Twist Gene Expression Research Articles

Advancing 3D Spheroid Research through 3D Scaffolds Made by Two-Photon Polymerization.

Three-dimensional cancer cell cultures have been a valuable research model for developing new drug targets in the preclinical stage. However, there are still limitations to these in vitro models. Scaffold-based systems offer a promising approach to overcoming these challenges in cancer research. In this study, we show that two-photon polymerization (TPP)-assisted printing of scaffolds enhances 3D tumor cell culture formation without additional modifications. TPP is a perfect fit for this task, as it is an advanced 3D-printing technique combining a μm-level resolution with complete freedom in the design of the final structure. Additionally, it can use a wide array of materials, including biocompatible ones. We exploit these capabilities to fabricate scaffolds from two different biocompatible materials-PEGDA and OrmoClear. Cubic spheroid scaffolds with a more complex architecture were produced and tested. The biological evaluation showed that the human ovarian cancer cell lines SKOV3 and A2780 formed 3D cultures on printed scaffolds without a preference for the material. The gene expression evaluation showed that the A2780 cell line exhibited substantial changes in CDH1, CDH2, TWIST, COL1A1, and SMAD3 gene expression, while the SKOV3 cell line had slight changes in said gene expression. Our findings show how the scaffold architecture design impacts tumor cell culture 3D spheroid formation, especially for the A2780 cancer cell line.

Co-expression of Twist and Snai1: predictor of poor prognosis and biomarker of treatment resistance in untreated prostate cancer.

Prostate cancer (PCa) remains one of the most complex tumors in men. The assessment of gene expression is expected to have a profound impact on cancer diagnosis, prognosis, and treatment decisions. The aim of this study was to determine the utility of the epithelial-mesenchymal transition (EMT) transcription factors Twist and Snai1 in the treatment of naïve prostate cancer. We analyzed formalin-fixed paraffin-embedded (FFPE) prostate tissues from 108 PCa patients and 20 control biopsies using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and 2-ΔΔCtmethods for Twist and Snail gene expression. The expression of Twist and Snai1 mRNA was significantly overexpressed in primary tissues of PCa patients compared with controls using ROC curve. Statistical analysis showed that the mRNAs of these two genes expression Snai1 and Twist were positively correlated with tumor development and prognostic parameters as Gleason score (p < 0.001; r = 0.707) and (p < 0.001; r = 0.627) respectively. The results of Kaplan-Meier analysis showed that mRNA expression of Snai1 and Twist genes expression were significant predictors of poor overall survival (OS) (Log rank p < 0.001) and progression-free survival (PFS) of patients (Log rank p < 0.001). Furthermore, our results showed that the expression of Snai1 and Twist genes expression in primary tissues of PCa patients could predict resistance to androgen deprivation therapy (p < 0.001) and resistance to the acidic drugs abirateroneor enzalutamide (p < 0.001). However, these two transcription factors failed to predict taxanes resistance at the time of diagnosis (p > 0.05). These results suggest that Snai1 and Twist are overexpressed during the onset and progression of PCa malignancies and may be theranostic markers of resistance to ADT, abiraterone, or enzalutamide therapy.

Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures.

Melanoma is characterized by high metastatic potential favored by the epithelial-to-mesenchymal transition (EMT), leading melanoma cells to exhibit a spectrum of typical EMT markers. This study aimed to analyze the expression of EMT markers in A375 and BLM melanoma cell lines cultured in 2D monolayers and 3D spheroids using morphological and molecular methods. The expression of EMT markers was strongly affected by 3D arrangement and revealed a hybrid phenotype for the two cell lines. Indeed, although E-cadherin was almost undetectable in both A375 and BLM cells, cortical actin was detected in A375 2D monolayers and 3D spheroids and was strongly expressed in BLM 3D spheroids. The mesenchymal marker N-cadherin was significantly up-regulated in A375 3D spheroids while undetectable in BLM cells, but vimentin was similarly expressed in both cell lines at the gene and protein levels. This pattern suggests that A375 cells exhibit a more undifferentiated/mesenchymal phenotype, while BLM cells have more melanocytic/differentiated characteristics. Accordingly, the Zeb1 and 2, Slug, Snail and Twist gene expression analyses showed that they were differentially expressed in 2D monolayers compared to 3D spheroids, supporting this view. Furthermore, A375 cells are characterized by a greater invasive potential, strongly influenced by 3D arrangement, compared to the BLM cell line, as evaluated by SDS-zymography and TIMPs gene expression analysis. Finally, TGF-β1, a master controller of EMT, and lysyl oxidase (LOX), involved in melanoma progression, were strongly up-regulated by 3D arrangement in the metastatic BLM cells alone, likely playing a role in the metastatic phases of melanoma progression. Overall, these findings suggest that A375 and BLM cells possess a hybrid/intermediate phenotype in relation to the expression of EMT markers. The former is characterized by a more mesenchymal/undifferentiated phenotype, while the latter shows a more melanocytic/differentiated phenotype. Our results contribute to the characterization of the role of EMT in melanoma cells and confirm that a 3D cell culture model could provide deeper insight into our understanding of the biology of melanoma.

