7291

TWIST1

MSX2 is thought to be a regulator of organ development and a downstream target of the ras signaling pathway; however, little is known about the role of MSX2 in the development of pancreatic cancers, most of which harbor a K-ras gene mutation. Therefore, we examined whether the presence of MSX2 correlates with the malignant behavior of pancreatic cancer cells. BxPC3 pancreatic cancer cells that stably overexpress MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration, and anchorage-independent cell growth were enhanced in MSX2-expressing cells. Injection of MSX2-expressing cells into the pancreas of nude mice resulted in a significant increase in liver metastases and peritoneal disseminations compared with injection of control cells. Microarray analysis revealed a significant induction of Twist 1 expression in cells that express MSX2. When MSX2 was inactivated in pancreatic cancer cells following transfection with an MSX2-specific small interfering RNA, Twist 1 was down-regulated. Immunohistochemistry of human pancreatic carcinoma tissue revealed that MSX2 was frequently expressed in cancer cells, and that increased expression of MSX2 significantly correlated with higher tumor grade, vascular invasion, and Twist 1 expression. These data indicate that MSX2 plays a crucial role in pancreatic cancer development by inducing changes consistent with epithelial to mesenchymal transition through enhanced expression of Twist 1.

BACKGROUND: tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. RESULTS: To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. CONCLUSIONS: Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.

The epithelial-mesenchymal transition (EMT), one of the main mechanisms underlying development of cancer metastasis, induces stem-like properties in epithelial cells. Bmi1 is a polycomb-group protein that maintains self-renewal, and is frequently overexpressed in human cancers. Here, we show the direct regulation of BMI1 by the EMT regulator, Twist1. Furthermore, Twist1 and Bmi1 were mutually essential to promote EMT and tumour-initiating capability. Twist1 and Bmi1 act cooperatively to repress expression of both E-cadherin and p16INK4a. In patients with head and neck cancers, increased levels of both Twist1 and Bmi1 correlated with downregulation of E-cadherin and p16INK4a, and was associated with the worst prognosis. These results suggest that Twist1-induced EMT and tumour-initiating capability in cancer cells occurs through chromatin remodelling, which leads to unfavourable clinical outcomes.

Twist1, a basic helix-loop-helix transcription factor, promotes breast tumor cell epithelial-mesenchymal transition (EMT), invasiveness, and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (Ser 68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This Ser 68 is phosphorylated by p38, c-Jun N-terminal kinases (JNK), and extracellular signal-regulated kinases1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in human embryonic kidney 293 and breast cancer cells. Prevention of Ser 68 phosphorylation by an alanine (A) mutation (Ser 68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of mitogen-activated protein kinases (MAPK) by an active Ras protein or TGF-beta treatment significantly increases Ser 68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, whereas blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and uninduced Ser 68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness on stimulation with TGF-beta or the active Ras and paclitaxel resistance compared with the same cells expressing the Ser 68A-Twist1 mutant. Importantly, the levels of Ser 68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor-negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1.

BACKGROUND: Twist1 is known to be involved in epithelial-mesenchymal transition (EMT) and tumor progression. However, its specific role is different depending on the type of cancer. The functional role and clinical significance of Twist1 in esophageal squamous cell carcinoma (ESCC) have been rarely studied. METHODS: We examined Twist1 expression in a total of 199 samples using immunohistochemistry and real-time reverse-transcription polymerase chain reaction (RT-PCR). We also studied the association of Twist1 with EMT of ESCC using tissue samples and in vitro models. In addition, we examined the changes in the whole-genome expression profiles of two ESCC cell lines after inducing upregulation of Twist1. RESULTS: Twist1 expression was increased in ESCC compared with that of normal esophagus (P < 0.01). We found that expression of Twist1 was strongly associated with poor prognosis on both univariate analysis [relative risk (RR) = 3.019, P = 0.000] and multivariate analysis (RR = 3.131, P = 0.000). In addition, we found that Twist1 expression was strongly correlated with EMT in ESCC tissues. Upregulation of Twist1 induced other EMT inducers in all three esophageal cancer cell lines investigated (TE1, TE8, and TE10). Twist1 also enhanced the migratory and invasive abilities of all three cell lines. Finally, microarray analysis of gene expression in the three ESCC cell lines revealed that Twist1 upregulation affected the expression of mainly cytoskeleton and extracellular matrix-related genes, which are directly related to cell morphology and movement. CONCLUSIONS: Our study indicates that Twist1 is associated with EMT of esophageal squamous cell carcinoma and may be used as a diagnostic and prognostic biomarker in ESCC.

epithelial mesenchymal transition (EMT) plays an important role in the development of metastases. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are regulated by transcription factors, such as SNAIL, SLUG, and TWIST. We examined the prognostic value of these factors as well as E-cadherin and N-cadherin, in a well-described large cohort of breast cancer patients treated with primary surgery. Analyses were stratified by estrogen receptor (ER) status, because of its crucial role in the regulation of these transcription factors. SNAIL, SLUG, and TWIST expression were examined on a TMA containing 575 breast tumors using immunohistochemistry. Nuclear expression was quantified using a weighted histoscore and classified as high versus low expression, based on the median histoscore. High expression of SNAIL, SLUG, and TWIST was seen in 54, 50, and 50% of tumors, respectively. The level of SNAIL (P = 0.014) and TWIST (P = 0.006) expression was associated with a worse patient relapse-free period, specifically in patients with ER-positive tumors (interaction Cox proportional hazards P = 0.039). Combining both factors resulted in an independent prognostic factor with high discriminative power (both low versus either high: HR 1.15; both low versus both high HR 1.84; P = 0.010). Co-expression of SNAIL-TWIST was associated with low-E-cadherin and high-N-cadherin expression, especially in ER-positive tumors (P = 0.009), suggesting that, through interactions with ER, SNAIL and TWIST may regulate E- and N-cadherin expression, thereby inducing EMT. Our results are indicative that SNAIL and TWIST play a crucial role in EMT through regulation of E- and N-cadherin expression, exclusively in ER-positive breast cancer patients.

cancer metastasis is the major cause of cancer-related death despite significant improvements in multimodal cancer therapy. epithelial-mesenchymal transition (EMT), a major mechanism of cancer metastasis, is a process that generates cells with stem cell-like properties (cancer stemness). cancer stemness is a concept that describes a minor population of cells (cancer stem cells) residing within a tumour that are able to self-renew and are resistant to conventional therapy. The mechanisms delineating the generation of cancer stemness and its connection to cancer metastasis remain largely unknown. Twist1 is an EMT regulator and increased Twist1 expression, which has prognostic significance in various human cancers, has been widely reported. Bmi1 is a critical component of polycomb repressive complex (PRC) 1, which maintains self-renewal and stemness. Bmi1 is frequently overexpressed in different types of human cancers and can induce drug resistance (Table 2). Recent studies have shown that Twist1 directly activates Bmi1 expression and that these two molecules function together to mediate cancer stemness and EMT. These results present a unique mechanism of EMT-induced cancer metastasis and stemness. Further investigation of the mechanisms of EMT-mediated cancer metastasis and stemness will contribute to the management and treatment of metastatic cancers.CI - (c) 2011 The Authors. Clinical and Experimental Pharmacology and Physiology (c) 2011 Blackwell Publishing Asia Pty Ltd.

EMT (epithelial-mesenchymal transition), a major mechanism of cancer metastasis, is a process that generates cells with stem-like properties. These stem-like cells in tumours are described as cancer stem cells. The link between EMT and cancer stemness is well documented without detailed mechanistic proof. Bmi1 belongs to the PRC1 (polycomb repressive complex 1) maintaining self-renewal and stemness together with EZH2 (enhancer of zeste homologue 2), which is a component of PRC2. Bmi1 is frequently overexpressed in different types of human cancers. Recent demonstration of an EMT regulator, Twist1, directly regulating the expression of Bmi1 provides a mechanistic explanation of the relationship between EMT and cancer stemness. The functional interdependence between Twist1 and Bmi1 provides a fresh insight into the common mechanism mediating EMT and cancer stemness. This observation is also confirmed using head and neck cancer patient samples. These results provide a critical mechanism of Twist1-induced EMT and cancer stemness in cancer cells through chromatin remodelling. The role of hypoxia and microRNAs in regulating EMT and cancer stemness is also discussed.CI - (c) The Authors Journal compilation (c) 2011 Biochemical SocietyFAU - Wu, Kou-Juey

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor beta activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.

Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT). The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33) and showed that TWIST1 expression was linked to EGFR mutations (P<0.001), to low CDH1 expression (P<0.05) and low disease free survival (P = 0.044). To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

This study aimed to investigate the expression of Twist in gastric cancer tissues and its correlation between Twist and the epithelial-mesenchymal transition (EMT). By means of RT-PCR and Western blot, the mRNA and protein expressions of Twist, E-cadherin, and Vimentin in 61 gastric cancer tissues and adjacent normal tissues were detected. The positive rates of Twist, E-cadherin, and Vimentin mRNA expression in gastric cancer tissues were 73.9. 40.6, and 60.9 %, respectively; compared to the expression of these genes in adjacent normal tissues (2.9, 75.4, and 27.5 %), the differences were significant (p < 0.05). The E-cadherin protein expression level in gastric cancer tissues was significantly lower than that in the adjacent normal tissues (p < 0.05). After the transfection of Twist siRNA into the MKN45 cells, the protein expression of Twist was significantly reduced (p < 0.05), the protein expression of E-cadherin was significantly increased, and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group (p < 0.05). Twist may be associated with the epithelial-mesenchymal transition in gastric cancer and the tumorigenesis, invasion, and metastasis of gastric cancer.

Twist1 is a transcription factor that is involved in epithelial-mesenchymal transition by suppressing intercellular adhesion. In this study, we aimed to determine the role of Twist1 in the regulation of claudin-4 expression and investigate its specific mechanisms and clinical implications using human esophageal carcinoma cell lines and tissues. As a result, up-regulation of Twist1 decreased both gene and protein expression levels of endogenous claudin-4 and the suppression was mediated by direct binding of Twist1 to the canonical E-box in the promoter region of claudin-4. In addition, there was a significant inverse correlation of claudin-4 with Twist1 in esophageal cancer tissues. High Twist1 and low claudin-4 expression was associated with the poorest prognosis and was more highly correlated with adverse outcome than any other subgroup with statistical significance (p=0.001). Our results indicate that Twist1 induces the repression of claudin-4 expression during the epithelial-mesenchymal transition in esophageal carcinoma.CI - Copyright (c) 2012 Elsevier Inc. All rights reserved.

