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TWIST1 is an important basic helix‐loop‐helix protein linked to multiple physiological and pathological processes. Although TWIST1 is believed to be involved in vascular pathogenesis, its effects on homeostasis of smooth muscle cells (SMCs) remain poorly understood. Here, we show that TWIST1 protein levels were significantly elevated during SMC phenotypic switching in vivo and in vitro. TWIST1 overexpression promoted phenotypic switching of SMCs, while siRNA targeting of TWIST1 prevented cell transition. Mechanistically, TWIST1 decreased the level of microRNA‐143/145, which governs smooth muscle marker gene transcription. In addition, TWIST1 repressed p68 mRNA and protein expression, a crucial modulator of SMC behavior and microRNA biogenesis. Our co‐immunoprecipitation assay demonstrated a previously unrecognized molecular interaction between TWIST1 and p68 protein. Finally, we found that TWIST1 triggered SMC phenotypic switching and suppressed microRNA‐143/145 expression by promoting the proteasomal degradation of p68. These data suggest a novel role of TWIST1 in the regulation of SMC homeostasis by modulating p68/microRNA‐143/145 axis., Increased TWIST1 expression leads to the suppression of the DDX5/microRNA‐143/145 pathway in VSMC, which induces phenotypic switching of VSMC and promotes the progression of vascular remodeling. Therefore, TWIST1 might be a attractive therapeutic target for inhibiting phenotype‐related vascular disorders.

BackgroundBreast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated.MethodsWe investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and its functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for oestrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1,VIM,CD44,CD24), integrins beta 1 (ITGB1), alpha 2 (ITGA2) andILK. Integrin andILKexpression in epidermal growth factor (EGF)-induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/− EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed.ResultsThe ED03 (ER+/PR−/HER2−/lobular) and EDW01 (ER+/PR−/HER2−/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passages 4–5, corresponding with a decrease in E-cadherin. At passages 6–7,VIMwas upregulated along withITGB1andITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined.ILK,ITGB1andITGA2mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in whichCDH1was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration.ConclusionOur data suggest that despite an increase inITGA2andITGB1gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Protein arginine methyltransferase 5 (PRMT5) is an important type II arginine methyltransferase that can play roles in cancers in a highly tissue-specific manner, but its role in the carcinogenesis and metastasis of head and neck squamous cell carcinoma (HNSCC) remains unclear. Here, we detected PRMT5 expression in HNSCC tissues and performed series of in vivo and in vitro assays to investigate the function and mechanism of PRMT5 in HNSCC. We found that PRMT5 was overexpressed in dysplastic and cancer tissues, and associated with lymph node metastasis and worse patient survival. PRMT5 knockdown repressed the malignant phenotype of HNSCC cells in vitro and in vivo. PRMT5 specific inhibitor blocked the formation of precancerous lesion and HNSCC in 4NQO-induced tongue carcinogenesis model, prevented lymph node metastasis in tongue orthotopic xenograft model and inhibited cancer development in subcutaneous xenograft model and Patient-Derived tumor Xenograft (PDX) model. Mechanistically, PRMT5-catalyzed H3R2me2s promotes the enrichment of H3K4me3 in the Twist1 promoter region by recruiting WDR5, and subsequently activates the transcription of Twist1. The rescue experiments indicated that overexpressed Twist1 abrogated the inhibition of cell invasion induced by PRMT5 inhibitor. In summary, this study elucidates that PRMT5 inhibition could reduce H3K4me3-mediated Twist1 transcription and retard the carcinogenesis and metastasis of HNSCC.

MYB proto-oncogene-like 1 (MYBL1) has been reported to be a strong activator of transcription and plays an important role in the development of cancer. However, the precise biological function and molecular mechanism of MYBL1 in hepatocellular carcinoma (HCC) cells remain unclear. In the present study, we found that the expression of MYBL1 was markedly overexpressed in HCC cell lines and HCC samples, respectively. Moreover, MYBL1 expression positively correlated with tumor progression and inversely correlated with patient survival in 368 human HCC tissue samples. Overexpression of MYBL1 induced, whereas knockdown of MYBL1 reduced, HCC proliferation and metastasis both in vitro and in vivo. Furthermore, we demonstrated that HCC patients with high MYBL1 expression had significantly shorter overall and poorer disease-free survival than those with low MYBL1 expression. MYBL1 transcriptionally upregulated TWIST1 expression by directly targeting the TWIST1 promoter. More importantly, the in vitro analysis was consistent with the significant correlation between MYBL1 and TWIST1 expression observed in a large cohort of human HCC specimens. Taken together, our results demonstrate that MYBL1 plays an important role in HCC growth and metastasis and reveal a plausible mechanism for upregulation of TWIST1 in HCC., Graphical Abstract, Corresponding authors and colleagues demonstrate that MYBL1 transcriptionally upregulated TWIST1 expression by directly targeting the TWIST1 promoter and subsequently enhances growth and metastasis of hepatocellular carcinoma cells, which establish MYBL1 as a potential diagnosis biomarkers and may serve as a putative target for HCC diagnosis and therapy.

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 x 10(6) 67NR and 1 x 10(5) 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15-30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.

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

TG-interacting factor 1 (TGIF1) exerts inhibitory effects on transforming growth factor-beta (TGF-β) signaling by suppressing Smad signaling pathway at multiple levels. TGIF1 activity is important for normal embryogenesis and organogenesis, yet its dysregulation can culminate in tumorigenesis. For instance, increased expression of TGIF1 correlates with poor prognosis in triple-negative breast cancer patients, and enforced expression of TGIF1 facilitates Wnt-driven mammary tumorigenesis, suggesting that TGIF1 might function as an oncoprotein. Quite surprisingly, TGIF1 has recently been shown to function as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC), possibly owing to its ability to antagonize the pro-malignant transcription factor Twist1. In this article, we will briefly elaborate on the biological and clinical significance of the unique tumor-suppressive function of TGIF1 and its functional interaction with Twist1 in the context of PDAC pathogenesis and progression.