MicroRNA-122 Is More Effective than Rapamycin in Inhibition of Epithelial-mesenchymal Transition and mTOR Signaling Pathway in Triple Negative Breast Cancer.

The fundamental mechanism responsible for the aggressiveness of metastatic cancers such as triple-negative breast cancer (TNBC) is the epithelial-mesenchymal transition (EMT). In cancer microenvironments, the Phosphoinositide 3-kinases (PI3K)-Akt- mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating the EMT mechanism. The current study focuses on the impacts of rapamycin, a newly retargeted chemotherapeutic agent against mTOR, and MicroRNA (miR)-122 on the aggressive behavior of TNBC. The half-maximal inhibitory concentration (IC50) of rapamycin on 4T1 cells was determined using an MTT assay. Also, miR-122 was transiently transfected into 4T1 cells to study its effect on the pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression level of central mTOR and EMT-related cascade genes. Moreover, cell mobility and migration were evaluated using scratch and migration assays, respectively. Both rapamycin and miR-122 significantly decreased the expression levels of PI3K, AKT, and mTOR, as well as ZeB1 and Snail genes. However, no significant change was observed in Twist gene expression. Furthermore, scratch and migration assays revealed that the migration of 4T1 cells was markedly reduced, especially following miR-122 induction. Our experimental findings and gene enrichment studies indicated that miR-122 mainly operates on multiple metabolic pathways, as well as EMT and mTOR, while rapamycin has restricted targets in cancer cells. Consequently, miR-122 can be considered a potential cancer microRNA therapy option, which can be validated in the future in animal studies to demonstrate its efficacy in cancer control.

Genistein Inhibits Proliferation and Metastasis in Human Cervical Cancer Cells through the Focal Adhesion Kinase Signaling Pathway: A Network Pharmacology-Based In Vitro Study in HeLa Cells

Previous studies have provided evidence that genistein exerts a therapeutic effect on different tumor cells. However, the mechanism of action of genistein against cervical cancer cells remains largely unknown. The aim of this study was to comprehensively decipher the anti-metastatic effect and molecular mechanism of genistein action on cervical cancer cells. We developed an integrated strategy from genotype to phenotype, combining network pharmacology and a transcriptome screening approach, to elucidate the underlying mechanism of action of genistein against human cervical cancer cells. In silico studies predicted that the focal adhesion pathway may be an important signaling cascade targeted by genistein treatment. Using RNA sequencing analysis, representative genes of the focal adhesion pathway were demonstrated to be significantly downregulated. Phenotypic studies revealed that genistein demonstrated strong anti-proliferative and anti-metastatic activity in HeLa cells. Moreover, genistein modulated this activity in a concentration-dependent manner. Genistein also inhibited both the activation and gene expression of FAK (Focal Adhesion Kinase) and paxillin. In addition, vimentin and β-catenin protein expression, and Snail and Twist gene expression, were strongly inhibited by genistein. Our findings provide strong evidence for a pleiotropic effect of genistein on cervical cancer cells, mediated through the focal adhesion pathway.

INVERSE EXPRESSION BETWEEN TGF-β1 AND TWIST IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE AFTER CHOLESTEROL DEPLETION

Objectives To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). Study Design SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. Results The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Conclusions Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology. To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology.

Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS. Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6193.