BACKGROUND: The high mobility group protein A2 (HMGA2) is an architectural transcription factor that plays an important role in the development and progression of many malignant neoplasms. High expression of HMGA2 in gastric cancer correlates with invasiveness of cancer and is an independent prognostic factor. The reason for this might be HMGA2 promoting epithelial-mesenchymal transitions (EMT), which is the key process of metastasis for some underlying mechanisms. AIMS: This study was designed to test whether HMGA2 participates in the EMT and to further understand the underlying mechanisms of EMT promoted by HMGA2. METHODS: We examined the cell biology and molecular biology changes after overexpression and knockdown HMGA2 of gastric cancer cells in vitro and vivo. To further understand the underlying mechanisms of EMT promoted by HMGA2, based on our previous study, we examined the changes of target genes of HMGA2 after overexpression and knockdown HMGA2 of gastric cancer cells. RESULTS: The results indicated that overexpressing HMGA2 enabled enhancing the oncogenic properties of gastric epithelial origin cell in vitro and in vivo. Furthermore, our study showed that HMGA2 was able to elicit EMT and regulate several genes which are closely related to the Wnt/beta-catenin pathway by directly binding to their promoter thereby activating the Wnt/beta-catenin pathway. CONCLUSIONS: The Wnt/beta-catenin pathway activated by HMGA2 might be the underlying mechanism of EMT in gastric cancer cells.

UNLABELLED: Lipocalin-2 (Lcn2) is preferentially expressed in hepatocellular carcinoma (HCC). However, the functional role of Lcn2 in HCC progression is still poorly understood, particularly with respect to its involvement in invasion and metastasis. The purpose of this study was to investigate whether Lcn2 is associated with the epithelial-mesenchymal transition (EMT) in HCC and to elucidate the underlying signaling pathway(s). Lcn2 was preferentially expressed in well-differentiated HCC versus liver cirrhosis tissues, and its expression was positively correlated with the stage of HCC. The characteristics of EMT were reversed by adenoviral transduction of Lcn2 into SH-J1 cells, including the down-regulation of N-cadherin, vimentin, alpha-smooth muscle actin, and fibronectin, and the concomitant up-regulation of CK8, CK18, and desmoplakin I/II. Knockdown of Lcn2 by short hairpin RNA (shRNA) in HKK-2 cells expressing high levels of Lcn2 was associated with EMT. Epidermal growth factor (EGF) or transforming growth factor beta1 (TGF-beta1) treatment resulted in down-regulation of Lcn2, accompanied by an increase in Twist1 expression and EMT in HCC cells. Stable Lcn2 expression in SH-J1 cells reduced Twist1 expression, inhibited cell proliferation and invasion in vitro, and suppressed tumor growth and metastasis in a mouse model. Furthermore, EGF or TGF-beta1 treatment barely changed EMT marker expression in SH-J1 cells ectopically expressing Lcn2. Ectopic expression of Twist1 induced EMT marker expression even in cells expressing Lcn2, indicating that Lcn2 functions downstream of growth factors and upstream of Twist1. CONCLUSION: Together, our findings indicate that Lcn2 can negatively modulate the EMT in HCC cells through an EGF (or TGF-beta1)/Lcn2/Twist1 pathway. Thus, Lcn2 may be a candidate metastasis suppressor and a potential therapeutic target in HCC.CI - Copyright (c) 2013 by the American Association for the Study of Liver diseases.

Although many researches have been undertaken to disclose the mechanisms of chemoresistance, the mechanisms remain unclear. The aim of this study is to elucidate the role of miR-181a-Twist1 pathway in the chemoresistance of tongue squamous cell carcinoma (TSCC). We found that cisplatin-induced chemoresistance in TSCC cell lines underwent EMT (epithelial-mesenchymal transition) and was accompanied by enhancing metastatic potential (migration and invasion in vitro), miR-181a downregulation and Twist1 upregulation. Functional analyses indicated that miR-181a reversed chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Twist1 was confirmed as a direct miR-181a target gene by luciferase reporter gene assays. Twist1 knockdown by siRNA led to a reversal of the chemoresistance, inhibited EMT and metastatic potential in TSCC cells. Our study demonstrates that miR-181a-Twist1 pathway may play an important role in the development of cisplatin-chemoresistance, with EMT and an increase the metastatic potential of TSCC cells.CI - Copyright (c) 2013 Elsevier Inc. All rights reserved.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer deaths worldwide. In recent studies, a crucial link has been discovered between the acquisition of metastatic traits and tumour-initiating abilities in cancer cells during the epithelial-mesenchymal transition (EMT). Herein, we demonstrated that the ectopic expression of TWIST1, the EMT regulator, in HNSCC FaDu cells triggered EMT and resulted in the acquisition of a mesenchymal phenotype. Moreover, FaDu-pFLAG-TWIST1 cancer cell populations that were induced to EMT displayed an increased proportion of cells with the CD44 marker, which is associated with tumour initiation. Interestingly, we found that emodin treatment reduced the tumour-initiating abilities and inhibited cell migration and invasion in FaDu-pFLAG-TWIST1 cells. Emodin directly inhibited TWIST1 expression, upregulated E-cadherin mRNA and protein expression, and downregulated vimentin mRNA and protein expression. Moreover, we found that emodin inhibited TWIST1 binding to the E-cadherin promoter and repressed E-cadherin transcription activity. We also found that emodin inhibited TWIST1-induced EMT by inhibiting the beta-catenin and Akt pathways. More interestingly, emodin significantly inhibited TWIST1-induced invasion in vivo. Therefore, emodin might be applicable to anticancer therapy and could be a potential new therapeutic drug for HNSCC.CI - Copyright (c) 2013 Elsevier Ltd. All rights reserved.

Kruppel-like factor 17 (KLF17), a member of the KLF transcription factor family, has been shown to inhibit the epithelial-mesenchymal transition (EMT) and tumor growth. However, the expression, the cellular function and the mechanism of KLF17 in endometrioid endometrial cancer (EEC; a dominant type of endometrial cancer) remain elusive. Here, we report that among the KLF family members, KLF17 was consistently upregulated in EEC cell lines compared with immortalized endometrial epithelial cells. Overexpression of KLF17 in EEC cell lines induced EMT and promoted cell invasion and drug resistance, resulting in increased expression of TWIST1. In contrast, KLF17 suppression reversed EMT, diminished cell invasion, restored drug sensitivity and suppressed TWIST1 expression. Luciferase assays, site-directed mutagenesis and transcription factor DNA-binding analysis demonstrated that KLF17 transactivates TWIST1 expression by directly binding to the TWIST1 promoter. Knockdown of TWIST1 prevented KLF17-induced EMT. Consistent with these results, both KLF17 and TWIST1 levels were found to be elevated in EECs compared with normal tissues. KLF17 expression positively correlated with tumor grade but inversely correlated with estrogen and progesterone receptor expression. Thus, KLF17 may have an oncogenic role during EEC progression via initiating EMT through the regulation of TWIST1.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT. METHODS: The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC). RESULTS: MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3'UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens. CONCLUSIONS: Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is a well-established entity among pancreatic neoplasms that ranges from low-grade dysplasia to invasive carcinoma. epithelial-mesenchymal transition (EMT) contributes to tumor progression in various cancers. Moreover, Notch signaling is one of the important upstream effectors of EMT promotion. Currently, it is unclear whether EMT causes pathological progression of IPMN. AIM: We evaluated the expression of EMT-promoting transcription factors Twist and B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) in IPMN. METHODS: Patients who underwent resections at our institute and its affiliated hospital were enrolled in this study (n = 35). Protein expression of EMT markers Twist, Bmi1, Jagged1, and E-cadherin in resected specimens was investigated by immunohistochemistry. expression of these proteins was compared with the clinicopathological factors and patient survival. RESULTS: Positive expression of Twist and Bmi1 was observed in 40.0% and 42.9% of IPMNs, respectively. Twist and Bmi1 expression was significantly higher in IPMNs with high-grade dysplasia (P < 0.05) and invasive carcinoma (P < 0.05) than that in IPMNs with low-grade dysplasia. High expression of Twist was correlated with Jagged1 expression and inversely correlated with expression of E-cadherin (P = 0.06 and P < 0.05, respectively). In survival analyses, the recurrence rate was significantly higher in the group that showed simultaneous high expression of Twist and Bmi1 (P < 0.05). CONCLUSIONS: expression of Twist and Bmi1 is associated with aggressiveness and poor prognoses of IPMN through EMT promotion that might be induced by Notch signaling.CI - (c) 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

The pattern of secreted factors in the tumor microenvironment has been shown to initiate tumor epithelial-mesenchymal transition (EMT); however, little is known about their interplay undergoing this phenotypic switch. In this study, we revealed obvious coactions of cytokine IL-6 and chemokine CCL2 during EMT induction. We found that IL-6 effectively induced EMT and promoted tumor cell invasion, which could be markedly enhanced by addition of CCL2 in a CCR2-dependent manner. IL-6 and CCL2 induced each other and cooperatively elicited STAT3 phosphorylation; conversely, STAT3 regulated the production of IL-6 and CCL2, thus constituting a positive feedback loop to maintain and amplify STAT3 signaling, consequently promoting additional EMT events. Furthermore, CCL2 greatly enhanced IL-6-induced EMT events mainly by upregulating the expression of Twist. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-centered loop and markedly suppressed Twist expression as well as IL-6/CCL2-mediated EMT induction. Thus, our findings highlighted the synergy of the two secreted factors of tumor microenvironment, in regulating transformed properties of non-small cell lung cancer (NSCLC).

tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

AIM: To explore the correlation between Twist-related protein (Twist)1, fibroblast growth factor receptor (FGFR)2 and gastric adenocarcinoma differentiation and progression. METHODS: We evaluated Twist1 and FGFR2 in 52 gastric adenocarcinoma samples by immunohistochemistry and quantitative real time polymerase chain reaction, and analyzed the correlation between Twist1, FGFR2 and cancer differentiation. We also detected Twist1 and FGFR2 expression in gastric adenocarcinoma cell lines, and evaluated Twist1 influence on FGFR2 expression. In addition, we studied the role of FGFR2 in Twist1-promoted cancer progression, including proliferation, invasion and epithelial-mesenchymal transition (EMT). RESULTS: Twist1 and FGFR2 were detected in almost all the gastric adenocarcinoma samples. Twist1 (P = 0.0213) and FGFR2 (P = 0.0310) mRNA levels had a significant association with gastric adenocarcinoma differentiation. Moreover, Twist1 and FGFR2 expression in poorly differentiated cells (SNU-1 and SNU-16) was notably higher than in well-differentiated cells (MKN-7 and MKN-28). In poorly differentiated gastric adenocarcinomas, FGFR2 mRNA level was significantly positively correlated with Twist1 mRNA level (P = 0.004). Twist1 was proved to promote FGFR2 by regulating Twist1 expression by knockdown and overexpression. Additionally, Twist1 could induce proliferation, invasion and EMT in gastric cancer; of these, FGFR2 was required for invasion and EMT, rather than proliferation. CONCLUSION: Twist1 and FGFR2 are highly associated with differentiation of gastric adenocarcinoma; Twist1 can facilitate invasion and EMT in gastric adenocarcinoma via promotion of FGFR2 expression.