Pulmonary sarcomatoid carcinomas (PSCs) are a rare subtype of non‑small‑cell lung cancer and are typically biphasic neoplasms. No effective treatment for PSCs is currently available in clinical practice. The expression of the epithelial‑mesenchymal transition (EMT) transcription factors, Twist1, Slug and Snail, as well as the EMT phenotype and vasculogenic mimicry (VM) were analysed in 41 PSC and 79 pulmonary squamous carcinoma (PSCC) samples. Compared with the PSCCs, the PSCs exhibited an EMT phenotype and VM, and they also exhibited an increased expression of the Twist1, Slug, Snail and VM markers. Twist1 expression was associated with metastasis and TNM stage. Twist1‑positive patients exhibited a poorer prognosis for overall survival (OS) than those with Twist1‑negative PSCs. Transforming growth factor β1 (TGFβ1) was used to induce an EMT transition in a PSCC cell line. SK‑MES‑1 cells treated with TGFβ1 exhibited an increased expression of Twist1. The EMT phenotype, VM and increased migratory and invasive abilities were induced following TGFβ1 treatment. Importantly, in cells treated with TGFβ1, the EMT phenotype was reversed, VM marker expression was decreased, and the migratory and invasive ability of the PSCC cell line was decreased following Twist1 knockdown. Collectively, this study provides a new perspective of Twist1 in the aggressiveness of PSCs. The identification of Twist1 as an independent marker of poor prognoses may lead to the development of novel strategies for improving the treatment of patients with PSC.

Abstract Lung adenocarcinoma associated transcript 1 (LUADT1) has been reported as an oncogenic long non-coding RNA (lncRNA) in lung adenocarcinoma, while its roles in small cell lung cancer (SCLC) are unknown. Our RNA interaction bioinformatics prediction showed that LUADT1 could form strong base pairing with miR-15a-3p, which is a tumor-suppressive miRNA that can target Twist1. We found that LUADT1 and Twist1 were upregulated in SCLC, while miR-15a-3p was downregulated in SCLC. However, LUADT1 was posively correlated with Twist1 but was not significnatly correlated with miR-15a-3p. Overexpression experiments showed that and LUADT1 and miR-15a-3p did not significantly affect the expression of each other. Moreover, LUADT1 overexpression mediated the upregualtion of Twist1, and miR-15a-3p overexpression played an oppsoite role. Transwell assays showed that LUADT1 and Twist1 overexpression mediated the increased rate of cell invasion and migration, while miR-15a-3p overexpression mediated the decreased rate of cell invasion and migration. In addition, miR-15a-3p overexpression played an oppsoite role and attenuated the effects of LUADT1 overexpression. Therefore, LUADT1 may sponge miR-15a-3p to upregulate Twist1 in SCLC, thereby promoting cancer cell invasion and migration. Trial registration 2017GZH-1-201,746,382, registered at Jan 02,2017.

PurposeThe basic helix-loop-helix transcription factor (bHLH) transcription factor Twist1 plays a key role in embryonic development and tumorigenesis. p53 is a frequently mutated tumor suppressor in cancer. Both proteins play a key and significant role in breast cancer tumorigenesis. However, the regulatory mechanism and clinical significance of their co-expression in this disease remain unclear. The purpose of this study was to analyze the expression patterns of p53 and Twist1 and determine their association with patient prognosis in breast cancer. We also investigated whether their co-expression could be a potential marker for predicting patient prognosis in this disease.MethodsTwist1 and mutant p53 expression in 408 breast cancer patient samples were evaluated by immunohistochemistry. Kaplan-Meier Plotter was used to analyze the correlation between co-expression of Twist1 and wild-type or mutant p53 and prognosis for recurrence-free survival (RFS) and overall survival (OS). Univariate analysis, multivariate analysis, and nomograms were used to explore the independent prognostic factors in disease-free survival (DFS) and OS in this cohort.ResultsOf the 408 patients enrolled, 237 (58%) had high mutant p53 expression. Two-hundred twenty patients (53.9%) stained positive for Twist1, and 188 cases were Twist1-negative. Furthermore, patients that co-expressed Twist1 and mutant p53 (T+P+) had significantly advanced-stage breast cancer [stage III, 61/89 T+P+ (68.5%) vs. 28/89 T-P- (31.5%); stage II, 63/104 T+P+ (60.6%)vs. 41/104 T-P- (39.4%)]. Co-expression was negatively related to early clinical stage (i.e., stages 0 and I; P = 0.039). T+P+ breast cancer patients also had worse DFS (95% CI = 1.217–7.499, P = 0.017) and OS (95% CI = 1.009–9.272, P = 0.048). Elevated Twist1 and mutant p53 expression predicted shorter RFS in basal-like patients. Univariate and multivariate analysis identified three variables (i.e., lymph node involvement, larger tumor, and T+P+) as independent prognostic factors for DFS. Lymph node involvement and T+P+ were also independent factors for OS in this cohort. The total risk scores and nomograms were reliable for predicting DFS and OS in breast cancer patients.ConclusionsOur results revealed that co-expression of mutant p53 and Twist1 was associated with advanced clinical stage, triple negative breast cancer (TNBC) subtype, distant metastasis, and shorter DFS and OS in breast cancer patients. Furthermore, lymph nodes status and co-expression of Twist1 and mutant p53 were classified as independent factors for DFS and OS in this cohort. Co-evaluation of mutant p53 and Twist1 might be an appropriate tool for predicting breast cancer patient outcome.