Parthenolide reverses the epithelial to mesenchymal transition process in breast cancer by targeting TGFbeta1: In vitro and in silico studies

AimsBreast cancer metastasis is the leading cause of mortality among breast cancer patients. Epithelial to mesenchymal transition (EMT) is a biological process that plays a fundamental role in facilitating breast cancer metastasis. The present study assessed the efficacy of parthenolide (PTL Tanacetum parthenium) on EMT and its underlying mechanisms in both lowly metastatic, estrogen-receptor positive, MCF-7 cells and highly metastatic, triple-negative MDA-MB-231 cells. Main methodsMCF-7 and MDA-MB-231 cells were treated with PTL (2 μM and 5 μM). Cell viability was determined by MTT (3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was analyzed by the FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. The monolayer wound scratch assay was employed to evaluate cancer cell migration. Proteins were separated and identified by Western blotting. Gene expression was analyzed by quantitative real-time PCR. Key findingsPTL treatment significantly reduced cell viability and migration while inducing apoptosis in both cell lines. Also, PTL treatment reverses the EMT process by decreasing the mesenchymal marker vimentin and increasing the epithelial marker E-cadherin compared to the control treatment. Importantly, PTL downregulates TWIST1 (a transcription factor and regulator of EMT) gene expression, concomitant with the reduction of transforming growth factor beta1 (TGFβ1) protein and gene expression in both cell lines. Additionally, molecular docking studies suggest that PTL may induce anticancer properties by targeting TGFβ1 in both breast cancer cell lines. SignificanceOur findings provide insights into the therapeutic potential of PTL to mitigate EMT and breast cancer metastasis. These promising results demand in vivo studies.

Pengaruh Askorbat dan Hipoksia Terhadap Penghambatan Migrasi Sel Serta Penurunan Ekspresi Gen HIF-1α dan Twist pada Lini Sel Kanker Payudara MCF-7

Hypoxia at cellular level can induce the particular changes of certain gene expression. HIF-1α stability can support cancer cell migration through the regulation of TWIST expression. Hypoxic condition will maintain HIF-1α accumulation due to a decreased hydroxylases activity. The expression of HIF-1α is known to be reduced transcriptionally and translationally by increasing ascorbate under hypoxic. However, the role of ascorbate in inhibiting cell migration and its effect on the downregulation of HIF-1α and TWIST gene expression have not been clearly understood. Therefore, this study is aimed to observe the effect of ascorbate and hypoxia that regulate the inhibition of HIF-1α and TWIST. Scratch assay method was used to observe cell migration. RT-PCR method was used to detect HIF-1α and TWIST gene expression. The results showed that ascorbate and hypoxia inhibit MCF-7 cell migration within 34 hours. Under normoxic, ascorbate with the concentration of 0.40 mM (IC50) suppressed cell migration significantly with an average migration rate (Vm) relative to control of 1,28 µm/hour and a percentage of wound area closure of 19,37%. Under hypoxic, ascorbate with the concentration of 1,20 mM (IC75) inhibited cell migration with an average Vm of 0,93 µm/hour and reduced the percentage of wound area up to 14,2%. Relative expression of target genes under hypoxic was compared with normoxic condition. The results showed that ascorbate with the concentration of 1,20 mM (IC75) downregulated HIF-1α gene expression up to ±6,97x and TWIST gene up to ±3,25x under hypoxic condition. In summary, ascorbate and hypoxia can inhibit cell migration and downregulate HIF-1α and TWIST gene expression in MCF-7 cell.

High Expression of TWIST-1 in the Chronic Myeloid Leukemia Patients with T315I Mutation

Background: Among the known ABL mutations in chronic myeloid leukemia (CML), T315I is of particular importance. The T315I mutation may develop resistant cells that increase disease progression. TWIST-1 expression is impaired in patients with increased drug resistance. Objectives: The current study aimed to measure the expression of TWIST-1 gene in CML patients to investigate its association with T315I mutation. Methods: Peripheral blood samples were taken from 40 CML patients. The expression of TWIST-1 and BCR-ABL1 genes was quantified by real-time polymerase chain reaction (PCR). The gene expression was evaluated by REST software. cDNA was used for amplification refractory mutation system (ARMS)-PCR reaction. Results: Of the 40 patients (age range: 19 - 72 years) participating in the study, 23 (57.7%) were female, and 17 (42.5%) were male. The expression of TWIST-1 gene was 43 ± 184.09-fold. The T315I mutation was detected in 3 (7.5%) patients. Conclusions: According to our results, the TWIST-1 gene expression in patients with T315I mutation was significantly higher than patients without that mutation.