The transcription factor sex determining region Y-box protein 5 (SOX5) plays important roles in various types of cancers. However, the expression and function of SOX5 in hepatocellular carcinoma (HCC) have not been elucidated. Here, we found that SOX5 is significantly up-regulated in HCC tissues and cell lines. Gain- and loss-of-function studies demonstrated that SOX5 promoted HCC cell migration and invasion. In addition, we revealed that SOX5 is linked to epithelial-mesenchymal transition (EMT) by regulation of Twist1. Our results indicate for the first time that SOX5 is a novel regulator of EMT in HCC and may be a potential therapeutic target for HCC metastasis.

epithelial-mesenchymal transition (EMT) is an important process implicated in tumor invasion and metastasis. Twist1 is a transcription factor that induces EMT, including E-cadherin suppression and cancer cell migration and invasion; hence it promotes cancer metastasis. Twist1 directly or indirectly regulates the expression of various genes and cellular functions involved in cancer progression. However, the underlying mechanisms remain largely unknown. In this study, we investigated the molecular basis for Twist1-mediated invasion and EMT. In human cancer cells, Twist1 was found to directly upregulate transcription of the mesenchymal gene integrin alpha5 in an E-box-independent, but activating protein-1 (AP-1) element-dependent, manner. Twist1 activated the integrin alpha5 promoter by interacting with and activating the transcription factor AP-1, composed of c-Jun and activating transcription factor-2 (ATF-2); it also enhanced the nuclear presence of ATF-2. AP-1 was critical for Twist1-induced cancer cell invasion, primarily through the induction of integrin alpha5, which activated c-Jun N-terminal kinase and focal adhesion kinase-signaling activities. Using data from The cancer Genome Atlas, we found that Twist1 expression positively correlates with integrin alpha5 expression in human colorectal cancers. These findings suggest that cooperation between Twist1 and AP-1 represents a novel mechanism for EMT and tumor invasiveness. This study supports further investigation into the molecular basis underlying the diverse Twist1-mediated functions that occur during tumor progression.CI - (c) The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

epithelial-to-mesenchymal transition (EMT) is critical for the invasion and metastasis of hepatocellular carcinoma (HCC). However, to date, the association of signal transducer and activator of transcription 3 (STAT3) with EMT, and its mediated tumor invasion and metastasis in HCC, remain elusive. We investigated the relationship between STAT3 activation and EMT, and the underlying mechanisms involved in HCC progression. By stable transfection, we successfully overexpressed STAT3 in low metastatic SMMC7721 cells and silenced STAT3 expression in high metastatic MHCC97H cells. The EMT-associated molecular HCC cell changes were analyzed by real-time PCR, western blotting and immunocytochemical methods. The EMT-mediated HCC cell invasion and migration were evaluated by a Transwell cell invasion and cell migration assay, respectively. The interaction between STAT3 and Twist (a key EMT inducer) was evaluated by dual-luciferase reporter assay. In the present study, we found that STAT3 overexpression significantly reduced E-cadherin and beta-cadherin, and it enhanced N-cadherin and vimentin expression in the SMMC7721 cells. STAT3 knockdown significantly increased E-cadherin and beta-cadherin, and it decreased N-cadherin and vimentin expression in the MHCC97H cells. Meanwhile, a dual-luciferase reporter assay revealed that STAT3 may bind the Twist promoter, mediate its transcriptional activity, and then promote the EMT process in HCC cells. STAT3 activation-mediated EMT also evidently enhanced HCC cell invasion and migration. In summary, the present study demonstrated for the first time that STAT3 may cooperate with Twist to mediate EMT and induce HCC invasion and metastasis. Activated STAT3, Twist, and EMT markers may serve as potential molecular targets in the prevention and/or treatment of HCC invasion and metastasis.

p27 restrains normal cell growth, but PI3K-dependent C-terminal phosphorylation of p27 at threonine 157 (T157) and T198 promotes cancer cell invasion. Here, we describe an oncogenic feedforward loop in which p27pT157pT198 binds Janus kinase 2 (JAK2) promoting STAT3 (signal transducer and activator of transcription 3) recruitment and activation. STAT3 induces TWIST1 to drive a p27-dependent epithelial-mesenchymal transition (EMT) and further activates AKT contributing to acquisition and maintenance of metastatic potential. p27 knockdown in highly metastatic PI3K-activated cells reduces STAT3 binding to the TWIST1 promoter, TWIST1 promoter activity and TWIST1 expression, reverts EMT and impairs metastasis, whereas activated STAT3 rescues p27 knockdown. Cell cycle-defective phosphomimetic p27T157DT198D (p27CK-DD) activates STAT3 to induce a TWIST1-dependent EMT in human mammary epithelial cells and increases breast and bladder cancer invasion and metastasis. Data support a mechanism in which PI3K-deregulated p27 binds JAK2, to drive STAT3 activation and EMT through STAT3-mediated TWIST1 induction. Furthermore, STAT3, once activated, feeds forward to further activate AKT.

epithelial-mesenchymal transition (EMT) has an established role in promoting tumor progression and the acquisition of therapeutic resistance. Here, the EMT phenotype was detected in cisplatin-resistant ovarian cancer tissues and cell lines, and correlated with decreased miR-186 expression, increased Twist1 expression, chemoresistance and poor prognosis in epithelial ovarian cancer (EOC) patients. Introducing miR-186 into EOC cells led to a reduction in twist family bHLH transcription factor 1 (Twist1) expression along with morphological, functional and molecular changes consistent with mesenchymal-to-epithelial transition, G1 cell-cycle arrest and enhanced cell apoptosis, which consequently rendered the cells more sensitive to cisplatin in vitro and in vivo. Furthermore, luciferase reporter and rescue assay results showed that the EMT and drug resistance reversal in response to miR-186 was mediated by Twist1. Collectively, these findings implicate miR-186 as an attractive candidate for overcoming chemoresistance in ovarian cancer therapy.

BACKGROUND: Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue. METHODS: This study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score. RESULTS: CTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT. CONCLUSIONS: In this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.

Aggressive tumor cells can mimic embryonic vasculogenic networks and form vasculogenic mimicry (VM). Preliminary studies demonstrated that hypoxia can promote VM formation; however, the underlying mechanism remains unclear. The present study aimed to investigate the role of the Twist1Bmi1 connection in hypoxiainduced VM formation and the underlying mechanism. In the in vitro experiments, western blot analysis demonstrated that hypoxia upregulated the expression of Twist1, Bmi1, epithelialmesenchymal transition (EMT) markers, stem cell markers and VMassociated markers. The 3D culture assay showed that hypoxia promoted VM formation in hepatocellular carcinoma (HCC) cell lines. Using transfection and in vitro cell experiments, the Twist1Bmi1 connection was confirmed to have an important role in inducing EMT, cell stemness and VM formation. In the in vivo experiments, the murine hypoxia models were established via incomplete femoral artery ligation and the mechanism by which hypoxia promoted Twist1 and Bmi1 expression and led to VM formation was demonstrated by immunohistochemistry staining and endomucin/periodic acid Schiff doublestaining. In conclusion, hypoxia upregulate the expression of Twist1 and Bmi1, and these two proteins have an important role in inducing EMT and cancer cell stemness, which contributed to VM formation.

As reported, the CC chemokine receptor 7 (CCR7) trigger a series of signaling cascades in the epithelial-mesenchymal transition (EMT) of some malignancies. Meanwhile, Twist promotes EMT in pancreatic ductal adenocarcinoma (PDAC) progression. Here, effects of Twist on CCR7-induced EMT in the PDAC were investigated in detail. The immunohistochemistry was used to detect the expression of Twist, and then, in vitro assays were applied. The expression rate of Twist was 72.0 % in PDAC samples and closely correlated with tumor-node-metastasis (TNM) stage and invasion. When PDAC cell line PANC1 was subjected to CCL19 stimulation, the expression of p-ERK, p-AKT, Twist, N-cadherin, MMP9, and alpha-smooth muscle actin (alpha-SMA) was induced, while the GSK1120212, BEZ235, and MK2206 prohibited the increase of Twist and EMT biomarkers. For another thing, the si-Twist treatment attenuated CCL19-stimulated EMT occurrence, migration, and invasion phenotypes of PANC1 cells. In conclusion, CCR7 pathway up-regulates Twist expression via ERK and PI3K/AKT signaling to manage the EMT of PDAC. Our work allows for clinical gene or protein-targeted regimen of PDAC patients in the near future.

epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P<0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-beta promoted EMT by upregulating the expression of Twist through the TGF-beta/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-beta/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.

