AKR1C1 Contributes to Cervical Cancer Progression via Regulating TWIST1 Expression.

Cervical cancer (CC) is a common gynecological malignancy, accounting for 10% of all gynecological cancers. Recently, targeted therapy for CC has shown unprecedented advantages. To improve CC patients' prognosis, there are still urgent needs to develop more promising therapeutic targets. Aldo-keto reductase 1 family member C1 (AKR1C1) is a type of aldosterone reductase and plays a regulatory role in a variety of key metabolic pathways. Several studies indicated that AKR1C1 was highly expressed in a series of tumors, and participated in the progression of these tumors. However, the possible effects of AKR1C1 on CC progression remain unclear. Herein, we revealed AKR1C1 was highly expressed in human CC tissues and correlated with the clinical characteristics of patients with CC. AKR1C1 could regulate the proliferation and invasion of cervical cancer cells in vitro. Further experiments showed that AKR1C1 could regulate TWIST1 expression and AKT pathway. In summary, we confirmed the involvement of AKR1C1 in CC progression, and therefore AKR1C1 may have the potential to be a molecular target for CC treatment.

α-Linolenic acid inhibits the migration of human triple-negative breast cancer cells by attenuating Twist1 expression and suppressing Twist1-mediated epithelial-mesenchymal transition.

α-Linolenic acid (ALA), an essential fatty acid, has anticancer activity in breast cancer, but the mechanism of its effects in triple-negative breast cancer (TNBC) remains unclear. We investigated the effect of ALA on Twist1, which is required to initiate epithelial-mesenchymal transition (EMT) and promotes tumor metastasis, and Twist1-mediated migration in MDA-MB231, MDA-MB468 and Hs578T cells. Twist1 protein was constitutively expressed in these TNBC cells, particularly MDA-MB-231 cells. Treatment with 100 µM ALA and Twist1 siRNA markedly decreased the Twist1 protein level and cell migration. Moreover, ALA transiently attenuated the nuclear accumulation of STAT3α as well as Twist1 mRNA expression. Treatment with ALA significantly attenuated the phosphorylation of JNK, ERK and Akt and decreased the phosphorylation of Twist1 at serine 68 in MDA-MB-231 cells. ALA accelerated Twist1 degradation in the presence of cycloheximide, whereas the ubiquitination and degradation of Twist1 by ALA was suppressed by MG-132. Pretreatment with ALA mimicked Twist1 siRNA, increased the protein expression of epithelial markers such as E-cadherin, and decreased the protein expression of mesenchymal markers including Twist1, Snail2, N-cadherin, vimentin, and fibronectin. Our findings suggest that ALA can be used not only to abolish EMT but also to suppress Twist1-mediated migration in TNBC cells.

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats.

Malignant mesothelioma (MM) is one of the most lethal tumors in humans. The onset of MM is linked to exposure to asbestos, which generates reactive oxygen species (ROS). ROS are believed to be derived from the frustrated phagocytosis and the iron in asbestos. To explore the pathogenesis of MM, peritoneal MM was induced in rats by the repeated intraperitoneal injection of iron saccharate and nitrilotriacetate. In the present study, we used microarray techniques to screen the microRNA (miR) expression profiles of these MM. We observed that the histological subtype impacted the hierarchical clustering of miR expression profiles and determined that miR-199/214 is a distinctive feature of iron saccharate-induced sarcomatoid mesothelioma (SM). Twist1, a transcriptional regulator of the epithelial-mesenchymal transition, has been shown to activate miR-199/214 transcription; thus, the expression level of Twist1 was examined in iron-induced and asbestos-induced mesotheliomas in rats. Twist1 was exclusively expressed in iron saccharate-induced SM but not in the epithelioid subtype. The Twist1-miR-199/214 axis is activated in iron saccharate-induced and asbestos-induced SM. The expression levels of miR-214 and Twist1 were correlated in an asbestos-induced MM cell line, suggesting that the Twist1-miR-199/214 axis is preserved. MeT5A, an immortalized human mesothelial cell line, was used for the functional analysis of miR. The overexpression of miR-199/214 promoted cellular proliferation, mobility and phosphorylation of Akt and ERK in MeT5A cells. These results indicate that miR-199/214 may affect the aggressive biological behavior of SM.

CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.

Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully reacquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally nonfunctional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a subclassification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

MAML1 promotes ESCC aggressiveness through upregulation of EMT marker TWIST1.

BACKGROUND: Mastermind-like 1 (MAML1) is the main transcriptional co-activator of Notch signaling pathway. It plays essential roles in several pathways including MEF2C, p53, Nf-кB and Wnt/ß-catenin. TWIST1 is known as a regulator of epithelial mesenchymal transition (EMT), which is considered as a primary step in promotion of tumor cell metastasis. Since concomitant expression of these genes was observed in tumors, our aim in this study was to elucidate the linkage between MAML1 and TWIST1 co-overexpression in esophageal squamous cell carcinoma (ESCC). RESULTS: While MAML1 silencing significantly down-regulated TWIST1, its ectopic expression up-regulated TWIST1 expression in both mRNA and protein levels in KYSE-30 cells. Expression of mesenchymal markers was increased significantly after MAML1 and TWIST1 ectopic expression, while epithelial markers expression was significantly decreased after silencing of both genes. Concomitant protein expression of MAML1 and TWIST1 was significantly observed in ESCC patients. Enforced expression of TWIST1 had no impact on MAML1 gene expression in KYSE-30 cells. CONCLUSION: The results clearly suggest transcriptional regulation of TWIST1 by MAML1 transcription factor in ESCC cells KYSE-30. Since TWIST1 is known as an EMT inducing marker, our results may revealed the mastermind behind TWIST1 function and introduced MAML1 as an upstream master regulator of TWIST1 and EMT in KYSE-30 cells.

Expression of Selected Epithelial-Mesenchymal Transition Transcription Factors in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. The aim of this study was to analyze the expression of SNAIL, SLUG, TWIST1, TWIST2, ZEB1, and ZEB 2 in primary tumor and the correlation with morphological and clinical characteristics of EC. The study included 158 patients with EC after surgical treatments: total hysterectomy and lymphadenectomy. The percentages of EC specimens testing positively for the EMT transcription factors were 84.5% for SNAIL, 92.2% for SLUG, 10.9% for TWIST1, 100% for TWIST2, 89% for ZEB1, and 98% for ZEB2. The expression of SLUG in patients with FIGO stage III or IV, type II EC, myometrial invasion ≥ 50% of the uterine wall thickness, and adnexal involvement and in patients with distant metastases was significantly higher. SLUG and ZEB2 expressions were identified as significant predictors of higher FIGO stages (III or IV) on univariate analysis. The overexpression of SLUG was a significant predictor of more aggressive type II EC, myometrial invasion ≥ 50% of the uterine wall thickness, and distant metastases on both univariate and multivariate analysis. Moreover, the overexpression of SLUG and ZEB2 was shown to be significant predictors of adnexal involvement on univariate analysis. ZEB 2 overexpression was identified in multivariate analysis as another independent predictor associated with a lesser likelihood of type II EC. Both univariate and multivariate analyses demonstrated that SLUG expression was the only predictor of 5-year survival in the study group. The overexpression of SLUG was associated with a significant increase in mortality hazard on univariate analysis and was shown to be a highly significant predictor of death on multivariate analysis. Conclusions. Selected proteins of the EMT pathway play a role in endometrial carcinogenesis; SLUG and ZEB2 expressions in the primary tumor might predict clinical outcomes in EC and drive therapeutic decisions regarding adjuvant treatment in patients with this malignancy.

Activation of TGF-ß1 Pathway by SCUBE3 Regulates TWIST1 Expression and Promotes Breast Cancer Progression.