Normal and cancer fibroblasts differentially regulate TWIST1, TOX and cytokine gene expression in cutaneous T-cell lymphoma

BackgroundMycosis fungoides (MF) is a primary cutaneous T-cell lymphoma (CTCL) that transforms from mature, skin-homing T cells and progresses during the early stages in the skin. The role of the skin microenvironment in MF development is unclear, but recent findings in a variety of cancers have highlighted the role of stromal fibroblasts in promoting or inhibiting tumorigenesis. Stromal fibroblasts are an important part of the cutaneous tumor microenvironment (TME) in MF. Here we describe studies into the interaction of TME-fibroblasts and malignant T cells to gain insight into their role in CTCL.MethodsSkin from normal (n = 3) and MF patients (n = 3) were analyzed for FAPα by immunohistochemistry. MyLa is a CTCL cell line that retains expression of biomarkers TWIST1 and TOX that are frequently detected in CTCL patients. MyLa cells were cultured in the presence or absence of normal or MF skin derived fibroblasts for 5 days, trypsinized to detached MyLa cells, and gene expression analyzed by RT-PCR for MF biomarkers (TWIST1 and TOX), Th1 markers (IFNG, TBX21), Th2 markers (GATA3, IL16), and proliferation marker (MKI67). Purified fibroblasts were assayed for VIM and ACTA2 gene expression. Cellular senescence assay was performed to assess senescence.ResultsMF skin fibroblast showed increased expression of FAP-α with increasing stage compared to normal. Normal fibroblasts co-cultured with MyLa cells suppressed expression of TWIST1 (p < 0.0006), and TOX (p < 0.03), GATA3 (p < 0.02) and IL16 (p < 0.03), and increased expression of IFNG (p < 0.03) and TBX21 (p < 0.03) in MyLa cells. In contrast, MyLa cells cultured with MF fibroblasts retained high expression of TWIST1, TOX and GATA3. MF fibroblasts co-culture with MyLa cells increased expression of IL16 (p < 0.01) and IL4 (p < 0.02), and suppressed IFNG and TBX21 in MyLa cells. Furthermore, expression of MKI67 in MyLa cells was suppressed by normal fibroblasts compared to MF fibroblasts.ConclusionSkin fibroblasts represent important components of the TME in MF. In co-culture model, normal and MF fibroblasts have differential influence on T-cell phenotype in modulating expression of Th1 cytokine and CTCL biomarker genes to reveal distinct roles with implications in MF progression.

Effect of TWIST1 Gene on the Proliferation and Apoptosis of Human Glioma Cell Line TJ861 by Regulating Mammalian Target of Rapamycin Signaling Pathway