Overexpression of Twist, a highly conserved basic helix-loop-helix transcription factor, is associated with epithelial-mesenchymal transition (EMT) and predicts poor prognosis in various kinds of cancers, including breast cancer. In order to further clarify Twist's role in breast cancer, we detected Twist expression in breast cancer tissues by immunohistochemistry. Twist expression was observed in 54% (220/408) of breast cancer patients and was positively associated with tumor size, Ki67, VEGF-C and HER2 expression. Conversely, Twist was negatively associated with estrogen receptor (ER), progesterone receptor (PgR) and E-cadherin expression. Patients with Twist expression had a poorer prognosis for 30-month disease free survival (DFS) (82.9%) than patients with negative Twist (92.3%). Overexpression of Twist led to dramatic changes in cellular morphology, proliferation, migratory/invasive capability, and expression of EMT-related biomarkers in breast cancer cells. Moreover, we show that Twist serves as a driver of tumorigenesis, as well as an inducer of EMT, at least in part, through activation of the Akt and extracellular signal-regulated protein kinase (ERK) pathways which are critical for Twist-mediated EMT. Our results demonstrate that Twist expression is an important prognostic factor in breast cancer patients.

epithelial-mesenchymal transition (EMT) is regulated by multiple signal transduction pathways. Twist-1 is one of the most important transcription factors in these pathways. In a previous study, we found that Bcl-2 enhanced the role of Twist-1 in EMT. Coexpression of Twist-1 and Bcl-2 may play an import role in vasculogenic mimicry (VM) through regulation of EMT. Moreover, regulators of EMT and VM are known to be important targets for microRNAs (miRNAs). To better understand how these critical pathways are induced by coexpression of Twist-1 and Bcl-2, we performed a comprehensive comparative bioinformatics analysis using microarrays on HCCs that overexpressed Twist-1 and Bcl-2. Eleven miRNAs associated with coexpression of Twist-1 and Bcl-2 were selected from the comprehensive analysis of miRNA microarray and ChIP-seq analysis. Changes in miRNAs were associated with significant differences in the expression of genes involved in signal transduction pathways related to processes including tumor invasion, metastasis, angiogenesis, and tumor cell shape. We confirmed the role of Twist-1 and Bcl-2 coexpression in HCC cells using wound healing assays, invasion assays, and 3D Matrigel assays. Furthermore, the role of miR-27a as a crucial regulator of EMT and VM was confirmed in HCC cells by RT-PCR and western blot analysis. These findings provide evidence that Bcl-2 enhances the role of Twist-1 in VM and EMT through miRNAs.CI - Copyright (c) 2015 Elsevier Inc. All rights reserved.

Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-beta, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity pathways Analysis showed that integrin beta1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells.CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved.

We have previously shown that complement component 3 (C3) is secreted by malignant epithelial cells. To understand the mechanism of upregulation of C3 expression in tumor cells, we studied the C3 promoter and identified that twist basic helix-loop-helix transcription factor 1 (TWIST1) binds to the C3 promoter and enhances its expression. Because TWIST1 mediates epithelial-mesenchymal transition (EMT), we studied the effect of C3 on EMT and found that C3 decreased E-cadherin expression on cancer cells and promoted EMT. We showed that C3-induced reduction in E-cadherin expression in ovarian cancer cells was mediated by C3a and is Kruppel-like factor 5 dependent. We investigated the association between TWIST1 and C3 in malignant tumors and in murine embryos. TWIST1 and C3 colocalized at the invasive tumor edges, and in the neural crest and limb buds of mouse embryos. Our results identified TWIST1 as a transcription factor that regulates C3 expression during pathologic and physiologic EMT.CI - Copyright (c) 2016 by The American Association of Immunologists, Inc.

Inhibition of epithelial-mesenchymal transition (EMT)-inducing transcription factors Twist and Snail prevents tumor metastasis but enhances metastatic growth. Here, we report an unexpected role of a tumor suppressor tristetraprolin (TTP) in inhibiting Twist and Snail without enhancing cellular proliferation. TTP bound to the AU-rich element (ARE) within the mRNA 3'UTRs of Twist1 and Snail1, enhanced the decay of their mRNAs and inhibited the EMT of cancer cells. The ectopic expression of Twist1 or Snail1 without their 3'UTRs blocked the inhibitory effects of TTP on the EMT. We also observed that TTP overexpression suppressed the growth of cancer cells. Our data propose a new model whereby TTP down-regulates Twist1 and Snail1 and inhibits both the EMT and the proliferation of cancer cells.

Twist1 is an essential transcription factor required to initiate epithelial-mesenchymal transition (EMT) and promote tumor metastasis. PAQR3 is a newly found tumor suppressor that is frequently downregulated in many types of human cancers. Downregulation of PAQR3 is associated with accelerated metastasis and poor prognosis of the patients with gastric cancers. In this study, we demonstrate that PAQR3 is actively involved in the degradation of Twist1 and whereby regulates EMT and metastasis of gastric cancer cells. PAQR3 overexpression reduces the protein level but not the mRNA level of Twist1. The protein stability and polyubiquitination of Twist1 are altered by PAQR3. PAQR3 forms a complex with Twist1 and BTRC, an E3 ubiquitin ligase. PAQR3 enhances the interaction between Twist1 and BTRC. Twist1 is mobilized from the nucleus to a proteasome-containing structure in the cytoplasm upon overexpression of PAQR3 and BTRC, which is required for PAQR3-induced degradation of Twist1. The Twist1 box domain of the Twist1 protein is required for the interaction of Twist1 with both PAQR3 and BTRC, indispensable for PAQR3-mediated degradation of Twist1. Both BTRC and Twist1 are required for the inhibitory effects of PAQR3 on migration and EMT phenotype of gastric cancers cells. Importantly, Twist1 is indispensable for the inhibitory effect of PAQR3 on metastasis of gastric cancer cells in vivo Collectively, these findings not only pinpoint that Twist1 mediates the modulatory function of PAQR3 on EMT and metastasis but also suggest that targeting Twist1 is a promising strategy to control metastasis of tumors with downregulation of PAQR3.CI - (c) The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Pancreatic cancer (PC) remains one of the most lethal types of cancer in adults. The purpose of this study was to determine the role of miR-1271 in regulation of epithelial mesenchymal transition (EMT) and metastasis of pancreatic cancer cells. miR-1271 was identified to be significantly down-regulated in PC tissues by miRNA array. Also, an increase of EMT-regulators ZEB1 and TWIST1 expression level is accompanied by a decrease of miR-1271. We showed that expression of miR-1271 was significantly down-regulated in PC tissues as compared with that in normal tissues. In addition, our results showed that miR-1271 expression levels were decreased while ZEB1 and TWIST1 expression levels were increased in detected PC cell lines. Moreover, ectopic expression of miR-1271 suppressed and antagomiR-1271 promoted proliferation, migration, and invasion in SW1990 and PANC-1 cells. Bioinformatics coupled with luciferase and Western blot assays also revealed that miR-1271 inhibited expression of ZEB1 and TWIST1, which are master regulators of tumor metastasis. Our study first indicates that miR-1271 functions as a suppressor in regulating of pancreatic cancer EMT by targeting ZEB1 and TWIST1, and it promise as a therapeutic target and prognostic marker for metastatic pancreatic cancer.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

UNLABELLED: Twist-1 and miRNAs have been reported to be associated with tumor metastasis and angiogenesis. However, the relationship between Twist-1 and miRNAs and the function of miRNAs remain largely undefined. We aimed to reveal the Twist-1-related miRNA expression profile and to determine whether Twist-1 functions in tumor metastasis and vasculogenic mimicry (VM) by regulating miRNA expression in hepatocellular carcinoma (HCC). Results showed that the expression of miR-27a-3p was consistently down-regulated in HCC cell lines and tissue samples displaying high expression of Twist-1. Both loss- and gain-of-function assays revealed suppressive effects of miR-27a-3p. Low miR-27a-3p expression was significantly associated with early metastasis in HCC. Subsequent investigations revealed that miR-27a-3p mediated the inhibition of epithelial-mesenchymal transition (EMT). Additional experiments showed that VE-cadherin is a direct target of miR-27a-3p and further demonstrated the critical role of miR-27a-3p in suppressing tumor metastasis and VM. CONCLUSIONS: Twist-1 up-regulation in HepG2 cells resulted in the differential expression of 18 miRNAs. Among them, miR-27a-3p deregulation contributed to VM and metastasis. The miR-27a-3p-mediated down-regulation of VE-cadherin and inhibition of EMT may be essential for Twist-1 to induce tumor metastasis and VM. Our findings highlight the importance of miR-27a-3p and suggest a promising new strategy for anti-HCC therapy.

UNLABELLED: Twist1 is well known to induce epithelial-mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) are involved in the EMT process and are associated with metastasis in hepatocellular carcinoma (HCC). In the present study, microRNA-26b-5p (miR-26b-5p) expression was consistently and significantly downregulated in HepG2-Twist1 HCC cell lines compared with HepG2-vector cell lines using microarrays (the HepG2-Twist1 cell line can stably express Twist1). miR-26b- 5p downregulation was directly mediated by Twist1 through binding to the promoter region of miR-26b-5p in HepG2-Twist1 cells by ChIP-seq technology. Both gain- and loss-of-function studies showed that miR-26b-5p dramatically suppressed EMT and the invasion ability of HCC cells in vitro. Using mouse models, tumors derived from miR- 26b-5p-overexpressed HCC cells exhibited a significant reduction in tumorigenicity compared with the control group. Subsequent investigation revealed that miR-26b-5p directly inhibited SMAD family member 1 (SMAD1) expression. miR-26b-5p repressed BMP4/Smad1 signaling following SMAD1 inhibition. Overexpression of SMAD1 reversed the function of miR-26b-5p. In human HCC tissues and mouse xenograft tumors, miR-26b-5p levels were inversely correlated with SMAD1 expression as well as metastasis. CONCLUSION: miR-26b-5p suppresses Twist1-induced EMT, invasion, and metastasis of HCC cells by targeting SMAD1 and BMP4/Smad1 signaling. This suggests a promising application for miR-26b-5p in anti-HCC therapy.

