Twist mRNA Research Articles

Silencing Twist gene leads to mesenchymal-to-epithelial transition and loss of stemlike properties in breast cancer

Objective To observe the effect of silencing Twist gene on mesenchymal-to-epithelial transition (MET) and stem-like properties in breast cancer. Methods The small interfering RNA (siRNA) vectors targeting Twist gene were transfected into MDA-MB-231 cells. The mRNA and protein expression levels of Twist and MET-associated genes (Slug, Snail and Vimentin) were detected by using real-time quantitative polymerase chain reaction(Real-time PCR) and Western blotting. Cell morphology was observed under microscope. The proportion of CD44+ CD24- cells was measured by flow cytometry. In vitro self-renewal capacity was evaluated by sphere formation assay. Tumor-initiating ability was measured by limited dilution assays. Results Compared to the control group, the Twist mRNA (0.24±0.04, P<0.01) and protein expression in siRNA group was significantly decreased. Cells with Twist silencing underwent MET reversal, showing an epithelial-like appearance, decreased mRNA expression of mesenchymal markers, including Slug (0.25±0.05, P<0.01), Snail (0.34±0.03, P<0.01) and Vimentin (0.44±0.04, P<0.01), increased mRNA expression of epithelial marker, E-cadherin (58.71±1.27, P<0.01), down-regulated protein levels of Slug, Snail and Vimentin, and up-regulated protein level of E-cadherin. The proportion of CD44+ CD24- cells in the si-Twist group and control group was (63.30±0.80)% and (86.50±0.70)% respectively (P<0.01). The sphere number in the si-Twist and control groups was (46.67±2.52) and (109.67±2.52) respectively (P<0.01). Twist silencing led to the decrease of about 6-folds in tumor-initiating ability in nude mice compared to the control (P<0.01). Conclusion Silencing Twist gene leads to MET and loss of self-renewal capacity and tumor-initiating ability in breast cancer. Key words: Breast cancer; Twist; Small interfering RNA; Mesenchymal-to-epithelial transition; Tumor stem cells

Silencing of SOX12 by shRNA suppresses migration, invasion and proliferation of breast cancer cells.

Sex determining region Y-box protein 12 (SOX12) is essential for embryonic development and cell fate determination. The role of SOX12 in tumorigenesis of breast cancer is not well-understood. Here, we found that SOX12 mRNA expression was up-regulated in human breast cancer tissues. To clarify the roles of SOX12 in breast cancer, we used lentiviral small hairpin RNAs (shRNAs) to suppress its expression in two breast cancer cells with relatively higher expression of SOX12 (BT474 and MCF-7). Our findings strongly suggested that SOX12 was critical for cell migration and invasion of breast cancer cells. We found that silencing of SOX12 significantly decreased the mRNA and protein levels of MMP9 and Twist, while notably increased E-cadherin. Moreover, SOX12 knockdown significantly inhibited the proliferation of breast cancer cells in vitro and the growth of xenograft tumors in vivo Flow cytometry analysis revealed that breast cancer cells with SOX12 knockdown showed cell cycle arrest and decreased mRNA and protein levels of PCNA, CDK2 and Cyclin D1. Taken together, SOX12 plays an important role in growth inhibition through cell-cycle arrest, as well as migration and invasion of breast cancer cells.

Negative Regulatory Role of TWIST1 on SNAIL Gene Expression.

Epithelial-mesenchymal transition (EMT) is crucial for specific morphogenetic movements during embryonic development as well as pathological processes of tumor cell invasion and metastasis. TWIST and SNAIL play vital roles in both developmental and pathological EMT. Our aim in this study was to investigate the functional correlation between TWIST1 and SNAIL in human ESCC cell line (KYSE-30). The packaging cell line GP293T was cotransfected with either control retroviral pruf-IRES-GFP plasmid or pruf-IRES-GFP-hTWIST1 and pGP plasmid. The KYSE-30 ESCC cells were transduced with produced viral particles and examined with inverted fluorescence microscope. DNA was extracted from transduced KYSE-30 cells and analyzed for copy number of integrated retroviral sequences in the target cell genome. The concentration of retroviral particles was determined by Real-time PCR. After RNA extraction and cDNA synthesis, the mRNA expression of TWIST1 and SNAIL was assessed by comparative real-time PCR amplification. Ectopic expression of TWIST1 in KYSE-30, dramatically reduces SNAIL expression. Retroviral transduction enforced TWIST1 overexpression in GFP-hTWIST1 nearly 9 folds in comparison with GFP control cells, and interestingly, this TWIST1 enforced expression caused a-7 fold decrease of SNAIL mRNA expression in GFP-hTWIST1 compared to GFP control cells. Inverse correlation of TWIST1 and SNAIL mRNA levels may introduce novel molecular gene expression pathway controlling EMT process during ESCC aggressiveness and tumorigenesis. Consequently, these data extend the spectrum of biological activities of TWIST1 and propose that therapeutic repression of TWIST1 may be an effective strategy to inhibit cancer cell invasion and metastasis.