To observe TWIST1 gene expression in human glioma and study the effect human glioma cell line TJ861 on the proliferation and apoptosis, and further explore its potential mechanism to provide some reference for the targeted treatment of glioma in the future. Detection of cancer tissue (Carcinoma tissue) in 55 patients with glioma by RT-PCR and Expression level of TWIST1 in normal and paracancerous tissues (Adjacent tissue), the human glioma cell line TJ861 was further divided into, Nonsense sequence group, (si-NS group), TWIST1 Inhibition group (si-TWIST1 group) and control group. The glioma cells of si-NS group and si-TWIST1 group were transfected with nonsense sequence and TWIST1 siRNA respectively by liposome transfection technology. Use CCK8 assay to test the cell proliferation ability of each group at 0, 12, 24, 36, 48 and 72 hours; 48 hours after siRNA transfection, The ability of DNA replication in each group was detected by EdU staining; Apoptosis related protein expression, in each group, was analyzed by Western blot; TUNEL staining was used to test the apoptosis rate of each group; In the end, We studied TWIST1 effect knocking down on mTOR protein expression in human glioma cells and mTOR protein expression in cancer and adjacent tissues. TWIST1 expression in glioma cells was higher, compared with normal tissues (P &lt;0.05); After transfection of TWIST1 siRNA into human glioma cell line TJ861 in vitro, CCK8 showed glioma cells proliferation ability in si-TWIST1 group at 12, 24, 36, 48 and 72 hours was lower, compared with the control group (P &lt;0.05); After siRNA transfection at 48 hours, the DNA replication ability of glioma cells decreased significantly (P &lt;0.05) with EdU staining; The inhibition of TWIST1 increased Bax expression in glioma cells, and inhibited Bcl-2 expression (P &lt; 0.05) with Western blot; TUNEL staining further confirmed that the apoptosis level of glioma cells in the si-TWIST1 group was higher, compared with the control group (P &lt;0.05). Finally, we found that mTOR protein expression in glioma was higher, compared with adjacent tissues. in vitro experiments showed that mTOR expression in glioma cells was decreased after the inhibition of TWIST1 (P &lt;0.05). TWIST1 expression level in glioma was increased. The inhibition of TWIST1 inhibits the proliferation of glioma by blocking the mTOR signal pathway, and promote the apoptosis of glioma.

Anticancer Effects of AKBA on Glioblastoma Cancer Cells Through Modulating TWIST1 and FOXM1 Expression Levels

Glioblastoma multiforme (GBM) is a highly aggressive and poor prognosis brain tumor with about 12- to 18-month median survival period and high mortality rate in human. Glioma cells are able to disseminate and migrate far away from the primary tumor and display treatment resistance. Therefore, upgrading our knowledge regarding the genetic aspects of GBM and also applying a safe and promising source of therapeutic agent to prevent GBM might bring some new hopes to treat this disease. TWIST1 and FOXM1 upregulations are associated with migration and metastasis in gliomas. 3-Acetyl-11-keto-β-boswellic acid (AKBA) has been shown to inhibit the growth of a wide variety of cancer cells, such as gliomas. The aim of this study was to investigate the possible inhibitory effects of AKBA on the TWIST1 and FOXM1 gene expression levels and the migration of glioblastoma cancer cells. U87MG cells were treated with or without 50 µM of AKBA. The expression levels of TWIST1 and FOXM1 were assessed using real-time PCR. The migration of cells in the presence or absence of AKBA was examined through the wound healing assay. AKBA significantly downregulated both TWIST1 and FOXM1 gene expression levels. The migration of U87MG cancer cells was significantly declined when AKBA was applied. AKBA potentially prevents FOXM1 and TWIST1 axis in glioblastoma cancer cells and also inhibits cell migration.

TWIST1 DNA methylation is a cell marker of airway and parenchymal lung fibroblasts that are differentially methylated in asthma

BackgroundMesenchymal fibroblasts are ubiquitous cells that maintain the extracellular matrix of organs. Within the lung, airway and parenchymal fibroblasts are crucial for lung development and are altered with disease, but it has been difficult to understand their roles due to the lack of distinct molecular markers. We studied genome-wide DNA methylation and gene expression in airway and parenchymal lung fibroblasts from healthy and asthmatic donors, to identify a robust cell marker and to determine if these cells are molecularly distinct in asthma.ResultsAirway (N = 8) and parenchymal (N = 15) lung fibroblasts from healthy individuals differed in the expression of 158 genes, and DNA methylation of 3936 CpGs (Bonferroni adjusted p value < 0.05). Differential DNA methylation between cell types was associated with differential expression of 42 genes, but no single DNA methylation CpG feature (location, effect size, number) defined the interaction. Replication of gene expression and DNA methylation in a second cohort identified TWIST1 gene expression, DNA methylation and protein expression as a cell marker of airway and parenchymal lung fibroblasts, with DNA methylation having 100% predictive discriminatory power. DNA methylation was differentially altered in parenchymal (112 regions) and airway fibroblasts (17 regions) with asthmatic status, with no overlap between regions.ConclusionsDifferential methylation of TWIST1 is a robust cell marker of airway and parenchymal lung fibroblasts. Airway and parenchymal fibroblast DNA methylation are differentially altered in individuals with asthma, and the role of both cell types should be considered in the pathogenesis of asthma.