Basic helix-loop-helix transcription factor Twist1 is a master regulator of epithelial-mesenchymal transition (EMT), a cellular program implicated in different stages of development as well as metastatic dissemination of carcinomas. Here, we show that Twist1 requires TGF-beta type-I receptor (TGFBR1)-activation to bind an enhancer region of downstream effector ZEB1, thereby inducing ZEB1 transcription and EMT. When TGFBR1-phosphorylation is inhibited, Twist1 generates a distinct cell state characterized by collective invasion, simultaneous proliferation and expression of endothelial markers. By contrast, TGFBR1-activation directs Twist1 to induce stable mesenchymal transdifferentiation through EMT, thereby generating cells that display single-cell invasion, but lose their proliferative capacity. In conclusion, preventing Twist1-induced EMT by inhibiting TGFbeta-signaling does not generally block acquisition of invasion, but switches mode from single-cell/non-proliferative to collective/proliferative. Together, these data reveal that transient Twist1-activation induces distinct cell states depending on signaling context and caution against the use of TGFbeta-inhibitors as a therapeutic strategy to target invasiveness.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in north American men, and most its related deaths are due to advanced and metastatic PCa. However, the molecular mechanisms underlying PCa progression are still unclear. Here we use a pair of prostate cell lines P69/M12, which have the same genetic background and the highly metastatic cell line M12 is a subline derived from P69, to identify the pathogenesis of PCa. We find that a key miRNA--miR186 is significantly reduced in M12 compared to that in P69. Further, we validate that miR186 is also downregulated in human PCa specimens, most significantly in the metastatic patient specimens. The low miR186 expression is correlated with poor patient survival. Through knockdown or overexpression of miR186 in PCa cell lines, we discover that miR186 strongly inhibits cell motility, invasive, soft-agar colony formation, 3D culture growth and vasculogenic mimicry (VM) formation capacity, as well as the epithelial-to-mesenchymal transition (EMT) process by downregulation of its target Twist1. Moreover, the inverse relationship between the expression levels of miR186 and Twist1 is confirmed in vivo tumor metastasis experiment and clinical specimens. Taken together, our findings demonstrate an important role of miR186/Twist1 axis in the regulation of PCa progression, suggesting a potential application of miR186/Twist1 in PCa treatment.

The epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific.

Colorectal cancer (CRC) with microsatellite instability (MSI) may exhibit impaired epithelial-mesenchymal transition (EMT), but little is known about the underlying mechanisms of this phenomenon. In this study, we investigated the role of Twist1 and its downstream signaling cascades in EMT induction according to MSI status. To investigate the effects of Twist1 on EMT induction according to MSI status, MSS LS513 and MSI LoVo colon cancer cell lines, which overexpress human Twist1, were generated. Twist1-induced EMT and its downstream signaling pathways were evaluated via in vitro and in vivo experiments. We found that Twist1 induced EMT markers and stem cell-like characteristics via AKT signaling pathways. Twist1 induced activation of AKT and suppression of glycogen synthase kinase (GSK)-3beta, which resulted in the activation of beta-catenin, increasing CD44 expression. In addition, Twist1 activated the AKT-induced NF-kappaB pathway, increasing CD44 and CD166 expression. Activation of both the AKT/GSK-3beta/beta-catenin and AKT/NF-kappaB pathways occurred in MSS LS513 cells, while only the AKT/GSK-3beta/beta-catenin pathway was activated in MSI LoVo cells. In conclusion, Twist1 induces stem cell-like characteristics in colon cancer cell lines related to EMT via AKT signaling pathways, and those pathways depend on MSI status.

BACKGROUND: Kruppel-like factor 4 (KLF4) exerts tumor suppressive or oncogenic functions in a cell-type-dependent manner, but its prognostic role in urothelial carcinoma of bladder (UCB) remains unclear. We aimed to determine how KLF4 regulates epithelial-mesenchymal transition (EMT) and predicts patient survival in UCB. PATIENTS AND METHODS: The roles of KLF4 and other EMT regulators in cancer progression were studied in UCB specimens of 398 patients, UCB cell lines. The results were validated by open-access The cancer Genome Atlas dataset. RESULTS: Over a median follow-up of 46.5 months, tissue microarray demonstrated that strong KLF4 expression was associated with higher risk toward metastasis and death (P<0.001). KLF4 expression positively correlated with TWIST1 and vimentin, and inversely correlated with E-cadherin expression. Metastasis-free survival was poorest in KLF4/TWIST1 coexpression group, followed by KLF4 or TWIST1 expression-alone group, and no-expression group (P<0.001). Multivariate analysis substantiated that KLF4/TWIST1 coexpression independently predicted overall mortality and metastasis risk with hazard ratios of 2.43 (95% CI: 1.65-3.64) and 7.54 (CI: 4.03-12.10). The cancer Genome Atlas dataset of bladder cancer also revealed a trend toward decreased overall survival in the high KLF4 expression group as compared to the low KLF4 group. In vitro, KLF4 is accompanied with decreased E-cadherin and beta-catenin expressions, increased vimentin and fibronectin expressions, and enhanced migration/invasion. KLF4 knockdown suppressed TWIST1 expression and inhibited EMT, migration and invasion, whereas enforced KLF4 overexpression activated TWIST1 expression and restored EMT and metastatic phenotype. Furthermore, TWIST1 knockdown abolished KLF4-faciliated EMT and metastatic feature without affecting KLF4 expression. CONCLUSIONS: KLF4 promotes TWIST1-mediated EMT and may represent a novel prognostic predictor in UCB.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Twist has been shown to cause treatment failure, cancer progression, and cancer-related death. However, strategies that directly target Twist are not yet conceivable. Here we reveal that K63-linked ubiquitination is a crucial regulatory mechanism for Twist activation. Through an E3 ligase screen and biochemical studies, we unexpectedly identified that RNF8 functions as a direct Twist activator by triggering K63-linked ubiquitination of Twist. RNF8-promoted Twist ubiquitination is required for Twist localization to the nucleus for subsequent EMT and CSC functions, thereby conferring chemoresistance. Our histological analyses showed that RNF8 expression is upregulated and correlated with disease progression, EMT features, and poor patient survival in breast cancer. Moreover, RNF8 regulates cancer cell migration and invasion and cancer metastasis, recapitulating the effect of Twist. Together, our findings reveal a previously unrecognized tumor-promoting function of RNF8 and provide evidence that targeting RNF8 is an appealing strategy to tackle tumor aggressiveness and treatment resistance.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

High expression of high mobility group protein A2 (HMGA2) is correlated with the invasiveness of gastric cancer and is an independent prognostic factor. The reason may be that HMGA2 promotes epithelial-mesenchymal transition (EMT) and the acquisition of tumor stem cell properties, yet the mechanism remains unclear. In this study, immunohistochemistry and western blot analysis revealed that the expression of HMGA2 and Twist-related protein 1 (TWIST1) in gastric carcinoma tissues was higher than that in the peritumoral tissues and that the expression levels of these two proteins were positively correlated. The protein expression levels of HMGA2 and TWIST1 were high in the poorly differentiated gastric cancer MKN-45 cells and were low in the moderately differentiated SGC-7901 cells. TWIST1 was inhibited after HMGA2 interference and was significantly increased after overexpression of HMGA2. Luciferase experiments showed that TWIST1 was a direct downstream target gene of HMGA2. The simultaneous interference of HMGA2 expression and the overexpression of TWIST1 in MKN-45 cells reversed the inhibitory effect of HMGA2 interference on the invasion and migration of gastric cancer cells, EMT and the expression of stemness markers. However, the simultaneous overexpression of HMGA2 and the interference of TWIST1 expression in the SGC-7901 cells reversed the promoter effect of HMGA2 overexpression on the invasiveness and migration of gastric cancer cells, EMT and the expression of stemness markers. In addition, animal experiments showed that TWIST1 overexpression reversed the inhibition of HMGA2 interference on the metastasis of MKN-45 cells. Therefore, HMGA2 regulates the EMT of gastric cancer cells and the acquisition of tumor stem cell properties through direct regulation of the downstream target gene TWIST1.

BACKGROUND: Associated with recent achievements in therapy for advanced lung adenocarcinoma, there will still be an unmet medical need for effective treatment of stage IIIb/IV, and the prognosis of lung cancer is not optimistic till now. OBJECTIVE: In order to obtain some essential evidences for a potential targeted therapy in lung adenocarcinoma, the effects of Ovol2 gene on epithelial-to-mesenchymal transition (EMT) was observed and the probable mechanisms were analyzed. METHODS: Ovol2 expression was previously evaluated by immunochemistry in lung adenocarcinoma tissue, and Ovol2 was overexpressed by lentivirus infection in A549 cells. Subsequently, the migration and invasion ability of A549 cells was tested by Transwell and Wound healing experiments. The mRNA level of genes correlated to EMT was detected by Real-time PCR, and the expression of reasonable makers was probed by Western Blot. Finally, rescue experiment, Luciferase assay, and chromatin immunoprecipitation assay were performed to explore the probable mechanisms. RESULTS: After treated with Ovol2 overexpression, the expression level of E-cadherin was increased, while the expression level of Vimentin and Twist1 was declined not only in the mRNA level but also in the protein level. Moreover, we found that Ovol2 represses transcription of Twist1 by binding to its promoter directly. Wound healing and Transwell assays indicate that the migration and invasion ability were downregulated by Ovol2 in A549 cells. CONCLUSION: Ovol2 can suppress migration and invasion ability of A549 cells, and prevent EMT by inhibition of Twist1 transcription directly.CI - Copyright (c) 2016. Published by Elsevier B.V.

Platelet-derived growth factor-D (PDGF-D) plays a crucial role in the progression of several cancers. However, its role in colorectal cancer (CRC) remains unclear. Our study showed that PDGF-D was highly expressed in CRC tissues and was positively associated with the clinicopathological features. Down-regulation of PDGF-D inhibited the tumor growth, migration and angiogenesis of SW480 cells in vitro and in vivo. Whereas up-regulation of PDGF-D promoted the malignant behaviors of HCT116 cells. Moreover, PDGF-D up-regulated the expression of Notch1 and Twist1 in CRC cells. In addition, PDGF-D expression promoted epithelial to mesenchymal transition (EMT), which was accompanied with decreased E-cadherin and increased Vimentin expression. Consistently, PDGF-D, Notch1, and Twist1 are obviously up-regulated in transforming growth factor-beta 1 (TGF-beta1) treated HCT116 cells. Since Notch1 and Twist1 play an important role in EMT and tumor progression, we examined whether there is a correlation between Notch1 and Twist1 in EMT status. Our results showed that up-regulation of Notch1 was able to rescue the effects of PDGF-D down-regulation on Twist1 expression in SW480 cells, whereas down-regulation of Notch1 reduced Twist1 expression in HCT116 cells. Furthermore, we found that Twist1 promoted EMT and aggressiveness of CRC cells. These results suggest that PDGF-D promotes tumor growth and aggressiveness of CRC, moreover, down-regulation of PDGF-D inactivates Notch1/Twist1 axis, which could reverse EMT and prevent CRC progression.