Abstract LB-352: p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells

Abstract Background: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism behind ovarian cancer metastasis is still not clear. Twist1 is associated with the increased ability of migration and tumor-initiation of ovarian cancer cells; therefore, understanding the regulation of Twist is critical for the prevention of metastatic disease. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We propose that p53 functions as a normal inhibitor of Twist1. We hypothesize that through their interaction, p53 promotes the proteasome-dependent degradation of Twist1 and consequently inhibits epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. Method: p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Co-immunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Result: Wild type p53 overexpression reduces Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to regulate Twist1 degradation. E3 ligase, Pirh2, forms complex with p53 and Twist1 to induce Twist1 degradation. In EOC cells, Twist1 is suppressed by proteasome-dependent degradation. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. The 9 samples with high levels of wild type p53 all showed low or no Twist1 expression. Conclusion: Our data revealed a mechanism by which a crucial EMT inducer, Twist1, is regulated by wild type p53 through enhancing its proteasome-dependent degradation. p53 promotes the formation of a Twist1-p53-Pirh2 complex and facilitates Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-352.

Mesoderm patterning and morphogenesis in the polychaete Alitta virens (Spiralia, Annelida): Expression of mesodermal markers Twist, Mox, Evx and functional role for MAP kinase signaling

Mesoderm represents the evolutionary youngest germ layer and forms numerous novel tissues in bilaterian animals. Despite the established conservation of the gene regulatory networks that drive mesoderm differentiation (e.g. myogenesis), mechanisms of mesoderm specification are highly variable in distant model species. Thus, broader phylogenetic sampling is required to reveal common features of mesoderm formation across bilaterians. Here we focus on a representative of Spiralia, the marine annelid Alitta virens, whose mesoderm development is still poorly investigated on the molecular level. We characterize three novel early mesodermal markers for A. virens – Twist, Mox, and Evx – which are differentially expressed within the mesodermal lineages. The Twist mRNA is ubiquitously distributed in the fertilized egg and exhibits specific expression in endomesodermal- and ectomesodermal-founder cells at gastrulation. Twist is expressed around the blastopore and later in a segmental metameric pattern. We consider this expression to be ancestral, and in support of the enterocoelic hypothesis of mesoderm evolution. We also revealed an early pattern of the MAPK activation in A. virens that is different from the previously reported pattern in spiralians. Inhibition of the MAPK pathway by U0126 disrupts the metameric Twist and Mox expression, indicating an early requirement of the MAPK cascade for proper morphogenesis of endomesodermal tissues.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc.

The interplay between microRNAs and Twist1 transcription factor: a systematic review.

Twist1 (also known as Twist) is a transcription factor that belongs to the family of basic helix-loop-helix (bHLH) proteins. It functions as a negative regulator of epithelial gene expression and a positive regulator of mesenchymal gene expression, thereby leading to induction of the epithelial mesenchymal transition (EMT), a process in which epithelial cells acquire the motile and migratory characteristics of mesenchymal cells. In addition to regulating the expression of protein-coding genes, Twist1 regulates the expression of microRNAs (miRNAs), adding a regulatory layer to EMT induction. Interestingly, the mRNA of Twist1 represents a downstream target of miRNAs, indicating an intricate network between miRNAs and Twist1. This network was shown to play multiple roles in cancer cell migration, invasion, and metastasis. The network can induce angiogenesis, protect cells from oncogene-induced apoptosis and senescence, enhance cancer cell resistance to conventional therapies, and increase cancer stem cell (CSC) populations. Recently, miRNAs have attracted considerable attention as potential promising tools in cancer therapies. Thus, this systematic review was conducted to clarify the reciprocal link between Twist1 and miRNAs in order to provide potential candidate miRNAs for diagnostic and therapeutic approaches in cancer treatment.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis—Sox9, Sox6, and Sox5—were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen—cartilage-specific ECM substances—were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML.Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients.Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test.Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile.Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MyoD transcription factor induces myogenesis by inhibiting Twist-1 through miR-206

