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.
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
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.
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.
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.
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.
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.
Twist1; Twist box; epithelial-mesenchymal transition; prostate cancer; metastasis; Hoxa9
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.
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation.
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.
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation
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.
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.
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.
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.
Twist1; development; differentiation; cancer; epithelial-mesenchymal transition; metastasis
Overexpression of Twist, a highly conserved basic helix-loop-helix transcription factor, is associated with epithelial-mesenchymal transition (EMT) and predicts poor prognosis in various kinds of cancers, including breast cancer. In order to further clarify Twist’s role in breast cancer, we detected Twist expression in breast cancer tissues by immunohistochemistry. Twist expression was observed in 54% (220/408) of breast cancer patients and was positively associated with tumor size, Ki67, VEGF-C and HER2 expression. Conversely, Twist was negatively associated with estrogen receptor (ER), progesterone receptor (PgR) and E-cadherin expression. Patients with Twist expression had a poorer prognosis for 30-month disease free survival (DFS) (82.9%) than patients with negative Twist (92.3%). Overexpression of Twist led to dramatic changes in cellular morphology, proliferation, migratory/invasive capability, and expression of EMT-related biomarkers in breast cancer cells. Moreover, we show that Twist serves as a driver of tumorigenesis, as well as an inducer of EMT, at least in part, through activation of the Akt and extracellular signal-regulated protein kinase (ERK) pathways which are critical for Twist-mediated EMT. Our results demonstrate that Twist expression is an important prognostic factor in breast cancer patients.
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.
TWIST1; Dermo-1; Focal Facial Dermal Dysplasia; skin development
Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.
Breast cancer; Twist1; tamoxifen; Twist1 degradation; lung metastasis
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.
cancer; brain tumor; neuron; oncogene; invasion
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.
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.
E-cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial-mesenchymal transition (EMT) mechanism-mediated invasion and metastasis, Twist1 plays an important role through its regulation of E-cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E-cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down-regulate E-cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.
human hepatocellular carcinoma; metastasis; invasion; MMPs
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.
We immunohistochemically investigated the relationship between their expression and clinicopathological factors including prognosis in surgical specimens of primary tumors in 166 patients with ESCC.
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.
Evaluation of Twist and E-cadherin expressions should be useful for determining tumor properties, including prognosis, in patients with ESCC.
Twist2 is a member of the basic helix-loop-helix (bHLH) family and plays a critical role in tumorigenesis. Growing evidence has proven that Twist2 is involved in tumor progression; however, the role of Twist2 in human kidney cancer and its underlying mechanisms remain unclear. Real-time polymerase chain reaction and Western blot analysis were used to detect the expression of Twist2 in kidney cancer cells and tissues. Cell proliferation, cell cycle, apoptosis, migration, and invasion assay were analyzed using the Cell Count Kit-8, flow cytometry, wound healing, and Transwell analysis, respectively. In this study, we showed that Twist2 was upregulated in human kidney cancer tissues compared with normal kidney tissues. Twist2 promoted cell proliferation, inhibited cell apoptosis, and augmented cell migration and invasion in human kidney-cancer-derived cells in vitro. Twist2 also promoted tumor growth in vivo. Moreover, we found that the knockdown of Twist2 decreased the levels of ITGA6 and CD44 expression. This result indicates that Twist2 may promote migration and invasion of kidney cancer cells by regulating ITGA6 and CD44 expression. Therefore, our data demonstrated that Twist2 is involved in kidney cancer progression. The identification of the role of Twist2 in the migration and invasion of kidney cancer provides a potential appropriate treatment for human kidney cancer.
kidney cancer; Twist2; ITGA6; CD44
Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated β1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the β1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the β1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR.