Background An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) play pivotal roles in cancer onset and development. LncRNA AFAP1-AS1 has been validated to be abnormally upregulated and play oncogenic roles in various malignant tumors. The biological role and mechanism of AFAP1-AS1 in OS (osteosarcoma) remains unclear. Methods Quantitative reverse transcription PCR (qRT-PCR) is applied to examine AFAP1-AS1 expression in OS tissues and OS cell lines. The function of AFAP1-AS1 in OS cells is investigated via in-vitro and in-vivo assays. Western bolt and rescue experiments are applied to detect the expression changes of key molecules including epithelial-mesenchymal transition markers and identify the underlying molecular mechanism. RNA immunoprecipitation is performed to reveal the interaction between AFAP1-AS1 and RhoC. Results AFAP1-AS1 expression is upregulated in human OS tissues and cell lines. AFAP1-AS1 knockdown induces OS cell apoptosis and cell cycle G0/G1 arrest, suppresses OS cells growth, migration, invasion, vasculogenic mimicry formation and epithelial-mesenchymal transition (EMT), and affects actin filament integrity. AFAP1-AS1 knockdown suppresses OS tumor formation and growth in nude mice. AFAP1-AS1 knockdown elicits a signaling inhibition including decreased levels of RhoC, ROCK1, p38MAPK and Twist1. Moreover, AFAP1-AS1 interacts with RhoC. Overexpression of RhoC can partly reverse AFAP1-AS1 downregulation-induced cell EMT inhibition. Conclusions AFAP1-AS1 is overexpressed in osteosarcoma and plays an oncogenic role in osteosarcoma through RhoC/ROCK1/p38MAPK/Twist1 signaling pathway, in which RhoC acts as the interaction target of AFAP1-AS1. Our findings indicated a novel molecular mechanism underlying the tumorigenesis and progression of osteosarcoma. AFAP1-AS1 could serve as a promising therapeutic target in OS treatment.

Procollagen C-proteinase enhancer protein (PCOLCE) was originally identified as an enhancer to facilitate the catalysis of procollagens by BMP1. PCOLCE participates in the reconstitution of extracellular and corneal repair. The elevation of PCOLCE in blood indicates that breast cancer has metastasized into the bones. However, direct research on PCOLCE has not been reported. Methods: ECM candidates were identified by RNA-seq analysis from 4 normal and 16 osteosarcoma tissues. The in vitro migration and invasion abilities of osteosarcoma cells were determined by a Transwell assay. A spontaneous metastatic osteosarcoma model was established to assess osteosarcoma metastasis in vivo. The N-linked glycosylated amino acids were identified by PNGase F treatment combined with Western blotting. The mechanism of TWIST1 regulating PCOLCE transcription was elucidated by luciferase, qPCR and ChIP assays. Results: PCOLCE was markedly up-regulated in human osteosarcoma tissues compared to its expression in noncancerous adjacent tissues; high PCOLCE expression in tissues correlated with a poor patient prognosis, and the knockdown of PCOLCE by shRNAs impaired the migration, invasion and lung metastasis of osteosarcoma cells. The overexpression of wild-type PCOLCE, but not its N29Q mutant, promoted migration, invasion and metastasis, indicating that the glycosylation of PCOLCE at Asn29 is necessary for its functions in osteosarcoma. TWIST1, a key transcription factor in metastasis, was also overexpressed in osteosarcoma tissues and positively correlated with either PCOLCE or its potential procollagen substrates, such as COL1A1, COL1A2, COL5A1, COL8A2 and COL10A1. Conclusion: Our findings are the first to provide evidence that PCOLCE plays a critical role in promoting the lung metastasis of osteosarcoma, and this up-regulation of PCOLCE by TWIST1 may lead to a new therapeutic strategy to treat patients with metastatic osteosarcoma.

Recently, microRNAs (miRNAs) have been reported to participate in multiple biological processes. However, the effects of miR-495 on gastric cancer (GC) remain unclear. The purpose of this study was to explore the functions of miR-495 in GC cell proliferation, metastasis, and apoptosis. SGC-7901 and BGC-823 cell lines were transfected with miR-495 mimic, miR-495 inhibitor, and negative controls (mimic control and inhibitor control). The expressions of miR-495, cell viability, migration, apoptosis, and apoptosis-related factors were examined by qRT-PCR, trypan blue staining, Transwell, flow cytometry, and Western blot, respectively. Simultaneously, key factor expression levels of EMT were detected by qRT-PCR and Western blot. The direct target of miR-495 was confirmed by dual-luciferase assay. Additionally, sh-Twist1, pc-Twist1, and corresponding controls were transfected into SGC-7901 and BGC-823 cells, and the protein levels of EMT-associated factors were detected by Western blot. miR-495 was downregulated in GC cells. miR-495 expression level was effectively overexpressed or suppressed in SGC-7901 and BGC-823 cells. Overexpression of miR-495 significantly decreased cell viability and migration, increased apoptosis, and inhibited the EMT process. Suppression of miR-495 showed contrary results. Twist1 was clarified as a target gene of miR-495, and Twist1 silencing obviously reduced the promoting effect of miR-495 suppression on these biological processes. Twist1 silencing significantly blocked the EMT process in both SGC-7901 and BGC-823 cells. miR-495 inhibited proliferation and metastasis and promoted apoptosis by targeting Twist1 in GC cells. These data indicated that miR-495 might be a novel antitumor factor of GC and provide a new method for the treatment of GC.