Background: Recently, signal peptide-CUB-EGF-like domain containing protein 3 (SCUBE3) has been found to be associated with the development of several cancers. However, the biological role of SCUBE3 in breast cancer progression has not been reported. Materials and Methods: Western blotting and quantitative real-time polymerase chain reaction were used to measure the expressions of SCUBE3, TGF-ß (transforming growth factor-ß) signaling pathway markers, and epithelial-mesenchymal transition markers. The influence of SCUBE3 on the breast cancer cell proliferation, invasion, and migration was detected using methyl thiazolyl tetrazolium, wound healing, colony formation, and transwell assay. The role of SCUBE3 in vivo was confirmed using tumor xenograft experiment. Results: SCUBE3 expression was markedly increased in breast cancer cells and tissues. Knockdown of SCUBE3 suppressed cell growth, invasion, and migration, while SCUBE3 overexpression promoted cell growth, invasion, and migration in breast cancer cells. In addition, TGF-ß1 and its downstream proteins were positively regulated by SCUBE3, and the promotion effect on TWIST1 expression induced by pcDNA3.1-SCUBE3 can be reversed by TGF-ß1 inhibitor in breast cancer cell lines. Moreover, silencing of SCUBE3 suppressed breast cancer cell growth and tumorigenesis through reducing TGF-ß1 in vivo. Conclusion: Knockdown of SCUBE3 downregulated TGF-ß1 and TWIST1 expression, thereby inhibiting breast cancer cell growth and tumorigenesis.

The RNA binding protein RBMS3 inhibits the metastasis of breast cancer by regulating Twist1 expression.

BACKGROUND: Metastasis remains the biggest obstacle for breast cancer treatment. Therefore, identification of specific biomarker of metastasis is very necessary. The RNA binding protein 3 (RBMS3) acts as a tumor suppressor in various cancers. Whereas, its role and underlying molecular mechanism in breast cancer is far from elucidated. METHODS: Quantitative real-time PCR and western blots were carried out to determine the expression of RBMS3 in breast cancer cells and tissues. Transwell and in vivo metastasis assay were conducted to investigate the effects of RBMS3 on migration, invasion and metastasis of breast cancer cells. Transcriptome sequencing was applied to screen out the differential gene expression affected by RBMS3. RNA immunoprecipitation assay combined with luciferase reporter assay were performed to explore the direct correlation between RBMS3 and Twist1 mRNA. RESULTS: RBMS3 was downregulated in breast cancer and ectopic expression of RBMS3 contributed to inhibition of cell migration, invasion in vitro and lung metastasis in vivo. Furthermore, RBMS3 negatively regulated Twsit1 expression via directly binding to 3'-UTR of Twist1 mRNA, and thereby decreased Twist1-induced expression of matrix metalloproteinase 2 (MMP2). Additionally, Twist1-induced cell migration, invasion and lung metastasis could be reversed by the upregulation of RBMS3. CONCLUSIONS: In summary, our study revealed a novel mechanism of the RBMS3/Twsit1/MMP2 axis in the regulation of invasion and metastasis of breast cancer, which may become a potential molecular marker for breast cancer treatment.

Gene expression of TWIST1 and ZBTB16 is regulated by methylation modifications during the osteoblastic differentiation of mesenchymal stem cells.

BACKGROUND: Osteoblastic differentiation of mesenchymal stem cells (MSCs) is the principal stage during the restoration and regeneration of bone tissue. Epigenetic modifications such as DNA methylation play a key role in the differentiation process of stem cells. In this study, the methylation status of the promoter region of ZBTB16 and Twist1 genes and their role in controlling osteoblastic differentiation in MSCs was investigated during the osteoblastic differentiation of MSCs. METHODS: The MSCs were cultured under standard conditions and differentiated into the osteoblasts. We had three treatment groups including 5-azacytidine (methylation inhibitor), metformin (Twist-inhibitor), and procaine (Wnt/ß-catenin inhibitor) and a non-treated group (control). Methylation level of DNA in the promoter regions was monitored by methylation specific-quantitative polymerase chain reaction (PCR). Also, the mRNA levels of key genes in osteoblastic differentiation were measured using real-time PCR. RESULTS: ZBTB16 gene expression was upregulated, and promoter methylation was decreased. For Twist1 messenger RNA (mRNA) level decreased and promoter methylation increased during osteoblastic differentiation of MSCs. 5-Azacytidine caused a significant reduction in methylation and increased the mRNA expression of ZBTB16 and Twist1. Metformin repressed the Twist1 expression, and therefore osteoblastic differentiation was increased. On the opposite side, procaine could block the WNT/ß-catenin signaling pathway, as a consequence the gene expression of key genes involved in osteoblastic differentiation was declined. CONCLUSION: We found that methylation of DNA in the promoter region of ZBTB16 and Twist1 genes might be one of the main mechanisms that controlling the gene expression during osteoblastic differentiation of MSCs. Also, we could find an association between regulation of Twist1 and ZBTB16 genes and osteoblastic differentiation in MSCs by showing the relation between their expression and some key genes involved in osteoblastic differentiation. In addition, we found a connection between the Twist1 expression level and osteoblastic differentiation by using a Twist-inhibitor (metformin).