glucose metabolism; Twist; PI3K/AKT; p53
p27 restrains normal cell growth, but PI3K-dependent C-terminal phosphorylation of p27 at threonine 157 (T157) and T198 promotes cancer cell invasion. Here, we describe an oncogenic feedforward loop in which p27pT157pT198 binds Janus kinase 2 (JAK2) promoting STAT3 (signal transducer and activator of transcription 3) recruitment and activation. STAT3 induces TWIST1 to drive a p27-dependent epithelial–mesenchymal transition (EMT) and further activates AKT contributing to acquisition and maintenance of metastatic potential. p27 knockdown in highly metastatic PI3K-activated cells reduces STAT3 binding to the TWIST1 promoter, TWIST1 promoter activity and TWIST1 expression, reverts EMT and impairs metastasis, whereas activated STAT3 rescues p27 knockdown. Cell cycle-defective phosphomimetic p27T157DT198D (p27CK-DD) activates STAT3 to induce a TWIST1-dependent EMT in human mammary epithelial cells and increases breast and bladder cancer invasion and metastasis. Data support a mechanism in which PI3K-deregulated p27 binds JAK2, to drive STAT3 activation and EMT through STAT3-mediated TWIST1 induction. Furthermore, STAT3, once activated, feeds forward to further activate AKT.
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.
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.
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.
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.
Twist proteins belong to the basic helix-loop-helix (bHLH) family of multifunctional transcriptional factors. These factors are known to use domains other than the common bHLH in protein-protein interactions. There has been much work characterizing the bHLH domain and the C-terminus in protein-protein interactions but despite a few attempts more focus is needed at the N-terminus. Since the region of highest diversity in Twist proteins is the N-terminus, we analyzed the conservation of this region in different vertebrate Twist proteins and study the sequence differences between Twist1 and Twist2 with emphasis on the glycine-rich regions found in Twist1. We found a highly conserved sequence motif in all Twist1 (SSSPVSPADDSLSNSEEE) and Twist2 (SSSPVSPVDSLGTSEEE) mammalian species with unknown function. Through sequence comparison we demonstrate that the Twist protein family ancestor was “Twist2-like” and the two glycine-rich regions found in Twist1 sequences were acquired late in evolution, apparently not at the same time. The second glycine-rich region started developing first in the fish vertebrate group, while the first glycine region arose afterwards within the reptiles. Disordered domain and secondary structure predictions showed that the amino acid sequence and disorder feature found at the N-terminus is highly evolutionary conserved and could be a functional site that interacts with other proteins. Detailed examination of the glycine-rich regions in the N-terminus of Twist1 demonstrate that the first region is completely aliphatic while the second region contains some polar residues that could be subject to post-translational modification. Phylogenetic and sequence space analysis showed that the Twist1 subfamily is the result of a gene duplication during Twist2 vertebrate fish evolution, and has undergone more evolutionary drift than Twist2. We identified a new signature motif that is characteristic of each Twist paralog and identified important residues within this motif that can be used to distinguish between these two paralogs, which will help reduce Twist1 and Twist2 sequence annotation errors in public databases.
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.
zoledronic acid; epithelial mesenchymal transition; self-renewal; triple negativebreast cancer; nuclear factor kappa B
The TWIST1 embryonic transcription factor displays biphasic functions during the course of carcinogenesis. It facilitates the escape of cells from oncogene-induced fail-safe programs (senescence, apoptosis) and their consequent neoplastic transformation. Additionally, it promotes the epithelial-to-mesenchymal transition and the initiation of the metastatic spread of cancer cells. Interestingly, cancer cells recurrently remain dependent on TWIST1 for their survival and/or proliferation, making TWIST1 their Achilles’ heel. TWIST1 has been reported to form either homodimeric or heterodimeric complexes mainly in association with the E bHLH class I proteins. These complexes display distinct, sometimes even antagonistic, functions during development and unequal prometastatic functions in prostate cancer cells. Using a tethered dimer strategy, we successively assessed the ability of TWIST1 dimers to cooperate with an activated version of RAS in human mammary epithelial cell transformation, to provide mice with the ability to spontaneously develop breast tumors, and lastly to maintain a senescence program at a latent state in several breast cancer cell lines. We demonstrate that the TWIST1-E12 complex, unlike the homodimer, is an oncogenic form of TWIST1 in mammary epithelial cells and that efficient binding of both partners is a prerequisite for its activity. The detection of the heterodimer in human premalignant lesions by a proximity ligation assay, at a stage preceding the initiation of the metastatic cascade, is coherent with such an oncogenic function. TWIST1-E protein heterodimeric complexes may thus constitute the main active forms of TWIST1 with regard to senescence inhibition over the time course of breast tumorigenesis.
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.
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.
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.
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).
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.
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.
Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.