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

PGC1α oppositely regulates cancer metastasis in melanoma, breast, and pancreatic cancer, however, little is known about its impact on lung cancer metastasis. Transcriptome and in vivo xenograft analysis show that a decreased PGC1α correlates with the epithelial–mesenchymal transition (EMT) and lung cancer metastasis. The deletion of a single Pgc1α allele in mice promotes bone metastasis of KrasG12D-driven lung cancer. Mechanistically, PGC1α predominantly activates ID1 expression, which interferes with TCF4-TWIST1 cooperation during EMT. Bioinformatic and clinical studies have shown that PGC1α and ID1 are downregulated in lung cancer, and correlate with a poor survival rate. Our study indicates that TCF4-TWIST1-mediated EMT, which is regulated by the PGC1α-ID1 transcriptional axis, is a potential diagnostic and therapeutic target for metastatic lung cancer.

Simple Summary Intracranial meningiomas are one of the most common primary brain tumors. Although mostly benign, a small portion may exhibit aggressive and malignant characteristics leading to higher recurrence and mortality rate. Detecting the molecular profiles and genes that are involved in meningioma progression can lead to better stratification of patients and more efficient and targeted treatments. The results of this study reveal the role of main actors of the Wnt signaling pathway (β-catenin) and epithelial to mesenchymal transition (E-cadherin, N-cadherin, TWIST1, SNAIL and SLUG) in progression of these tumors, potentially bringing novel biomarkers in diagnostics and molecular targets for therapeutic interventions. Abstract Epithelial to mesenchymal transition (EMT), which is characterized by the reduced expression of E-cadherin and increased expression of N-cadherin, plays an important role in the tumor invasion and metastasis. Classical Wnt signaling pathway has a tight link with EMT and it has been shown that nuclear translocation of β-catenin can induce EMT. This research has showed that genes that are involved in cadherin switch, CDH1 and CDH2, play a role in meningioma progression. Increased N-cadherin expression in relation to E-cadherin was recorded. In meningioma, transcription factors SNAIL, SLUG, and TWIST1 demonstrated strong expression in relation to E- and N-cadherin. The expression of SNAIL and SLUG was significantly associated with higher grades (p = 0.001), indicating their role in meningioma progression. Higher grades also recorded an increased expression of total β-catenin followed by an increased expression of its active form (p = 0.000). This research brings the results of genetic and protein analyzes of important molecules that are involved in Wnt and EMT signaling pathways and reveals their role in intracranial meningioma. The results of this study offer guidelines and new markers of progression for future research and reveal new molecular targets of therapeutic interventions.

Background Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial–mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. Methods A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. Results Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). Conclusions In this study, we identified a TWIST1–ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

Background Acute kidney injury (AKI), with a high morbidity and mortality, is recognized as a risk factor for chronic kidney disease (CKD). AKI-CKD transition has been regarded as one of the most pressing unmet needs in renal diseases. Recently, studies have showed that salt inducible kinase 1 (SIK1) plays a role in epithelial-mesenchymal transition (EMT) and inflammation, which are the hallmarks of AKI-CKD transition. However, whether SIK1 is involved in AKI-CKD transition and by what mechanism it regulates AKI-CKD transition remains unknown. Methods We firstly detected the expression of SIK1 in kidney tissues of AKI patients and AKI mice by immunohistochemistry staining, and then we established Aristolochic acid (AA)-induced AKI-CKD transition model in C57BL/6 mice and HK2 cells. Subsequently, we performed immunohistochemistry staining, ELISA, real-time PCR, Western blot, immunofluorescence staining and Transwell assay to explore the role and underlying mechanism of SIK1 on AKI-CKD transition. Results The expression of SIK1 was down-regulated in AKI patients, AKI mice, AA-induced AKI-CKD transition mice, and HK2 cells. Functional analysis revealed that overexpression of SIK1 alleviated AA-induced AKI-CKD transition and HK2 cells injury in vivo and in vitro. Mechanistically, we demonstrated that SIK1 mediated AA-induced AKI-CKD transition by regulating WNT/β-catenin signaling, the canonical pathway involved in EMT, inflammation and renal fibrosis. In addition, we discovered that inhibition of WNT/β-catenin pathway and its downstream transcription factor Twist1 ameliorated HK2 cells injury, delaying the progression of AKI-CKD transition. Conclusions Our study demonstrated, for the first time, a protective role of SIK1 in AKI-CKD transition by regulating WNT/β-catenin signaling pathway and its downstream transcription factor Twist1, which will provide novel insights into the prevention and treatment AKI-CKD transition in the future.

Simple Summary There is clinical evidence that ulcerated and inflammatory cell-infiltrated oral cancer is frequently associated with early metastases. Our results from genomic screening in patients with metastatic oral cancer identified specific changes in genes that regulate macrophage chemotaxis and drive tumor progression. This opens up potential therapeutic opportunities toward personalized medicine tailored to manage patients with advanced disease. Abstract Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a leading cause of cancer-related deaths. As HCC has a high mortality rate and its incidence is increasing worldwide, understanding and treating HCC are crucial for resolving major public health concerns. In the present study, wound healing screening assays were performed using natural product libraries to identify natural chemicals that can inhibit cancer cell migration. Glaucarubinone (GCB) showed a high potential for inhibiting cell migration. The anti-cancer effects of GCB were evaluated using the HCC cell line, Huh7. GCB showed anti-cancer effects, as verified by wound healing, cell migration, invasion, colony formation, and three-dimensional spheroid invasion assays. In addition, cells treated with GCB showed suppressed matrix metalloproteinase activities. Immunoblotting analyses of intracellular signaling pathways revealed that GCB regulated the levels of Twist1, a crucial transcription factor associated with epithelial-to-mesenchymal transition, and mitogen-activated protein kinase. The invasive ability of cancer cells was found to be decreased by the regulation of Twist1 protein levels. Furthermore, GCB downregulated phosphorylation of extracellular signal-regulated kinase. These results indicate that GCB exhibits anti-metastatic properties in Huh7 cells, suggesting that it could be used to treat HCC.