Twist-1 is mostly expressed during development and has been previously shown to control myogenesis. Because its regulation in muscle has not been fully exploited, the aim of this project was to identify micro (mi)RNAs in muscle that regulate Twist-1. miR-206, one of the most important muscle-specific miRNAs (myomiRs), was identified as a possible regulator of Twist-1 mRNA. Luciferase assays and transfections in human foetal myoblasts showed that Twist-1 is a direct target of miR-206 and that through this pathway muscle cell differentiation is promoted. We next investigated whether MyoD, a major myogenic transcription factor, regulates Twist-1 because it is known that MyoD induces expression of the miR-206 gene. We found that forced MyoD expression induced miR-206 upregulation and Twist-1 downregulation through binding to the miR-206 promoter, followed by increased muscle cell differentiation. Finally, experiments were performed in muscle cells from subjects with congenital myotonic dystrophy type 1, in which myoblasts fail to differentiate into myotubes. MyoD overexpression inhibited Twist-1 through miR-206 induction, which was followed by an increase in muscle cell differentiation. These results reveal a previously unidentified mechanism of myogenesis that might also play an important role in muscle disease.

Twist1 regulates the epithelial-mesenchymal transition via the NF-κB pathway in papillary thyroid carcinoma.

Expression of the oncogene Twist1 is correlated with tumor development and metastasis. Recent studies have suggested that the epithelial-to-mesenchymal transition (EMT) is necessary for tumor progression and metastases. Little is known concerning the role of Twist1 and EMT in thyroid cancer. In the present work, the expression levels of Twist1 and one marker of EMT, vimentin, were measured in papillary thyroid carcinoma (PTC). The results showed Twist1 expression to be correlated only with cancer lymph node metastases (P = 0.004) and not with other clinicopathological indicators. Moreover, Twist1 expression was positively correlated with the expression of vimentin (r = 0.408, P = 0.003). In vitro studies further indicated that reducing Twist1 expression using short hairpin RNA against Twist1 can decrease the invasive and metastatic properties of PTC cells and that the down-regulation of Twist1 can reverse EMT by increasing the expression of E-cadherin and down-regulating the expression of vimentin in the PTC cell line IHH-4. To investigate the effects on Twist1, the PTC cell lines TPC-1 and BCPAP were treated with TNF-α, resulting in Twist1 up-regulation that was dependent on NF-κB activation. After the inhibition of NF-κB activity with Bay11-7082, the Twist1 mRNA and protein levels could not be increased. The decline in the Twist1 mRNA and protein levels rendered the cancer cells less invasive. Thus, we conclude that Twist1 plays an important role in the EMT of PTC via the NF-κB pathway.

Abstract POSTER-THER-1426: Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer

Abstract INTRODUCTION/PURPOSE: The vast majority of epithelial ovarian cancer (EOC) patients present with advanced metastatic disease, but will respond well to first line chemotherapy consisting of a platinum drug and/or paclitaxel. Unfortunately, most of these patients will relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. Therefore, novel therapies that can eliminate disseminated and chemoresistant cells are urgently needed. Two potential targets to supplement current treatment are TWIST1 and TWIST2, related basic helix-loop-helix transcription factors vital in development and reactivated in many cancers. TWIST proteins regulate epithelial to mesenchymal transition (EMT), the process underlying metastatic spread. Furthermore, recent studies from multiple groups have correlated mesenchymal characteristics with a cancer stem cell phenotype, and with chemoresistance to a variety of agents. However, transcription factors are difficult to target with small molecule drugs due to their nuclear localization. Therefore, we employ siRNA to target TWIST mRNA, in order to reduce metastasis and re-sensitize cancer cells to conventional chemotherapeutic agents. METHODS: We have designed and validated two siRNAs against TWIST. We have also developed two nanoparticle-based delivery systems for siRNA which would be translatable from the bench to the clinic. The first is YTZ3-15, an amphiphilic generation three poly(amidoamine) (PAMAM) dendrimer, the second, a polyethylenimine (PEI) coated mesoporous silica nanoparticle (MSNP). Several EOC cell lines varying in their TWIST1 and TWIST2 expression were treated siRNA delivered via YTZ3-15 or MSNPs. Fluorescent microscopy was used to evaluate siRNA uptake. Western blotting and qRT-PCR were used to assay TWIST knockdown. Sulphorhodamine B and MTT cytotoxicity assays were used to test chemosensitivity before and after TWIST knockdown. TWIST1 levels in clinical samples were evaluated using IHC. RESULTS: Fluorescent microscopy demonstrated that all tested cell lines efficiently take up PAMAM dendrimers and MSNPs, delivering their siRNA cargo to the cell interior. Tracking of punctate signals over time showed slow release from endosomes into the cytosol. Both YTZ3-15 and MSNP delivery of siRNA resulted in robust TWIST knockdown lasting at least one week post-transfection. Furthermore, TWIST knockdown sensitized cells to chemotherapy compared to cells treated with non-targeting control siRNA. In clinical samples, nuclear TWIST1 staining correlated with tumor stage, with early stage tumors showing cytoplasmic staining, and staining in surrounding cells rather than tumor cells themselves. CONCLUSIONS: TWIST family proteins are promising targets to address the compound problems of metastasis and acquired drug resistance in EOC. Addition of TWIST inhibitors, such as the siRNA-based technologies we have developed, to a traditional treatment regimen has the potential to cure currently intractable disease. These combined therapies will drastically improve survival and provide new hope for patients with recurrent ovarian cancer. Citation Format: Cai Roberts, Gina Lowe, James Finlay, Joana Loeza, Thanh Dellinger, Ernest Han, Jeff Zink, Fuyu Tamanoi, Carlotta Glackin. Characterization and targeting of TWIST family proteins to prevent chemoresistance and metastasis in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1426.

Decreased mRNA and protein expression of TWIST1 in myocardial tissue of fetuses with ventricular septal defects.

Ventricular septal defect (VSD) is the most common type of congenital heart disease (CHD). The single gene mutations or absences that contribute to VSD development are well established; however, the aim of the present study was to measure gene expression variation between VSDs and normal fetal myocardial tissue. TWIST1, an important tumor biomarker, is a basic helix-loop-helix transcription factor that regulates cell proliferation, migration and differentiation in embryonic development and transformed tumor cells. Although growing evidence demonstrates that TWIST1 participates in a variety of human neoplastic diseases, the role of TWIST1 in VSD has remained elusive. Twenty-six VSD fetal myocardial tissue samples and 12 normal samples at matched gestational weeks (22-28 weeks) were included in the present study. Using reverse transcription quantitative polymerase chain reaction (PCR) and real-time PCR, it was demonstrated that TWIST1 mRNA was reduced by almost two-fold in the VSD samples compared with the normal samples. Western blot analysis also revealed that TWIST1 expression was decreased by ~three-fold (P=0.001) in the VSD samples compared with that in the normal samples. Of note, five complete ventricular (also called functionally univentricular or single ventricular) septal ageneses were identified among the specimens. For the five complete ventricular septal agenesis samples, similar results to those for other VSD fetal myocardial tissues were obtained. In conclusion, the results of the present study showed that TWIST1 mRNA and protein levels were reduced in VSDs. The present study was the first, to the best of our knowledge, to report that TWIST1 is not only a tumor biomarker, but may also be involved in the pathogenesis of VSD.

The mRNA Binding Protein AUF1 Controls the Regenerative Potential of Activated Skeletal Muscle Stem Cells