p53-Pirh2 Complex Promotes Twist1 Degradation and Inhibits EMT.

Epithelial-mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype.Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.

Expression of epithelial-mesenchymal transition regulators TWIST, SLUG and SNAIL in follicular thyroid tumours may relate to widely invasive, poorly differentiated and distant metastasis.

AIMS: To assess the expression of epithelial-mesenchymal transition (EMT) regulators in follicular thyroid tumours. METHODS AND RESULTS: The expression of E-cadherin (E-CAD) and transcription factors TWIST, SLUG and SNAIL in follicular thyroid tumours was examined by immunohistochemistry in tissue samples, including 18 follicular adenomas (FA), 12 minimally invasive follicular thyroid carcinomas (MI-FTC), 16 widely invasive follicular thyroid carcinomas (WI-FTC), 10 poorly differentiated follicular thyroid carcinomas (PDTC) and six anaplastic thyroid carcinomas (ATC). Metastatic tumour tissues from six of these cases were also examined. The results showed an increasing expression trend of EMT regulators in a panel of follicular tumour cases with a spectrum of morphological subtypes from low- to high-risk malignancy. The expression of EMT regulators was higher in the WI-FTC, PDTC and ATC groups but focal and lower in the FA and MI-FTC groups. Different expression intensity of E-CAD and EMT regulators at the tumour centre part and the invasive front (IF) was observed. The loss of E-CAD and expression of EMT regulators was significantly correlated with distant metastasis and vascular invasion (VI) in the well-differentiated follicular carcinoma (WD-FTC), and six tumours of metastatic sites also showed variables positive for EMT regulators. The disease-free survival analysis showed an apparent relationship between the expression of EMT regulators and the tumour disease-free outcomes in WD-FTC. CONCLUSIONS: Our study supported the role of EMT in the development of follicular thyroid carcinoma and indicated that EMT regulatory proteins may play an important role in WD-FTC that are widely invasive and exhibit distant metastasis.

Expression of Epithelial-Mesenchymal Transition Proteins in Pancreatic Anaplastic (Undifferentiated) Carcinoma.

OBJECTIVES: The aim of this study was to identify an association of pancreatic anaplastic carcinoma (APC) with the epithelial-mesenchymal transition (EMT). METHODS: Resected APCs (n = 24) were examined to assess components of APCs, including carcinomatous, transitional, and sarcomatous regions. Analysis was performed based on the immunoreactivity of E-cadherin and 3 EMT-related proteins: Slug (zinc finger protein SNAI2), Twist (Twist-related protein 1), and Zeb1 (zinc finger E-box-binding homeobox 1). Expression score was determined based on staining intensity and stained area of the target cells. Finally, we performed a hierarchical clustering based on the expression pattern of E-cadherin and EMT-related proteins of the sarcomatous component. RESULTS: The expression score of E-cadherin decreased in the order of sarcomatous > transitional > carcinomatous components (P < 0.01). Although there were significant differences in the immunohistochemical scores of Slug, Twist, and Zeb1 between carcinomatous and transitional components (P < 0.01), the significant difference in immunohistochemical score of Zeb1 between transitional and sarcomatous components was found (P < 0.05). Furthermore, APCs were divided into 2 subgroups based on the expression patterns of E-cadherin and EMT-related proteins (hierarchical clustering analysis). Consequently, these subgroups were distinguished by Twist expression. CONCLUSIONS: Epithelial-mesenchymal transition plays an essential role in the pathogenesis of APC.

miR-186 inhibits proliferation, migration, and epithelial-mesenchymal transition in breast cancer cells by targeting Twist1.