HPV16-positive cancers have a better clinical outcome that their non-HPV anatomical counterparts. Furthermore, the presence of HPV16 E2 RNA predicts a better outcome for HPV16-positive tumors; the reasons for this are not known., Human papillomaviruses (HPVs) are causative agents in around 5% of all cancers, including cervical and oropharyngeal. A feature of HPV cancers is their better clinical outcome compared with non-HPV anatomical counterparts. In turn, the presence of E2 predicts a better clinical outcome in HPV-positive cancers; the reason(s) for the better outcome of E2-positive patients is not fully understood. Previously, we demonstrated that HPV16 E2 regulates host gene transcription that is relevant to the HPV16 life cycle in N/Tert-1 cells. One of the genes repressed by E2 and the entire HPV16 genome in N/Tert-1 cells is TWIST1. Here, we demonstrate that TWIST1 RNA levels are reduced in HPV-positive versus HPV-negative head and neck cancer and that E2 and HPV16 downregulate both TWIST1 RNA and protein in our N/Tert-1 model; E6/E7 cannot repress TWIST1. E2 represses the TWIST1 promoter in transient assays and is localized to the TWIST1 promoter; E2 also induces repressive epigenetic changes on the TWIST1 promoter. TWIST1 is a master transcriptional regulator of the epithelial to mesenchymal transition (EMT), and a high level of TWIST1 is a prognostic marker indicative of poor cancer outcomes. We demonstrate that TWIST1 target genes are also downregulated in E2-positive N/Tert-1 cells and that E2 promotes a failure in wound healing, a phenotype of low TWIST1 levels. We propose that the presence of E2 in HPV-positive tumors leads to TWIST1 repression and that this plays a role in the better clinical response of E2-positive HPV tumors. IMPORTANCE HPV16-positive cancers have a better clinical outcome that their non-HPV anatomical counterparts. Furthermore, the presence of HPV16 E2 RNA predicts a better outcome for HPV16-positive tumors; the reasons for this are not known. Here, we demonstrate that E2 represses expression of the TWIST1 gene; an elevated level of this gene is a marker of poor prognosis for a variety of cancers. We demonstrate that E2 directly binds to the TWIST1 promoter and actively represses transcription. TWIST1 is a master regulator promoting EMT, and here, we demonstrate that the presence of E2 reduces the ability of N/Tert-1 cells to wound heal. Overall, we propose that the E2 repression of TWIST1 may contribute to the better clinical outcome of E2-positive HPV16-positive tumors.

TWIST1 is a basic helix-loop-helix transcription factor, and one of the master Epithelial-to-Mesenchymal Transition (EMT) regulators. We show that tumor suppressor miR-145-5p controls TWIST1 expression in an immortalized prostate epithelial cell line and in a tumorigenic prostate cancer-derived cell line. Indeed, shRNA-mediated miR-145-5p silencing enhanced TWIST1 expression and induced EMT-associated malignant properties in these cells. However, we discovered that the translational inhibitory effect of miR-145-5p on TWIST1 is lost in 22Rv1, another prostate cancer cell line that intrinsically expresses high levels of the CPEB1 cytoplasmic polyadenylation element binding protein. This translational regulator typically reduces TWIST1 translation efficiency by shortening the TWIST1 mRNA polyA tail. However, our results indicate that the presence of CPEB1 also interferes with the binding of miR-145-5p to the TWIST1 mRNA 3'UTR. Mechanistically, CPEB1 binding to its first cognate site either directly hampers the access to the miR-145-5p response element or redirects the cleavage/polyadenylation machinery to an intermediate polyadenylation site, resulting in the elimination of the miR-145-5p binding site. Taken together, our data support the notion that the tumor suppressive activity of miR-145-5p on TWIST1 translation, consequently on EMT, self-renewal, and migration, depends on the CPEB1 expression status of the cancer cell. A preliminary prospective study using clinical samples suggests that reconsidering the relative status of miR-145-5p/TWIST1 and CPEB1 in the tumors of prostate cancer patients may bear prognostic value.

Objective During hair follicle regeneration, hair follicle stem cells (HFSCs) are regulated by signals from dermal papilla cells (DPCs). Previously we found that Tcf4 could promote the proliferation of DPCs. In this study, we focused on whether and how the biological properties of Tcf4-induced DPCs were regulated by Twist1. Methods Twist1 was overexpressed or knocked down in DPCs following different adenovirus or lentivirus infection. Phase-contrast microscopy was used to observe the agglutinative growth of DPCs. The CCK-8 assay was used to test the proliferation of DPCs. Western blot and qPCR experiments were used to determine the expression of HGF, IGF-1, VEGF, c-myc, survivin, and CyclinD1 in DPCs. ELISAs were used to test the growth factors secreted by DPCs. Conditional medium culture was used to detect the inductive ability of DPCs. Co-immunoprecipitation and immunofluorescence were used to test the binding of Twist1, Tcf4, and β-catenin in DPCs. Immunofluorescence was also used to test the expression of Twist1, Tcf4, and KRT15 in hair follicles. Results Twist1 induced DPC agglutinative growth and proliferation. Twist1 upregulated the expression of downstream target genes downstream of Tcf4, c-myc, survivin, in Tcf4-induced DPCs, as well as the expression and secretion of growth factors HGF, IGF-1, VEGF, which had the ability to induce hair follicle growth. The conditional medium from Twist1-treated DPCs increased the expression of KRT40 and MSX2 in HaCaT cells. Twist1 and Tcf4 co-localized in DPCs both in vitro and in vivo. Anti-Twist1 precipitated Tcf4 and β-catenin. Conclusion These results indicate that Tcf4 and Twist1 play a synergistic role in regulating the hair follicle induction ability of DPCs. Twist1 functions by forming a ternary complex with Tcf4 and β-catenin. Thus, we report new data that elucidate whether and how Twist1 regulates some biological properties of DPCs.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis invivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