The acquisition of radioresistance by esophageal squamous carcinoma (ESC) cells during radiotherapy may lead to cancer recurrence and poor survival. Previous studies have demonstrated that ionizing radiation (IR) induces epithelial-mesenchymal transition (EMT) of ESC cells accompanied by increased migration, invasion, and radioresistance. However, the underlying molecular mechanisms of IR-induced EMT and radioresistance are not well established, hampering the development of potential solutions. To address this issue, we investigated the role of the IL-6/STAT3/TWIST signaling pathway in IR-induced EMT. We found not only the pathway was activated during IR-induced EMT but also STAT3 inhibition or Twist depletion reversed the EMT process and attenuated radioresistance. These results improve our understanding of the underlying mechanisms involved in IR-induced EMT and suggest potential interventions to prevent EMT-induced acquisition of radioresistance.

MicroRNA-361-5p (miR-361-5p) has been reported to be dysregulated in various human cancer types. However, the function of miR-361-5p in glioma remains unknown. In the present study, we aimed to investigate the biological functions of miR-361-5p in regulating glioma progression and the underlying molecular mechanism. We found that miR-361-5p was significantly decreased in glioma tissues and cell lines as detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Functional analysis revealed that miR-361-5p overexpression significantly inhibited glioma cell migration, invasion and epithelial-mesenchymal transition (EMT) whereas suppression of miR-361-5p showed opposite effects. Bioinformatic analysis showed that Twist1, a critical EMT inducer, was a predicted target of miR-361-5p which was validated by dual-luciferase reporter assay, RT-qPCR and western blot analysis. Further analysis indicated that miR-361-5p regulates the Twist1/Bmi-1 signaling axis. Rescue experiments showed that restoration of Twist1 expression significantly reversed the suppressive effect of miR-361-5p on cell migration, invasion and EMT. Taken together, the present study demonstrated an important role of miR-361-5p in glioma - which regulated the EMT of glioma cells by targeting and regulating Twist1. These findings provide novel insight into understanding the role and mechanism of miR-361-5p in regulating the biolo-gical behavior of glioma cells and suggest that miR-361-5p is a novel potential therapeutic target for glioma.

The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.

microRNA-489 (miR-489) is a novel cancer-related miRNAs and functions as a tumor suppressor in human cancers. While, the clinical significance of miR-489 and its role in colorectal cancer (CRC) remain rarely known. Here, we found that the levels of miR-489 in CRC tissues were significantly lower than those in matched tumor-adjacent tissues. Furthermore, decreased levels of miR-489 also observed in CRC cell lines compared to HIEC cells. Clinicopathological analysis revealed that miR-489 underexpression was positively correlated with advanced pT stage, pN stage and AJCC stage. Moreover, miR-489 low expressing CRC patients showed a obvious shorter survival. Functionally, miR-489 restoration inhibited cell migration and invasion as well as epithelial-mesenchymal transition (EMT) in HCT116 cells, while miR-489 loss facilitated these cellular processes in SW480 cells. In vivo experiments revealed that miR-489 overexpression reduced the number of metastatic nodules in nude mice liver. Notably, TWIST1 was recognized as a direct downstream target of miR-489 in CRC cells. Interestingly, TWIST1 restoration abrogated the effects of miR-489 on CRC cells with enhanced cell migration, invasion and EMT process. Furthermore, overexpression of long noncoding RNA cardiac hypertrophy-related factor (lncRNA CHRF) was inversely correlated with miR-489 expression in CRC tissues. CHRF knockdown increased the expression of miR-489 and suppressed EMT events of HCT116 cells, while CHRF overexpression showed opposite effects on miR-489 expression and EMT in SW480 cells. Taken together, this work support the first evidence that lncRNA CHRF-induced miR-489 loss facilitates metastasis and EMT process of CRC cells probably via TWIST1/EMT signaling pathway.

Inherent and acquired chemoresistance reduce the effectiveness of irinotecan in the treatment of metastatic colorectal cancer (CRC). However, the molecular mechanisms underlying this resistance process are still unclear. Twist1 is one of the master transcription factors of epithelial-mesenchymal transition (EMT). Our previous study indicated that Twist1 is overexpressed in colon cancer tissues, and demonstrated that Twist1 plays a crucial role in the chemoresistance of CRC. In the present study, we further investigated how Twist1 contribute to acquired resistance to irinotecan in colon cancer. The irinotecan-resistant cells were established by gradual adaptation of increasing irinotecan concentrations in LoVo cells, named LoVo/CPT-11R cells. Results showed that cell viabilities to different anticancer drugs were markedly increased in LoVo/CPT-11R cells compared to LoVo cells. Moreover, LoVo/CPT-11R cells displayed EMT, CSC-like cellular morphology and relative biomarkers were also significantly increased. In addition, overexpressed Twist1 LoVo cells were established by lentivirus transfection assay, named LoVo/Twist1 cells. Results showed that the LoVo/Twist1 cells perform a distinctly decreased sensitivity to irinotecan, downregulated expression of E-cadherin, upregulated expression of cluster of differentiation 44 (CD44), and a significant enhancement of invasion and migration potential by regulation of MMP2 compared with control cells. In contrast, the inhibition of Twist1 transfected with siRNA could enhance the irinotecan sensitivity in LoVo/CPT-11R cells and downregulate the expression of vimentin and CD44. Our data provide evidence that EMT and CSC-like phenotype induced by Twist1 contribute to acquire resistance to irinotecan and enhanced migration and invasion in colon cancer.

MicroRNAs (miRs) play key roles in cancers, yet the potential molecular mechanisms of miR-539 on esophageal squamous cell carcinoma (ESCC) are not well understood. Utilizing informatics screening, Twist-related protein 1 (TWIST1) was hypothesized to be a possible target gene of miR-539. Since the melanoma-associated antigen (MAGE) A4 is reported to be upregulated by TWIST1, this study aimed to examine the biological functions and mechanism involving TWIST1 and MAGE4 of miR-539 in ESCC. miR-539 mimics or scrambled miRs were transfected into human ESCC TE3 cells to interfere with the expression of miR-539. Then qRT-PCR and Western blot analyses were performed to determine the expression levels of epithelial-mesenchymal transition (EMT)-related factors at mRNA and protein levels. The association between miR-539 and TWIST1 as well as TWIST1 and MAGEA4 was evaluated. The connection of miR-539 and TWIST1-MAGEA4 during the EMT progress of ESCC was also explored. Our data demonstrated that miR-539 inhibited the EMT of TE3 cells by downregulating TWIST1, and TWIST1 was a target of miR-539. Moreover, MAGEA4 was positively correlated with TWIST1, and its knockdown inhibited EMT in TE3 cells. Collectively, miR-539 could inhibit EMT in TE3 cells through TWIST1-mediated regulation of MAGEA4. All these findings suggested that miR-539 may be involved in the progression of ESCC and could be a new therapeutic target for this disease.

In this study, we investigated the underlying mechanism of the phosphoinositide 3-kinase/Akt- and Wnt/beta-catenin-mediated promotion of epithelial-to-mesenchymal transition by epigenetic regulation of histone acetylation in gastric cancer. First, we used immunohistochemistry to detect the expression of phosphorylated Akt, phosphorylated glycogen synthase kinase 3 beta, and beta-catenin in gastric cancer tissues and adjacent tissues. In addition, we confirmed that the phosphoinositide 3-kinase/Akt and Wnt/beta-catenin signaling pathways were correlated with tumorigenesis, progression, and maintenance of gastric cancer using the phosphoinositide 3-kinase inhibitor LY294002 and an inhibitor of the beta-catenin/TCF4 complex, FH535. epithelial-to-mesenchymal transition-related gene expression was measured by western blotting and quantitative real-time polymerase chain reaction assays. Furthermore, we detected the acetylation of histone H3 lysine 4 and lysine 27 using the FH535 and LY294002 inhibitors at different concentrations for 24 and 48 h. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction was performed to detect the specific binding of H3K27ac to the promoter of the epithelial-to-mesenchymal transition-related factor, Twist. Taken together, abnormal activation of the phosphoinositide 3-kinase/Akt and Wnt/beta-catenin signaling pathway was correlated with the gastric cancer progression and contributed to epithelial-to-mesenchymal transition regulation by controlling histone acetylation.

In this study, we aimed to explore new downstream effectors of TWIST1/2 in inducing epithelial-to-mesenchymal transition in gastric cancer. Bioinformatic data mining was performed using data in The cancer Genome Atlas Stomach Adenocarcinoma. Survival curves were generated using Kaplan-Meier plotter. Gastric cancer cell lines (AGS and SGC-7901) were used as in vitro cell model to investigate the regulative effect of TWIST1/2 on epithelial membrane protein 3 expression and the progression of epithelial-to-mesenchymal transition. Results showed that TWIST1 and TWIST2 are usually co-upregulated in patients with primary gastric cancer. High TWIST1 expression is associated with worse overall survival (hazard ratio = 1.26; 95% confidence interval = 1.06-1.49; p = 0.007) and also worse first progression-free survival (hazard ratio = 1.47; 95% confidence interval = 1.18-1.82; p < 0.0001). Similarly, high TWIST2 expression is associated with unfavorable overall survival (hazard ratio = 1.71; 95% confidence interval = 1.32-2.22; p < 0.0001) and progression-free survival (hazard ratio = 1.99; 95% confidence interval = 1.45-2.72; p < 0.0001). epithelial membrane protein 3 is negatively correlated to CDH1 expression (Pearson's r = -0.46) but is positively correlated to VIM expression (Pearson's r = 0.83). Knockdown of epithelial membrane protein 3 significantly increased E-cadherin but significantly decreased Vimentin expression in AGS cells. Gastric cancer patients with metastasis have significantly higher epithelial membrane protein 3 expression than the cases without metastasis. In addition, high epithelial membrane protein 3 expression is associated with worse overall survival (hazard ratio = 2.59; 95% confidence interval = 2.06-3.26; p < 0.0001) and also worse progression-free survival (hazard ratio = 2.21; 95% confidence interval = 1.78-2.74; p < 0.0001). In conclusion, epithelial membrane protein 3 is a downstream effector of TWIST1/2 in inducing epithelial-to-mesenchymal transition in gastric cancer. epithelial membrane protein 3 upregulation might be associated with gastric cancer metastasis and is a potential indicator of unfavorable overall survival and progression-free survival in gastric cancer patients.