Androgen up-regulation of Twist1 gene expression is mediated by ETV1.

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.

The Effects of DNA Methylation on Medulloblastoma Cell Survivability and Development

Establish the role epigenetic methylation plays in the segregation of Medulloblastoma subgroups, through pathway analysis in the literature and cancer genome Atlas publicly available data. The goal is to develop an in-vitro assay to study therapeutic responses for subgroups 3 and 4. Functional analysis assays will be used to determine a mechanism for chemoresistance in specific subgroups. In the current study, we assessed Medulloblastoma group 3 (D341) and Group 4 (D283) cell lines using the DNA isolation kit and angiogenesis kits which allows for amplification via PCR and visualize tube formation for tumor nutrients. Twist 1 fended were measured and recorded with the methylation Gold. Group 3 MB cell lines treated with chemotherapy reagents demonstrate altered methylation profiles, angiogenic tube formation increase, a decrease in apoptosis, and underwent phenotypic changes to overcome resistance. Gel electrophoresis results indicated consistent Twist-1 expression in the DAOY+cisplatin cell line in comparison to the control. Cells treated with chemotherapeutic agents evade the mechanisms of apoptosis resulting in angiogenesis. We identified a possible correlation between cisplatin treatment, alteration of methylation patterns, and an increase of Twist-1 gene expression. Twist-1 previously identified to be involved in angiogenesis and cisplatin resistance (Roberts et al. 2016), this supports our hypothesis that alterations in methylation patterns result in an increase in overall chemoresistance.

TWIST1 Gene Expression as a Biomarker for Predicting Primary Doxorubicin Resistance in Breast Cancer.

Doxorubicin is one of the most commonly used chemotherapeutic agents for adjuvant chemotherapy of breast cancer. In the studies focused on finding biomarkers to predict the response of the patients and tumors to the drugs used, the Twist transcription factor has been suggested as a candidate biomarker for predicting chemo-resistance of breast tumors. In this study, we aimed to investigate the relationship between TWIST transcription factor expression and the effectiveness of doxorubicin treatment on directly taken primary tumor samples from chemotherapy-naive breast cancer patients. Twenty-six primary breast tumor samples taken from 26 different breast cancer patients were included in this study. Adenosine triphosphate tumor chemo-sensitivity assay (ATP-TCA) has been used to determine tumor response to doxorubicin and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used for analyzing the TWIST1 gene expression of tumors. There was a significant difference in TWIST gene expression between responder and non responder tumors (p <0.05). The TWIST gene expression of the drug-resistant group was higher than the responsive group. This difference was not dependent on the histopathological features of tumors. In conclusion, compatible with earlier studies that have been performed with cell lines, the current study supports the role of higher TWIST gene expression as a biomarker for predicting the response of breast tumors to chemo-therapeutic agent doxorubicin.

CASC15 promotes epithelial to mesenchymal transition and facilitates malignancy of hepatocellular carcinoma cells by increasing TWIST1 gene expression via miR-33a-5p sponging

Long noncoding RNA cancer susceptibility candidate 15 (CASC15) facilitates progression of hepatocellular carcinoma (HCC) cells, but the molecular mechanisms remain unknown. CASC15 co-expressing genes were explored in the Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset. Putative co-expressing genes were analyzed by Gene Ontology and biological pathway enrichment analysis. CASC15 overexpression or knockdown and TWIST1 overexpression or knockdown in HCC cells was achieved by lentiviral transduction or plasmid transfection. Interaction between CASC15 and microRNA-33a-5p (miR-33a-5p) was verified by argonaute 2-RNA Immunoprecipitation (AGO2-RIP) and luciferase reporter assays. HCC cell malignancy was determined by cell proliferation, apoptosis, migration and invasion assays. Tumorigenicity was evaluated by xenograft assay. Epithelial-to-mesenchymal transition (EMT) of HCC cells was assessed by Western blot. TWIST1, sex-determining region Y-related high-mobility group box 4 (Sox4) and Versican were found as putative CASC15 co-expressing genes. CASC15 positively regulated TWIST1 gene expression as well as protein level of Sox4 and Versican in HCC cells. Positive correlation in expression between CASC15 and TWIST1 mRNA was verified in 42 pairs of HCC pathologic and adjacent tissue specimens. CASC15 upregulated TWIST1 gene expression in HCC cells by sponging miR-33a-5p. CASC15 promoted EMT in HCC cells by increasing N-cadherin and Vimentin protein level while decreasing that of E-cadherin through TWIST1. CASC15 facilitated HCC cell proliferation, migration and invasion while reducing cell apoptosis through TWIST1. CASC15 facilitated the tumorigenicity of HCC cells in vivo. a CASC15 could promote EMT and facilitate malignancy of HCC cells by increasing TWIST1 gene expression via miR-33a-5p sponging.