OBJECTIVE: Breast cancer (BC) is the most prevalent malignancy in women worldwide. Our study aimed to investigate the expression and biological effect of miR-186 in BC. METHODS: Expression of miR-186 was determined by quantitative reverse transcription PCR. Kaplan-Meier curves were calculated for the survival data analysis. Functional assays were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound healing assay. Protein expression was analyzed by Western blot. RESULTS: miR-186 was downregulated in BC tissues and cells. Downregulation of miR-186 was associated with tumor metastasis and a poor overall survival in patients with BC. Overexpression of miR-186 inhibited BC cells proliferation, migration, and epithelial-mesenchymal transition process; while suppression of miR-186 exhibited an opposite effects on BC cells. In addition, Twist1 was identified as a direct target of miR-186 in BC and restoration of Twist1 attenuated the biological effect of miR-186 on BC cells. CONCLUSION: Our findings suggest that miR-186 functions as a tumor suppressor by targeting Twist1 in BC. miR-186 may serve as a novel biomarker in BC diagnosis or a new therapeutic target in BC treatment.

miR-532-5p is a prognostic marker and suppresses cells proliferation and invasion by targeting TWIST1 in epithelial ovarian cancer.

OBJECTIVE: Dysregulated miR-532-5p has been observed in epithelial ovarian cancer (EOC). However, the potential biological function and clinical significance have not been fully explained. The study aimed to investigate the prognostic value and potential role of miR-532-5p in EOC. PATIENTS AND METHODS: MiR-532-5p and Twist homolog 1 (TWIST1) mRNA expression were examined using quantitative real-time PCR. The correlation of miR­532-5p expression with clinicopathological factors was statistically analyzed. Kaplan-Meier analysis and Cox proportional hazards regression models were explored to reveal the correlations of miR-532-5p expression with survival of patients. Cell Counting Kit-8, colony formation and transwell invasion assays were performed to evaluate the effects of miR-532-5p on cell proliferation and invasion, respectively. MiR­532-5p target genes were confirmed using luciferase activity, RT-PCR and Western blot assays. RESULTS: We found that miR-532-5p was significantly decreased in EOC tissue and cell lines, and its expression levels were highly correlated with grade (p = 0.011), FIGO stage (p = 0.004) and distant metastasis (p = 0.008). In addition, overall patient survival for those with high miR-532-5p expression was significantly longer than those patients with low miR-532-5p expression (p = 0.0058). Multivariate regression analysis identified miR-532-5p down-regulation as an independent unfavorable prognostic factor in EOC patients. Function assays showed that overexpression of miR-532-5p inhibited proliferation, colony formation and invasion of EOC cells. Mechanistic investigations confirmed TWIST1 as a direct target of miR-532-5p. Further in vitro assay indicated that restored expression of TWIST1 dampened miR-532-5p-mediated suppression of tumor progression. CONCLUSIONS: Our data suggested that miR-532-5p may act not only as a novel prognostic marker, but also as a potential target for molecular therapy of EOC.

SOX6 is downregulated in osteosarcoma and suppresses the migration, invasion and epithelial-mesenchymal transition via TWIST1 regulation.

Transcription factor SOX6 (SOX6) has been reported to serve essential roles in numerous types of cancers. However, the expression and functions of SOX6 in osteosarcoma (OS) have not been analyzed. In the present study, the patterns of SOX6 expression in OS cell lines and tissues were investigated by reverse transcription­quantitative polymerase chain reaction and western blotting. The results of the present study revealed that SOX6 was notably downregulated in OS tissues and cell lines. Subsequently, gain­ and loss­of­function studies demonstrated that SOX6 inhibited OS cell migration and invasion. In addition, SOX6 may have suppressed epithelial­mesenchymal transition via twist­related protein 1 (TWIST1) modulation. Chromatin immunoprecipitation (ChIP), quantitative ChIP and dual luciferase activity assays were used to confirm the binding of SOX6 to the promoter region of TWIST1. Additionally, colony formation assays and Cell Counting Kit­8 assays demonstrated that SOX6 suppressed cell proliferation. The findings of the present study indicated that SOX6 serves as a tumor suppressor in OS and may be a potential therapeutic target for OS.