AU‐rich mRNA binding factor 1 (AUF1) is a promoter of mRNA decay. AUF1‐/‐ mice show loss of skeletal muscle mass and muscle weakness similar to limb girdle muscular dystrophy (LGMD). A mutation in a family cohort affected with LGMD was association‐mapped to the chromosomal location of human AUF1. We found that AUF1 levels increase in satellite cells following injury. We compared the rate of skeletal muscle regeneration between wild type (WT) and AUF1‐/‐ mice. 15 days following injury WT mice show repair while the AUF1‐/‐ mice show no regeneration. Mouse fiber culture studies show that AUF1‐/‐ satellite cells are unable to express the late stage myogenic regulatory factor, myogenin. We also observed a loss of Pax7, an early satellite cell marker, in AUF1‐/‐ satellite cells. This suggests that in the absence of AUF1 satellite cells cannot differentiate or return to quiescence. To understand the molecular role of AUF1, we completed studies using C2C12 cells. When AUF1 is partially silenced we observe delayed myogenesis due to reduced expression of Myogenin. The expression of nascent Myogenin was also reduced leading us to examine Myogenin regulating transcription factors. When AUF1 is partially silenced there is a 2.5 fold increase in Twist1, a Myogenin transcriptional repressor. Using RNA immuno‐precipitation we identified a direct interaction between AUF1 and Twist1 mRNA. This suggests that AUF1 mediated decay of Twist1 mRNA is crucial for the ability of activated satellite cells to express Myogenin and complete regeneration. Without AUF1 the satellite cell population maintains a “stem‐like” phenotype and depletes the quiescent population.

MyoD transcription factor induces myogenesis by inhibiting Twist-1 through miR-206.

Twist-1 is mostly expressed during development and has been previously shown to control myogenesis. Because its regulation in muscle has not been fully exploited, the aim of this project was to identify micro (mi)RNAs in muscle that regulate Twist-1. miR-206, one of the most important muscle-specific miRNAs (myomiRs), was identified as a possible regulator of Twist-1 mRNA. Luciferase assays and transfections in human foetal myoblasts showed that Twist-1 is a direct target of miR-206 and that through this pathway muscle cell differentiation is promoted. We next investigated whether MyoD, a major myogenic transcription factor, regulates Twist-1 because it is known that MyoD induces expression of the miR-206 gene. We found that forced MyoD expression induced miR-206 upregulation and Twist-1 downregulation through binding to the miR-206 promoter, followed by increased muscle cell differentiation. Finally, experiments were performed in muscle cells from subjects with congenital myotonic dystrophy type 1, in which myoblasts fail to differentiate into myotubes. MyoD overexpression inhibited Twist-1 through miR-206 induction, which was followed by an increase in muscle cell differentiation. These results reveal a previously unidentified mechanism of myogenesis that might also play an important role in muscle disease.

Tamoxifen inhibits ER-negative breast cancer cell invasion and metastasis by accelerating Twist1 degradation.

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.

The basic helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates Twist1 through an E-box element

Differentiated embryo chondrocyte 2 (DEC2/Sharp-1/Bhlhe41), a basic helix-loop-helix (bHLH) transcription factor, has been shown to regulate the transcription of target genes by binding to their E-box elements. We identified a possible DEC2-response element (consensus E-box: CACGTG) in the promoter region of Twist1. Forced expression of DEC2 significantly repressed Twist1 promoter activity under normoxia and under hypoxia as assessed by a luciferase reporter assay. In addition, over-expression of DEC2 repressed Twist1 mRNA expression assessed by quantitative real-time PCR. Site-directed mutagenesis studies showed that mutagenesis of the consensus E-box sequence eliminated the ability of DEC2 to reduce the Twist1 promoter activity. Chromatin immunoprecipitation (ChIP) assays confirmed that the DEC2-mediated repression is primarily achieved by binding to the E-box in the Twist1 promoter. Knockdown of DEC2 by siRNA significantly attenuated the repression of Twist1 expression. DEC2 and Twist1 exhibit inversed protein expression patterns during development of mouse tongue embryo tissue. Given the fact that DEC2 protein is emerging as an important regulator in a vast array of cellular events, including cell differentiation, maturation of lymphocytes and the molecular clock, our study elucidates an important mechanism by which DEC2 regulates cellular function by modulating the expression of Twist1.

Epstein-Barr virus latent membrane protein 2A suppresses the expression of HER2 via a pathway involving TWIST and YB-1 in Epstein-Barr virus-associated gastric carcinomas.

