies evaluated lung cancer, three evaluated head and neck cancer, two evaluated breast cancer, two evaluated esophageal cancer, two evaluated liver cancer and one each evaluated osteosarcoma, bladder, cervical and ovarian cancer. A total of 2006 patients were enrolled in these studies, and the median trial sample size was 118 patients. Twist-1 expression was associated with worse overall survival (OS) at both 3 years (hazard ratio “HR” for death = 2.13, 95% CI = 1.86 to 2.45, p < 0.001) and 5 years (HR for death = 2.01, 95% CI = 1.76 to 2.29, p < 0.001). Expression of Twist-1 is associated with worse survival in carcinoma.
doi:10.3390/ijms151221621
PMCID: PMC4284667  PMID: 25429425
Twist-1; immunohistochemistry; tumor; prognosis; meta-analysis
24.  Twist1 Directly Regulates Genes That Promote Cell Proliferation and Migration in Developing Heart Valves 
PLoS ONE  2011;6(12):e29758.
Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC) valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive extracellular matrix (ECM) molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs) containing E-box consensus sequences that are potential Twist1 binding sites. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation (ChIP) assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo, which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1-responsive regulatory sequences.
doi:10.1371/journal.pone.0029758
PMCID: PMC3248441  PMID: 22242143
25.  Epidermal Growth Factor Receptor Cooperates with Signal Transducer and Activator of Transcription 3 to Induce Epithelial-Mesenchymal Transition in Cancer Cells via Up-regulation of TWIST Gene Expression 
Cancer research  2007;67(19):9066-9076.
Aberrant epidermal growth factor receptor (EGFR) signaling is a major cause of tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether deregulated EGFR pathway is involved in epithelial-mesenchymal transition (EMT), an early event that occurs during metastasis of cancers of an epithelial origin. Here, we show that EGF induces EGFR-expressing cancer cells to undergo a transition from the epithelial to the spindle-like mesenchymal morphology. EGF reduced E-cadherin expression and increased that of mesenchymal proteins. In search of a downstream mediator that may account for EGF-induced EMT, we focused on transcription repressors of E-cadherin, TWIST, SLUG, and Snail and found that cancer cells express high levels of TWIST and that EGF enhances its expression. EGF significantly increases TWIST transcripts and protein in EGFR-expressing lines. Forced expression of EGFR reactivates TWIST expression in EGFR-null cells. TWIST expression is suppressed by EGFR and Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) inhibitors, but not significantly by those targeting phosphoinositide-3 kinase and MEK/ERK. Furthermore, constitutively active STAT3 significantly activates the TWIST promoter, whereas the JAK/STAT3 inhibitor and dominant-negative STAT3 suppressed TWIST promoter. Deletion/mutation studies further show that a 26-bp promoter region contains putative STAT3 elements required for the EGF-responsiveness of the TWIST promoter. Chromatin immunoprecipitation assays further show that EGF induces binding of nuclear STAT3 to the TWIST promoter. Immunohistochemical analysis of 130 primary breast carcinomas indicates positive correlations between non-nuclear EGFR and TWIST and between phosphorylated STAT3 and TWIST. Together, we report here that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression.
doi:10.1158/0008-5472.CAN-07-0575
PMCID: PMC2570961  PMID: 17909010

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