Benzo(a)pyrene (BaP), an important toxic component of cigarette smoke, can cause lung cancer and lead to the progression of lung cancer. In the present study, we investigated the effect of BaP on the migration of lung adenocarcinoma A549 cells. BaP (1 microM) promoted the migration of A549 cells in a time-dependent manner and upregulated the expression of the Twist family BHLH transcription factor 1 (Twist1). BaP also induced upregulation of the mesenchymal markers N-cadherin and vimentin and downregulation of the epithelial marker E-cadherin. When the expression of Twist1 was knocked down in A549 cells that were treated with BaP for 4 weeks (A549BaP-4w), the expression of Twist1 decreased, which inhibited the migration capacity of A549BaP-4w cells, the expression of N-cadherin and vimentin was downregulated and the expression of E-cadherin was upregulated. In addition, morphological observations of A549BaP-4w cells revealed that the epithelial characteristics of A549 cells became mesenchymal characteristics. When the expression of Twist1 was knocked down, the A549BaP-4w cells were transformed back to cells with epithelial characteristics. In conclusion, the results from the present study indicate that BaP enhances the epithelial-mesenchymal transition-associated migration of lung adenocarcinoma A549 cells by upregulating Twist1.

In cancer cells, intracellular Ca(2+) homeostasis is altered, and this is involved in tumor initiation, progression, and metastasis. However, little is known about the underlying mechanisms. Here, we report that transient receptor potential channel 5 (TrpC5), a receptor-activated non-selective Ca(2+) channel, is correlated with tumor metastasis in colon cancer patients. Moreover, in colon cancer cells, overexpression of TrpC5 caused a robust rise in the concentration of ([Ca(2+)]i), decreased E-cadherin, and increased mesenchymal biomarker expression, then promoted cell migration, invasion, and proliferation. Interestingly, we found that TrpC5 mediated hypoxia-inducible factor 1alpha (HIF-1alpha) expression, activating Twist to promote the epithelial-mesenchymal transition (EMT). Notably, patients with high expression of TrpC5 displayed poorer overall and metastasis-free survival. Taken together, our findings demonstrate that TrpC5 induces the EMT through the HIF-1alpha-Twist signaling pathway to promote tumor metastasis in colon cancer.CI - (c) 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Esophageal cancer is a common gastrointestinal cancer, with a very high mortality rate in patients with metastasis. Swainsonine, a cytotoxic fungal alkaloid, has been shown to inhibit cell growth in esophageal cancer. In the present study, we explored the effects of swainsonine on cell invasion and metastasis in esophageal cancer cells. Human esophageal carcinoma cells were treated with different doses of swainsonine, and then cell viability, invasion, and apoptosis were measured. The mRNA and protein expressions of Twist1, apoptosis- and EMT-related factors, and PI3K/AKT pathway factors were detected by qRT-PCR and Western blot. Swainsonine had no effect on esophageal cancer cell viability and apoptosis, but it significantly decreased cell invasion in a dose-dependent manner. Swainsonine increased the expression of E-cadherin but decreased the expression of N-cadherin, vimentin, ZEB1, and snail in a dose-dependent manner, thereby inhibiting EMT. Last, we found that swainsonine inhibits cell invasion and EMT in the esophageal carcinoma cells by downregulation of Twist1 and deactivation of the PI3K/AKT signaling pathway.

BACKGROUND/AIM: The transcription factors Twist, Snail, Slug, ZEB1 and ZEB2 regulate epithelial-mesenchymal transition (EMT) and their expression has been associated with a poor prognosis in several cancer entities. The aim of this analysis was to investigate in parallel the expression of all of these transcription factors in head and neck squamous cell carcinomas (HNSCCs) in order to gain insight into their possible co-expression. MATERIALS AND METHODS: tumor tissue samples were immunohistochemically stained using antibodies against these transcription factors. The staining intensity and cellular distribution of the immunoreactivity was recorded. RESULTS: In general, transcription factor immunoreactivity was noted in the nucleus of both cancer and stromal cells. The highest immunoreactivity was observed for Twist. Snail, Slug, ZEB1 and ZEB2 showed a much lesser immunoreactivity in cancer cells and they were expressed independently from each other. CONCLUSION: Twist is the major transcription factor active in HNSCC; the other transcription factors of EMT seem to be of less importance in this tumor entity.CI - Copyright(c) 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Messenchymal to epithelial transition (MET) is a significant physiological phenomenon involved in embryogenesis and cancer. This study aims at investigating the mechanism of microRNA-20a (miR-20a) mediated regulation of mesenchymal to epithelial transition and identification of its direct target genes in breast cancer cell-line, MDA-MB-231. Reduced migratory and invasive property, altered cellular morphology along with reduced capability for attachment to basement membrane was acquired by over-expression of miR-20a in invasive MDA-MB-231 cell-line initially expressing low level of this micro-RNA, indicating direct correlation between abundance of miR-20a and metastatic property. The switch from mesenchymal to epithelial cells mediated by miR-20a involved post-transcriptional down-regulation of twist1, which in turn controls downstream epithelial markers like E-cadherin, claudin and mesenchymal markers like N-cadherin, fibronectin, the crucial players of mesenchymal to epithelial transition (MET). Furthermore, another key component, TGF-beta and one of its receptors (TGFBR2) were found to be down-regulated by miR-20a. Additionally, reporter assay established that post-transcriptional down-regulation of TGFBR2 occurred through direct binding of miR-20a to its 3'UTR, thus abrogating the TGF-beta signaling pathway resulting in inhibition of MET. Delineating the underlying molecular mechanism of miR-20a-mediated MET and defining the target genes will help us to introduce a miRNA-mediated effective therapeutic strategy against breast cancer.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

OBJECTIVE: To investigate the relationship between tumor-associated macrophages (TAMs), neovascularization, and tumor cell migration in oral squamous cell carcinoma (OSCC) of an African subpopulation. MATERIALS AND METHODS: Twenty OSCC paraffin blocks underwent immunohistochemistry to TAM1 (CCR7), TAM2 (CD206), Twist, E-cadherin, N-cadherin, and CD34. The relative percentage of CCR7 + and CD206 + cells to overall immune cell population was calculated for three high power fields and an average was taken. TAM-related microvessel density (MVD) was determined as the mean of the three recorded values. Cases that had no CD34 + vessels adjacent to the TAMs region were regarded as having an MVD score of 0. RESULTS: Ten cases (50%) expressed greater CCR7 activity than CD206, seven cases (35%) expressed approximately equal activity of CCR7 and CD206, while three cases (15%) expressed greater activity of CD206 than CCR7. Twist expression was strong in some cases with strong N-cadherin and weak E-cadherin, but the expression of Twist was not consistently high in all cases that expressed strong N-cadherin and weak E-cadherin. CONCLUSIONS: TAMs distribution suggested antitumor activity and the potential for tumor metastasis was only partly due to Twist-mediated epithelial-mesenchymal transition.

The present study investigated the role of the Twist gene in epithelial-mesenchymal transition (EMT) and its effects on the invasion and metastasis of malignant tumors. In vitro, we transfected SW480, HCT116 and HT29 cells with recombinant plasmids, pTracer-CMV/BSD-Twist and pGenesil1.2-Twist-shRNA, to influence expression of Twist. The transfection efficacy of the plasmids in the cell lines was confirmed by flow cytometry. The relative mRNA and protein expression levels of Twist, E-cadherin and vimentin in the transfected cells were detected by RT-PCR and western blotting, respectively. In addition, migration and invasion were assessed by Transwell assays. In vivo, we established a xenogenic liver metastasis mouse model by intrasplenic injection with transfected SW480, HCT116 or HT29 human colon cancer cells and used hematoxylin and eosin (H&E) staining to demonstrate the effective establishment of the model. The relative mRNA levels of Twist and vimentin were detected by RT-PCR. In vitro, RT-PCR and western blotting showed higher relative mRNA and protein expression levels of Twist and vimentin in cell lines transfected with the recombinant, highly expressed Twist plasmid than in non-transfected cell lines (P<0.05), while E-cadherin was inhibited (P<0.05). After transfection with the plasmid pGenesil1.2-Twist-shRNA, the relative mRNA and protein levels of Twist and vimentin were markedly inhibited in the HCT116 cells (P<0.05), and the levels of E-cadherin were not changed (P>0.05), along with inhibition of the migration and invasion abilities of the cell line (P<0.01). In vivo, relative mRNA levels of Twist and vimentin in both the liver and spleen of the mouse model were higher in the groups that were injected with one of the three cell lines transfected with pTracer-CMV/BSD-Twist than in the groups injected with cells transfected with pGenesil1.2-Twist-shRNA (P<0.05). In conclusion, upregulation of Twist gene expression can promote EMT molecular events. Interfering with the Twist gene can effectively silence Twist gene expression in HCT116 cells and consequently inhibit colon cancer cell migration and invasion.

The epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Previous work reported that the transcription factors Twist plays an important role in hypoxia-induced EMT and renal fibrosis. Recent evidence indicates that miR-214 was regulated by Twist in many fibrotic diseases, but their role in hypoxia-induced EMT and renal fibrosis remains unknown. Here, we found that hypoxia significantly upregulated the expression of miR-214-3p in HK-2cells, unilateral ureteral obstruction (UUO) nephropathy and patients with chronic kidney disease. Knockdown of miR-214-3p reversed the EMT of renal tubular epithelial cells (TECs) and alleviated fibrosis in the UUO mouse in vivo, while the overexpression of miR-214-3p promoted EMT phenotype and expression of fibrotic factors in TECs under hypoxic condition. In addition, Twist was also observed increased gradually with the prolongation of hypoxia, and it positively correlated with the expression of miR-214-3p in HK-2cells transfected with Twist-overexpression or Twist-siRNA plasmid. Moreover, miR-214-3p negatively regulated the expression of epithelial cadherin (E-cadherin) by binding the E-cadherin 3' UTR under hypoxic condition. Overall, hypoxia-induced activation of Twist/miR-214/E-cadherin axis is involved in the EMT of TECs, and anti-miR-214 may be an attractive strategy to ameliorate the progression of renal fibrosis.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

The pathogenesis and the underlying mechanism of endothelial-mesenchymal transition in prostate cancer remain unclear. Plasmacytoma variant translocation 1 (PVT1), a novel long non-coding RNA maps to 8q24.21, and in many tumor studies the up-regulation of PVT1 has already been reported. PVT1 is closely related to tumor cell proliferation, invasion, and metastasis. In this study, we employed a combination of techniques to study the role of PVT1 in prostate cancer, which included bioinformatic analysis, Western blotting and cell migration assays of prostate cancer cell lines. We report that PVT1 promotes prostate cancer invasion and metastasis by modulating EMT. Furthermore, PVT1 can promote EMT by up-regulation of Twist1, a transcription factor associated with EMT. We then confirmed that PVT1 acts as a sponge for miRNA-186-5p and positively regulates Twist1 by a sponge effect. Therefore, this study has revealed a novel MECHANISM for the promotion of EMT in prostate cancer by PVT1. Our findings suggest that the PVT1/miR-186/Twist1 regulatory axis may be a new therapeutic target for prostate cancer.CI - Copyright (c) 2018. Published by Elsevier B.V.

Breast cancer is a heterogeneous disease, and triple-negative breast cancer (TNBC) continues to be a serious health problem. The potential involvement of lncRNAs in TNBC progression remains unexplored. Here, we demonstrated that LINC01638 is highly expressed in TNBC tissues and cells. LINC01638 maintains the mesenchymal traits of TNBC cells, including an enriched epithelial-mesenchymal transition (EMT) signature and cancer stem cell-like state. LINC01638 knockdown suppresses tumor proliferation and metastasis both in vitro and in vivo. LINC01638 overexpression predicts a poor outcome of breast cancer patients. Mechanistically, LINC01638 interacts with c-Myc to prevent SPOP-mediated c-Myc ubiquitination and degradation. C-Myc transcriptionally enhances MTDH (metadherin) expression and subsequently activates Twist1 expression to induce EMT. Our findings describe LINC01638-mediated signal transduction and highlight the crucial role of LINC01638 in TNBC progression.

Metastasis is the main cause of cancer mortality. However, the triggering mechanisms and regulation of epithelial-mesenchymal transition (EMT) factors in the commitment of metastasis have not been well characterized. Spermatogenic Zip 1 (SPZ1) acts as a proto-oncogene and an upstream regulator of EMT during tumorigenesis. Here we report that the HIV-1 Tat-interacting protein 60 kDa (Tip60) acetyltransferase mediates acetylation at lysine residues of SPZ1 at positions 369 and 374, and of TWIST1 at positions 73 and 76, which are required for SPZ1-TWIST1 complex formation and cancer cell migration in vitro and in vivo. Ectopic SPZ1 and TWIST1 expression, but not that of TWIST1 alone, enhanced vascular endothelial growth factor (VEGF) expression via the recruitment of bromodomain-containing protein 4 (BRD4), thus enhancing RNA-Pol II-dependent transcription and inducing metastasis. Neutralization of VEGF using humanized monoclonal antibodies such as Avastin, effectively abrogated the EMT and oncogenesis induced by the acetylated SPZ1-TWIST1 complex. Our findings highlight the importance of acetylation signaling in the SPZ1-TWIST1-BRD4 axis in the mediation of EMT and its regulation during tumor initiation and metastasis.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

Epsin 3 (EPN3) expression is limited to gastric parietal cells and wounded or pathological tissue rather than normal brain tissue, and although it has been identified as an oncogene in estrogen receptorpositive breast cancer and nonsmall cell lung cancer, its function in cancer is poorly understood. The present study aimed to investigate the association of EPN3 expression with the clinicopathological features of patients with glioma, as well as the effects of EPN3 on glioblastoma cells and the potential molecular mechanisms for its effects on glioblastoma cell behavior. EPN3 expression was assessed by immunohistochemistry in tissue samples from 167 patients with glioma, as well as by western blotting in 5 glioblastoma cell lines. The U87 and U251 glioblastoma cell lines were used to investigate the effects of EPN3 on glioblastoma cell invasion and migration through gain and loss of EPN3 expression experiments; expression levels were further investigated by reverse transcriptionquantitative polymerase chain reaction (RTqPCR) and western blot analyses. The results demonstrated that EPN3 expression levels were upregulated in highgrade glioma tissues compared with lowgrade tissues, and there were varying expression levels of EPN3 in the five glioblastoma cell lines. No significant differences were observed in EPN3 expression in relation to patient age, sex or tumor size. Overexpression of EPN3 promoted glioblastoma cell migration and invasion, which we hypothesized was through affecting epithelialmesenchymal transition (EMT). RTqPCR and western blotting revealed that EPN3 upregulation increased the expression of Notch1 intracellular domain, betacatenin, Slug, Twist and zincfinger Eboxbinding homeobox (ZEB)1. These results suggested that EPN3 enhances the migration and invasion of glioblastoma cells by activating the transcription factors Slug, Twist and ZEB1, but not Snail 1 or ZEB2, to induce EMT in glioma cells; EPN3 involvement in the Notch and WNT/betacatenin signaling pathways may contribute to this process.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that HCC has a poor prognosis. In the majority of cases, once metastatic, HCC is incurable. To identify an effective treatment for HCC, it is important to understand the underlying molecular mechanisms of HCCassociated occurrence, proliferation, metastasis and carcinogenesis. In the present study, the role of Upframeshift 1 (UPF1), a potential tumor suppressor, was investigated in the HCC cell lines. The expression levels of UPF1 in an HCC cell line were examined by reverse transcriptionquantitative polymerase chain reaction. The expression levels of 19 key proteins in numerous signaling pathways were detected via protein array analysis in the presence of UPF1 overexpression. The present study further investigated the effects of UPF1 expression levels on the epithelialmesenchymal transition (EMT) process by targeting Ecadherin, Ncadherin, Vimentin and Twistrelated protein 1 (Twist). The results of the present study revealed that UPF1 was significantly downregulated in an HCC cell line. The majority of the proteins exhibited upregulated expression levels in the presence of UPF1 overexpression in the HCC cell line, Huh7. Key proteins, including cluster of differentiation (CD)31 (platelet endothelial cell adhesion molecule1), Vimentin, CD44, PCNA, Ki67, NCadherin, Survivin, P53, Met and retinoblastoma exhibited a significant association with UPF1. Furthermore, western blotting indicated that the expression levels of Ncadherin, Vimentin and Twist were notably upregulated while UPF1 was overexpressed; however, Ecadherin was downregulated and opposing observations were reported with protein array analysis. In summary, Ecadherin expression levels were regulated by the manifold, and UPF1, a potential tumor suppressor, may promote the EMT process in Huh7 HCC cells. The findings of the present study suggested that UPF1 expression levels affected the EMT process by targeting Ecadherin, Ncadherin, Vimentin and Twist.

BACKGROUND/AIMS: TNF-alpha and TGF-beta associated epithelial-mesenchymal transition (EMT) occurs via NF-kappaB-dependent transcriptional upregulation of Twist1.Chrysin (ChR) is a major active ingredient ofpropolis, which inhibits various cancer cells and possesses anti-inflammatory activities. This study aimed to assess whether and how ChR inhibits proinflammatory cytokine-induced EMT phenotype and cancer stem-like cell (CSLC) features in the HeLa cell line. METHODS: HeLa cells were co-administered TNF-alpha (10.0 ng/mL) and TGF-beta (5.0 ng/mL) for 24h following TGF-beta (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0muM). Then, the levels of EMT-related factors, multi-potential transcription factors, and stem cell markers were analyzed by immunoblot. Wound healing and tumor sphere formation assays were performed to assess the migration and self-renewal capabilities of cells, respectively. Overexpression and/or knockdown of NF-kappaBp65 and/or Twsit1 were used to explore the molecular mechanisms. RESULTS: The results showed that ChR inhibited EMT and CSLC properties in HeLa cells administered TNF-alpha after prolonged TGF-beta treatment, in a concentration-dependent fashion. NF-kappaBp65 knockdown and ChR(10.0muM) cooperatively enhanced the inhibition of NF-kappaBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-kappaBp65 combined with ChR(10.0muM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-kappaBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-kappaBp65 knockdown and ChR. CONCLUSION: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-kappaB/Twist axis.CI - (c) Copyright by the Author(s). Published by Cell Physiol Biochem Press.

Hypoxia plays a pivotal role in tumor progression. The functions of hypoxia and subsequent Bcl-2/Twist1 activation in epithelial-mesenchymal transition (EMT) and vasculogenic mimicry (VM) formation are currently unclear. This study aimed to investigate the role of Bcl-2/Twist1 cooperation in hypoxia-induced EMT and VM formation. In in vitro experiments, we found that hypoxia resulted in co-overexpression of Bcl-2 and Twist1, facilitated Twist1 nuclear translocation and promoted EMT and VM formation. Co-overexpression of Bcl-2 and Twist1 under normoxia could also induce EMT and promote VM formation. Furthermore, blocking Bcl-2 or Twist1 attenuated the effects of hypoxia on EMT progress and VM formation in hepatocellular carcinoma cells. In in vivo experiments, the mechanism by which hypoxia promoted Bcl-2 and Twist1 co-overexpression and induced EMT process and VM formation was demonstrated using murine xenograft models. These results above suggest that hypoxia could activate the cooperation of Bcl-2 and Twist1, Bcl-2 plays an important role in assisting Twist1 nuclear translocation which could change the expression of a wide range of genes and lead to the induction of EMT and VM formation.CI - Copyright (c) 2015 Elsevier Inc. All rights reserved.

Twist1 is an epithelial-mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) Twist1 or tumor cell-specific Twist1 knockout (Twist1(TKO)). Twist1 knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in approximately 6% of the advanced WT tumor cells. Most of these Twist1(+) cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ERalpha and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin(+)/vimentin(+)). In advanced Twist1(TKO) tumor cells, Twist1 knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1(TKO) tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1(TKO) tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.CI - Published under the PNAS license.