To explore HER2 expression in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) and the possible mechanisms causing down-regulation of HER2 expression in EBVaGC, we first evaluated HER2 and LMP2A expression on a clinicopathological-features matched cohort including 78 EBVaGC and 216 EBV-negative gastric carcinoma (EBVnGC) cases by immunohistochemistry. Cases with high HER2 expression in EBVaGC were significantly less than in EBVnGC (5.1% versus 23.7%; p<0.001), and none of the 34 LMP2A+ EBVaGC showed high HER2 expression. Further, overexpressing LMP2A in EBV-negative SGC7901 cells significantly decreased HER2, TWIST and YB-1 mRNA by 36.1%±8.1%, 87.6%±14.0% and 83.8%±5.7%, and protein by 44%, 57% and 49%, respectively. Additionally, the nucleus/cytoplasm ratios of TWIST and YB-1 were also decreased by 85% and 80%, respectively. Silencing LMP2A by siRNA in EBV-positive SNU719 cells for 48 h significantly increased HER2, TWIST and YB-1 mRNA to 276.7%±14.6%, 1284.8%±38.2% and 332.0%±15.5% and protein to 212%, 457% and 232%, respectively. The nucleus/cytoplasm ratios of TWIST and YB-1 were up-regulated by 4.00- and 3.57-fold, respectively, following LMP2A down-regulation. Moreover, LMP2A+/HER2low EBVaGC cases presented the best overall survival compared with LMP2A-/HER2low and LMP2A-/HER2high cases (p=0.003, log-rank test). These results suggest that LMP2A may suppress the HER2 expression through the TWIST/YB-1 axis in EBVaGC.

Abstract 1126: Tristetraprolin inhibits Twist1-induced cancer cell migration

Abstract Epithelial-Mesenchymal Transition (EMT) enhances tumor cell migration. However, detail mechanisms for the regulation of EMT are not well known. Tristetraprolin (TTP) is an AU-rich element (ARE) binding protein that can promote the decay of the transcripts. This study demonstrates that TTP plays a critical role in EMT process. Overexpression of TTP in ovarian cancer cells decreased the expression of mesenchymal markers (N-cadherin and Vimentin), increased the expression of epithelial marker (E-cadherin), and inhibited cell migration and invasion activities. On the contrary, inhibition of TTP by siRNA decreased the expression of E-cadherin, increased the expression of N-cadherin and Vimentin, and promoted cell migration and invasion. The cDNA microarray analysis revealed that Twist1 was significantly down-regulated in TTP-overexpressed cells. Twist1 mRNA contains ARE within its 3′-UTR and TTP enhances the decay of Twist1 mRNA through binding to its 3′-UTR. In addition, TTP directly bound to the ARE of Twist1 mRNA by EMSA assay. Ectopic expression of Twist1 gene without 3′UTR blocked the inhibitory of TTP on the EMT. We also observed enhanced levels of Twist1 and reduced TTP levels in human ovarian adenocarcinoma. Collectively, our data indicate that TTP conducts anti-metastatic activity by down-regulating Twist1, presenting a novel mechanism of TTP-mediated regulation of tumor metastasis. Citation Format: Wha Ja Cho, Nal Ae Yoon, Mai-Tram Vo, Young Joo Min, Jeong Woo Park. Tristetraprolin inhibits Twist1-induced cancer cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2014-1126

Abstract 1057: LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes

Abstract Ovarian cancer is currently the most fatal gynecological cancer with a 5-year survival rate of only 45%. Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the genesis and the progression of ovarian cancer. In order to identify transcription factors that are induced by LPA, a commercial pathway reporter array was used. Hypoxia induced factor 1 (HIF-1α) induction was the most prominent response showing more than 150 fold transcriptional activation compared with control. LPA treatment under normoxic and hypoxic (1% O2) conditions increased HIF-1α protein level, which leads to epithelial-to-mesenchymal transition (EMT) in the ovarian cancer cell line OVCA432. We also found that stimulation of the OVCA432 cells with LPA at normoxic and hypoxic conditions led to a significant increase of the mRNA and protein expression of the EMT transcription factor Twist. Immunofluorescent imaging confirmed that Twist levels were significantly up-regulated and that Twist was translocated to the nucleus as early as 4 hours after stimulation with LPA in both a normoxic and hypoxic environment. Additionally, immunofluorescent imaging showed that expression of E-cadherin on the periphery of the cell as well as in the whole cell was greatly reduced when cells were stimulated with LPA in normoxic and hypoxic conditions. Finally, our study demonstrated that LPA-mediated induction of EMT required both gip2 and gep oncogenes. Overall, our study indicated that the downstream components regulated by oncogenic G-proteins could potentially be targeted in the future to prevent EMT in ovarian cancer. Citation Format: Ji Hee Ha, Jeremy Ward, Danny N. Dhanasekaran. LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2014-1057