Linkage between EMT and stemness state through molecular association between TWIST1 and NY-ESO1 in esophageal squamous cell carcinoma

Aberrant expression of cancer testis antigens (CTAs) is reported in tumors, especially those with stemness properties. A number of CTAs can induce epithelial mesenchymal transition (EMT) process and promote cancer stem cells (CSCs) characteristics. We aimed in this study to analyze the correlation between NY-ESO1 and TWIST1 in esophageal squamous cell carcinoma (ESCC), as well as their impact on EMT process. Gene expression profiling of NY-ESO1 and TWIST1 was performed in 43 esophageal tumors compared to their margin normal tissues of using qRT-PCR, and their correlation with clinicopathological variables of the patients was evaluated. In silico analysis of the NY-ESO1, epithelial and mesenchymal cell markers and also their promoter sequences was executed. ESCC cell lines KYSE-30 and YM-1 were transduced to ectopically express TWIST1 using a retroviral system, followed by qRT-PCR mRNA expression analysis to reveal the probable correlation among TWIST1, NY-ESO1 and EMT markers gene expression. Scratch assay was performed to estimate migration of TWIST1-induced cells. Overexpression of TWIST1 and NY-ESO1 mRNA was observed in 42% and 39.5% (P ˂ 0.05) of tumors, respectively. Expression of the genes was significantly correlated with each other (p = 0.005). TWIST1 and NY-ESO1 overexpression was significantly associated with stage of progression and size of tumors, respectively. A direct association between TWIST1 and NY-ESO1 mRNA expression was confirmed by induced ectopic expression of TWIST1 in ESCC cell lines KYSE-30 and YM-1. TWIST1-induced cells led to increase migration in ESCC cell line.Furthermore, significant up-regulation of EMT markers was observed following ectopic expression of TWIST1 in these cells. Based on our findings, it may be proposed that a vital association is exist between the EMT and the acquisition of cancer stemness state in tumor cells through the TWIST1/NY-ESO1 axis and it can be a critical hallmark in ESCC tumorigenesis.

Inhibitory effects of ursolic acid from Bushen Yijing Formula on TGF-β1-induced human umbilical vein endothelial cell fibrosis via AKT/mTOR signaling and Snail gene

The present study aimed to investigate the functional components from Bushen Yijing Formula and their inhibition of endothelial–mesenchymal transition (EndMT) and fibrosis in human umbilical vascular endothelial cells (HUVECs). HUVEC fibrosis was induced by treatment of transforming growth factor β (TGF-β) as the cellular model. Expression of EndMT biomarker gene and cofactors were determined by quantitative real-time-PCR, western blotting, and immunofluorescence. Angiogenesis capacity of vein endothelial cells was evaluated using tube formation assay. Ursolic acid and drug-contained serum ameliorated EndMT biomarker gene expression changes and angiogenesis capacity suppression induced by TGF-β treatment. Slug, Snail, and Twist gene expression and phosphorylation of mammalian target of rapamycin (mTOR) and AKT altered by TGF-β in HUVECs were suppressed by ursolic acid and drug-contained serum. Treatment with the mTOR signaling pathway inhibitor, rapamycin, inhibited the phosphorylation of mTOR and AKT, decreased Snail and Vimentin protein levels, and increased VE-cad protein levels. Overexpression of Snail gene promoted expression of EndMT-related genes and suppressed angiogenesis in HUVECs, which were attenuated by application of ursolic acid and drug-contained serum. Ursolic acid from Bushen Yijing Formula inhibits human umbilical vein endothelial cell EndMT and fibrosis, mediated by AKT/mTOR signaling and Snail gene expression.