Muscle cachexia is a catabolic response, usually takes place in various fatal diseases, such as sepsis, burn injury, and chronic kidney disease. Muscle cachexia is also a common co-morbidity seen in the vast majority of advanced cancer patients, often associated with low quality of life and death due to general organ dysfunction. The triggering events and underlying molecular mechanisms of muscle wasting are not yet clearly defined. Our recent study has shown that the ectopic expression of Twist1 in muscle progenitor cells is sufficient to drive muscle structural protein breakdown and attendant muscle atrophy, reminiscent of muscle cachexia. Intriguingly, muscle Twist1 expression is highly induced in cachectic muscles from several mouse models of pancreatic ductal adenocarcinoma (PDAC), raising the interesting possibility that Twist1 may mediate PDAC-driven muscle cachexia. Along these lines, both genetic and pharmacological inactivation of Twist1 function was highly significant at protecting against cancer cachexia, which translated into a significant survival benefit in the experimental PDAC animals. From a translational perspective, elevated expression of Twist1 is also detected in cancer patients with severe muscle wasting, implicating a role of Twist1 in cancer cachexia, and further providing a possible target for therapeutic attenuation of cachexia to improve cancer patient survival. In this article, we will briefly summarize how Twist1 acts as a master regulator of tumor-induced cachexia, and discuss the relevance of our findings to muscle wasting diseases in general. The mechanism of decreased muscle mass in various catabolic conditions is thought to rely on similar pathways, and, therefore, Twist1-induced cancer cachexia may benefit diverse groups of patients with clinical complications associated with loss of muscle mass and functions, beyond the expected benefits for cancer patients.

Purpose: E-cadherin is a calcium-dependent glycoprotein whose main role is cell-cell adhesion. Its transcriptional repressor TWIST1 is a basic helix-loop-helix (bHLH) protein that participates in gastrulation and formation of mesodermal tissues during embryogenesis. In adult tissues, the high expression of TWIST1 induces the epithelial-mesenchymal transition (EMT)-a process in which cells become motile and able to metastasize. In this paper, we investigated the involvement of E-cadherin and TWIST1 in the carcinogenesis of brain metastases originating from two different primary sites-breast and lung. ----- Methods: The localization and expression of E-cadherin and its transcriptional repressor TWIST1 were investigated using a DAB-labeled streptavidin-horseradish peroxidase immunohistochemical reaction and specific monoclonal antibodies against TWIST1 and E-cadherin. Image J software was used for semi-quantitative analysis while H-score served for statistical evaluations. ----- Results: Immunohistochemistry showed that the expression of E-cadherin was downregulated in 85.7% of brain metastases, while at the same time, 82.2% of them showed upregulated TWIST1. Statistical analysis confirmed a significant negative correlation between expressions of TWIST1 and E-cadherin (p = 0.001). When the brain metastases expression levels were compared to primary breast tumors in corresponding patients, E-cadherin showed higher expression in primary pairs compared to corresponding metastases. Consistent to its role, TWIST1 was downregulated in all primary tumor samples in comparison to corresponding metastases pairs (p = 0.034). ----- Conclusion: This research provides valuable data regarding molecular events involving two EMT key components that could give directions for new possibilities for brain metastases diagnosis and treatment.

Background Craniosynostosis is one of the major genetic disorders affecting 1 in 2,100–2,500 live newborn children. Environmental and genetic factors are involved in the manifestation of this disease. The suggested genetic causes of craniosynostosis are pathogenic variants in FGFR1, FGFR2, FGFR3, and TWIST1 genes. Methods In order to describe their major clinical characteristics and the presence of pathogenic variants, a sample of 36 Mexican patients with craniosynostosis diagnosed as: Crouzon (OMIM 123,500), Pfeiffer (OMIM 101,600), Apert (OMIM 101,200), Saethre‐Chotzen (OMIM 101,400), and Muenke (OMIM 602,849) was analyzed. Results In addition to craniosynostosis, most of the patients presented hypertelorism, midface hypoplasia, and abnormalities in hands and feet. To detect the pathogenic variants p.Pro252Arg FGFR1 (OMIM 136,350), p.Ser252Trp, p.Pro253Arg FGFR2 (OMIM 176,943), p.Pro250Arg, FGFR3 (OMIM 134,934), and p.Gln119Pro TWIST1 (OMIM 601,622), PCR amplification and restriction enzyme digestion were performed. Four and two patients with Apert presented the pathogenic variants p.Ser252Trp and p.Pro253Arg in FGFR2, respectively (with a frequency of 11.1% and 5.5%). The p.Pro250Arg pathogenic variant of FGFR3 was found in a patient with Muenke (with a frequency of 2.8%). The above percentages were calculated with the total number of patients. Conclusion The contribution of this work is discreet, since only 4 genes were analyzed and sample size is small. However, this strategy could be improved by sequencing the FGFR1, FGFR2, FGFR3, and TWIST1 genes, to determine different pathogenic variants. On the other hand, it would be important to include other genes, such as TCF12 (OMIM 600,480), MSX2 (OMIM 123,101), RAB23 (OMIM 606,144), and EFNB1 (OMIM 300,035), to determine their participation in craniosynostosis in the Mexican population., We describe the major clinical characteristics of Mexican patients with craniosynostosis. Analysis of FGFR1, FGFR2, FGFR3, and TWIST1 genes revealed a higher frequency of variants in Apert.