MAML1 and TWIST1 co-overexpression promote invasion of head and neck squamous cell carcinoma.

AIMS: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with considerable morbidity and mortality. Invasion and metastasis of HNSCC is a complex process involving multiple molecules and signaling pathways. Twist Family BHLH Transcription Factor 1 (TWIST1) and Mastermind-like 1 (MAML1) are essential in induction of epithelial-mesenchymal transition through direct regulation of implicated molecules in cellular adhesion, migration and invasion. Our aim in this study was to assess the clinical significance of MAML1 and TWIST1 expression in HNSCC, and elucidate the probable correlation between these genes to exhibit their possible associations with progression and metastasis of the disease. METHODS: The gene expression profile of MAML1 and TWIST1 was assessed in fresh tumoral compared to distant tumor-free tissues of 55 HNSCC patients using quantitative real-time Polymerase chain reaction (PCR). RESULTS: Significant overexpression of MAML1 and TWIST1 mRNA was observed in 49.1% and 38.2% (P Ë‚ 0.05) of tumor specimens, respectively. Overexpression of MAML1 was associated with vascular invasion (P = 0.048). Concomitant overexpression of MAML1 and TWIST1 was significantly correlated to each other (P = 0.004). Co-overexpression of the genes was significantly correlated to the various clinicopathological indices of poor prognosis including depth of tumor invasion (P < 0.01), lymphatic invasion and grade of tumor cell differentiation (P < 0.05). CONCLUSIONS: Significant correlation between MAML1 and TWIST1 in HNSCC was revealed. This study was the first report elucidating MAML1 clinical relevance in HNSCC. These new findings suggest an oncogenic role for concomitant expression of MAML1 and TWIST1 genes in HNSCC invasion and metastasis.

TMPRSS4 Upregulates TWIST1 Expression through STAT3 Activation to Induce Prostate Cancer Cell Migration.

Transmembrane protease serine 4 (TMPRSS4), a type-II transmembrane serine protease, is involved in the development and progression of wide range of tumors. However, the biological role of TMPRSS4 in prostate cancer remains obscure. Here, we investigated the effect of TMPRSS4 on proliferation and migration in prostate cancer and potential mechanisms. Our findings demonstrated over-expression of TMPRSS4 promoted the PC3 prostate cancer cells migration, which could be reversed by TMPRSS4 silencing. TMPRSS4 induced TWIST1 expression and followed progression of EMT along with upregulation of N-cadherin and downregulation of E-cadherin via STAT3 phosphorylation. Silencing TWIST1 significantly attenuated TMPRSS4-induced PC3 migration. Moreover, knockdown of STAT3 effectively attenuated TMPRSS4-induced TWIST1 expression and TWIST1 promoter activity. Taken together, we demonstrated a mechanistic cascade of TMPRSS4 up-regulating STAT3 activation and subsequent TWIST1 expression, leading to prostate cancer migration.

Reactivation of TWIST1 contributes to Ewing sarcoma metastasis.

BACKGROUND: Ewing sarcoma is a cancer of bone and soft tissue. Despite aggressive treatment, survival remains poor, particularly in patients with metastatic disease. Failure to treat Ewing sarcoma is due to the lack of understanding of the molecular pathways that regulate metastasis. In addition, no molecular prognostic markers have been identified for Ewing sarcoma to risk stratify patients. PROCEDURE: Ewing sarcoma patients were divided into high or low Twist1 gene expression and survival curves were generated using the R2 microarray-based Genomic Analysis platform (http://r2.amc.nl). Tumors from Ewing sarcoma patients were also evaluated for TWIST1 expression by immunohistochemistry. Ewing sarcoma xenografts were established to evaluate the role of TWIST1 in metastasis. The effects of Twist1 on migration and invasion were evaluated using migration and invasion assays in A673 and RDES cells. RESULTS: Twist1 expression was a negative prognostic marker for overall survival in a public Ewing sarcoma patient data set based on Twist1 mRNA levels and in patient tumor samples based on Twist1 immunohistochemistry. TWIST1 is detected in significantly higher percentage of patients with metastatic diseases than localized disease. Using Ewing sarcoma tumor xenografts in mice, we found that suppressing TWIST1 levels suppressed metastasis without affecting primary tumor development. Knockdown of Twist1 inhibited the migration and invasion capability, while overexpression of Twist1 promoted migration and invasion in Ewing sarcoma cells. CONCLUSION: These results suggest that TWIST1 promotes metastasis in Ewing sarcoma and could be used as a prognostic marker for treatment stratification; however, further validation is required in a larger cohort of patients.

Prognostic and clinicopathological value of Twist expression in breast cancer: A meta-analysis.

BACKGROUND: Despite initial indications that the transcription factor Twist could be used as a breast cancer prognostic marker, there still exists some controversy about its reliability. Thus, the aim of the present study was to assess the relationship between Twist expression and prognosis in breast carcinoma. MATERIALS AND METHODS: We identified eligible studies that reported an association between Twist expression and breast cancer prognosis by searching the literature in PubMed, Embase, the Cochrane Library, and Web of Science databases, through June 5, 2017. Studies investigating Twist protein or mRNA expression as well as reporting survival data in breast cancer were included. The pooled hazard ratio (HR) and odds radio (OR) with a 95% confidence interval (95% CI) were used to estimate associations. RESULTS: A total of 2,671 patients from seven included studies were assessed, and the data indicated that increased Twist expression significantly correlated with poor overall survival (OS) (HR, 1.15; 95% CI, 1.00-1.33; P = 0.04) in breast cancer. In addition, we also observed a significant correlation of elevated Twist expression with larger tumor size (OR, 1.92; 95% CI, 1.31-2.81; P = 0.0009), lymph node involvement (OR, 3.81; 95% CI, 1.16-12.54; P = 0.03), higher nuclear grade (OR, 1.45; 95% CI, 1.06-2.00; P = 0.02), and positive human epidermal growth factor receptor 2 (HER2) status (OR, 1.49; 95% CI, 1.06-2.09; P = 0.02). However, no correlation between Twist expression and disease-free survival (DFS), age, estrogen receptor (ER) status, and progesterone receptor (PR) status was observed. CONCLUSIONS: Our results demonstrate that Twist over-expression is a statistically significant indicator of OS in breast cancer. In addition, our meta-analysis shows that increased Twist expression is significantly associated with larger tumor size, lymph node involvement, higher nuclear grade, and positive HER2 status.

miR-326 regulates EMT and metastasis of endometrial cancer through targeting TWIST1.

OBJECTIVE: Endometrial carcinoma is the most common malignancy of the female genital tract. Therefore, there is an urgent need to understand the molecular mechanism of its metastasis. This study is aimed to explore the function and underlying mechanism of miR-326 in endometrial cancer (EC). PATIENTS AND METHODS: RT-PCR was used to evaluate the miR-326 expression in EC tissues and cell lines. The CKK-8 was used to detect the EC cells proliferation. Transwell assay was performed to evaluate the metastasis of EC cells. Targeted genes were predicted by a bioinformatics algorithm. Dual-luciferase reporter assays were performed to examine the regulation of putative miR-326 targets. The expression of TWIST1 and EMT-related proteins was assayed using Western blot. RESULTS: Our results proved that miR-326 expression was downregulated in EC cell lines and tissue samples. In vitro assays, our results indicated that over-expression of miR-326 inhibited cell proliferation, migration, invasion, and EMT. Moreover, Bioinformatics analysis revealed Twist homolog 1 (TWIST1), a putative tumor promoter, to be a potential target of miR-326. Results from a dual-luciferase reporter system supported TWIST1 as a direct target gene of miR-326. In addition, Western blot showed that over-expression of miR-326 resulted in decreased TWIST1 expression in EC cells. Final in vitro assays revealed that knockdown of TWIST1 inhibited EC cell, migration, invasion and EMT, suggesting that miR-326 exerted its tumor-suppressive role by targeting TWIST1. CONCLUSIONS: We demonstrate that miR-326 served as a tumor suppressor by targeting TWIST1, and may serve as a biomarker or therapeutic target for patients with EC.