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in preterm infants, which is characterized by alveolar and vascular dysplasia and increased vascular permeability. Hyperoxia is a critical factor in the pathogenesis of BPD, hyperoxia-induced acute lung injury (HALI) model has similar pathological manifestations as human BPD, therefore, may provide insight into the pathogenesis of human BPD. Studies have shown that Twist1 regulates pulmonary vascular permeability of LPS-induced lung injury through the Ang-Tie2 pathway. However, the effect of Twist1 pathway on vascular permeability in HALI has not been reported. Methods: We randomly exposed newborn rats to the room air or hyperoxia for 14 days. Lung histopathology, immunofluorescence, vascular permeability, mRNA and protein expression was assessed on day 1,7,14. Results: Our results verified that hyperoxia caused alveolar and vascular developmental disorders and increased pulmonary vascular permeability, which was consistent with previous findings. In hyperoxia-exposed rat lungs, the expressions of Twist1, Ang1, Tie1, Tie2, and pTie2 were significantly reduced, whereas the expression of Ang2 was significantly increased. Next, we observed a significant down-regulation of the Akt/Foxo1 pathway. Conclusion: In HALI, the pulmonary microvascular permeability was increased, accompanied by changes in Twist1-Tie2 pathway which combined to Angs, and downregulation of Tie1 and Akt/Foxo1 pathway.

The epithelial-mesenchymal transition (EMT) is a vital step in osteosarcoma (OS) progression toward metastasis, but the specific molecular events governing this process are incompletely characterized, with miRNAs having increasingly been found to regulate the EMT. In this study, We assessed levels of miR-22 and its target, Twist1, via real-time PCR (qRT-PCR). We further used functional proliferation assays, measures of cell morphology, and western blotting to assess the functional relevance of miR-22 in OS and confirmed Twist1 as a miR-22 target via luciferase reporter assay. We observed a significant decrease in miR-22 levels in OS tumor samples relative to normal tissue, with such downregulating being significantly associated with tumor histological grade. When overexpressed, miR-22 impaired OS cell proliferation and EMT progression. We found Twist1 to be a direct miR-22 target, with levels of miR-22 and Twist1 mRNA being inversely correlated in patient samples. When overexpressed, miR-22 suppressed Twist1 translation and thereby attenuated the EMT in OS cells. These results clearly demonstrate that miR-22 can regulate the EMT in OS cells via targeting Twist1, thus highlighting a potentially novel pathway that can be therapeutically targeted in order to treat OS.

Progression of actinic keratosis (AK) to cutaneous squamous cell carcinoma (cSCC) is rare. Most cases of AK remain as intraepidermal lesions, owing to the suppression of the epithelial-to-mesenchymal transition (EMT). Ovo-like transcriptional repressor 1 (OVOL1) and ovo-like zinc finger 2 (OVOL2) are important modulators of EMT in some tumors, but their roles in skin tumors remain elusive. This study elucidated the roles of OVOL1/2 in AK and cSCC using 30 AK/30 cSCC clinical samples, and an A431 human SCC cell line using immunohistochemistry and molecular biological approaches. Immunohistochemically, OVOL1/2 were upregulated in AK and downregulated in cSCC. Meanwhile, EMT-related factors, vimentin and zinc finger E-box binding homeobox 1 (ZEB1) were downregulated in AK and upregulated in cSCC. Moreover, ZEB1 expression was higher in tumors in which OVOL2 expression was low. Thus, we observed an inverse association between OVOL2 and ZEB1 expression in AK and cSCC. Although knockdown of OVOL1 or OVOL2 increased the mRNA and protein levels of ZEB1, only OVOL2 knockdown increased the invasive ability of A431. In conclusion, OVOL2 inhibits ZEB1 expression and may inhibit the promotion of AK into cSCC. OVOL2/ZEB1 axis may be a potential target for preventing the development of cSCC.

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).

Metastasis is a major cause of cancer mortality. We generated an autochthonous transgenic mouse model whereby conditional expression of MYC and Twist1 enables hepatocellular carcinoma (HCC) to metastasize in >90% of mice. MYC and Twist1 cooperate and their sustained expression is required to elicit a transcriptional program associated with the activation of innate immunity, through secretion of a cytokinome that elicits recruitment and polarization of tumor associated macrophages (TAMs). Systemic treatment with Ccl2 and Il13 induced MYC-HCCs to metastasize; whereas, blockade of Ccl2 and Il13 abrogated MYC/Twist1-HCC metastasis. Further, in 33 human cancers (n = 9502) MYC and TWIST1 predict poor survival (p=4.3×10−10), CCL2/IL13 expression (p, eLife digest Cancer develops when cells in the body gain mutations that allow them to grow and divide rapidly and uncontrollably. As the disease progresses these cancer cells develop the ability to spread around the body. This process of spreading, called metastasis, is responsible for most cancer-related deaths in humans, but no current treatments target it. Mutations that increase the levels of two proteins known as MYC and TWIST1 in cells cause many human cancers. In healthy adult cells, normal levels of MYC and TWIST1 act as key regulators that switch thousands of genes on or off. TWIST1 is known to control the movement and spread of cells in the embryo. However, it is not known how MYC and TWIST1 work together to promote the metastasis of cancer cells. To address this question, Dhanasekaran, Baylot et al. used mice to investigate the roles of MYC and TWIST1 in the metastasis of cancer cells. The experiments showed that these two proteins work together to reprogram mouse cancer cells to release signal molecules known as cytokines. These molecules convert immune cells known as macrophages to a tumor-friendly state that allows cancers cells to spread around the body. Inhibiting two cytokines known as CCL2 and IL13 prevented the cancer cells from moving. Further experiments analyzed tumor samples from around 10,000 human patients with 33 different cancers. This revealed that patients that had higher levels of MYC and TWIST1 proteins in their tumors also had increased levels of CCL2 and IL13, more activated macrophages and were less likely to recover from their cancer. The findings of Dhanasekaran, Baylot et al. suggest that MYC and TWIST1 may instigate metastasis in many human cancers, and therapies targeting specific cytokines may prevent these cancers from spreading around the body. Furthermore, screening blood for the levels of cytokines may help to identify the cancer patients who would benefit from such therapies.

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) represent the major cervical cancer histotypes. Both histotypes are caused by infection with high-risk HPV (hrHPV) and are associated with deregulated microRNA expression. Histotype-dependent expression has been observed for miR-9-5p, showing increased expression in SCC and low expression in AC. Here, we studied the regulation and functionality of miR-9-5p in cervical SCCs and ACs using cervical tissue samples and hrHPV-containing cell lines. Expression and methylation analysis of cervical tissues revealed that low levels of miR-9-5p in ACs are linked to methylation of its precursor genes, particularly miR-9-1. Stratification of tissue samples and hrHPV-containing cell lines suggested that miR-9-5p depends on both histotype and hrHPV type, with higher expression in SCCs and HPV16-positive cells. MiR-9-5p promoted cell viability and anchorage independence in cervical cancer cell lines SiHa (SCC, HPV16) and CaSki (metastasized SCC, HPV16), while it played a tumor suppressive role in HeLa (AC, HPV18). TWIST1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), was established as a novel miR-9-5p target. Our results show that miR-9-5p plays a dual role in cervical cancer in a histotype- and hrHPV type-dependent manner. MiR-9-5p mediated silencing of TWIST1 suggests two distinct mechanisms towards EMT in cervical cancer.

Epigenetic regulation is important for cancer progression; however, the underlying mechanisms, particularly those involving protein acetylation, remain to be fully understood. Here, we show that p300/CBP‐associated factor (PCAF)‐dependent acetylation of the transcription factor intestine‐specific homeobox (ISX) regulates epithelial–mesenchymal transition (EMT) and promotes cancer metastasis. Mechanistically, PCAF acetylation of ISX at lysine 69 promotes the interaction with acetylated bromodomain‐containing protein 4 (BRD4) at lysine 332 in tumor cells, and the translocation of the resulting complex into the nucleus. There, it binds to promoters of EMT genes, where acetylation of histone 3 at lysines 9, 14, and 18 initiates chromatin remodeling and subsequent transcriptional activation. Ectopic ISX expression enhances EMT marker expression, including TWIST1, Snail1, and VEGF, induces cancer metastasis, but suppresses E‐cadherin expression. In lung cancer, ectopic expression of PCAF–ISX–BRD4 axis components correlates with clinical metastatic features and poor prognosis. These results suggest that the PCAF–ISX–BRD4 axis mediates EMT signaling and regulates tumor initiation and metastasis., The PCAF–ISX–BRD4 axis is an important regulator of tumor metastasis and cell plasticity. PCAF‐mediated acetylation of the transcription factor ISX promotes translocation of ISX‐BRD4 to the nucleus to activate EMT genes and to induce metastasis.

Background Tumor cells transfer into endothelial cells by epithelial–endothelial transition (EET), which is characterized by vasculagenic mimicry (VM) in morphology. VM can change tumor microcirculation, progression, and metastasis. However, the molecular mechanisms of endothelial-like transition remain unclear. EET is a subtype of epithelial–mesenchymal transition (EMT). Twist1, a transcriptional regulatory factor of EMT, is an important factor that induces EET in hepatocellular carcinoma(HCC), but the upstream signal of Twist1 is unclear. Methods Expression plasmids, Ca mobilization, and three-dimensional cultures were evaluated. Western blot assay, reporter gene assay, and immunofluorescence staining were conducted. A murine xenograft model was established. Analyses of immunohistochemistry, patient samples, and complementary DNA (cDNA) microarrays were also performed. Results This study demonstrated that protease-activated receptor-1 (PAR1) can increase the expression of endothelial markers and enhance VM formation by upregulating Twist1 both in vitro and in vivo through thrombin binding. Thrombin not only activates PAR1 but also promotes PAR1 internalization in a time-dependent manner. Clinical pathological analysis further confirms that PAR1 expression is directly correlated with the endothelial marker expression, VM formation, and metastasis and indicates poor survival rate of patients with tumors. Conclusion PAR1 promotes EET through Twist1 in HCC. Electronic supplementary material The online version of this article (10.1186/s13046-018-0858-4) contains supplementary material, which is available to authorized users.

Background Metastasis-associated in colon cancer 1 (MACC1) has been reported to promote tumor cell invasion and metastasis. Cancer stem cells and epithelial-mesenchymal transition (EMT) have also been reported to promote tumor cell proliferation, invasion, and metastasis. KiSS-1, a known suppressor of metastasis, has been reported to be down-regulated in various tumors. However, the associations of MACC1, CD44, Twist1, and KiSS-1 in colonic adenocarcinoma (CAC) invasion and metastasis remain unclear. The purpose of this study is to investigate the roles of MACC1, CD44, Twist1, and KiSS-1 in CAC invasion and metastasis and their associations with each other and with the clinicopathological characteristics of CAC patients. Methods Immunohistochemistry and multivariate analysis were carried out to explore the expression of MACC1, CD44, Twist1, and KiSS-1 in 212 whole-CAC-tissue specimens and the corresponding normal colon mucosa tissues. Demographic, clinicopathological, and follow-up data were also collected. Results The results of this study showed MACC1, CD44, and Twist1 expression to be up-regulated, and KiSS-1 expression was down-regulated in CAC tissues. Positive expression of MACC1, CD44, and Twist1 was found to be positively correlated with invasion, tumor grades, and lymph- node-metastasis (LNM) stages and tumor-node-metastasis (TNM) stages for patients with CAC. Positive expression of KiSS-1 was inversely associated with invasion, tumor size, LNM stage, and TNM stage. The KiSS-1-positive expression group had significantly more favorable OS than did the KiSS-1-negative group. Univariate analysis indicated that overexpression of MACC1, CD44, and Twists1 was negatively associated with longer overall survival (OS) time, and there was a positive relationship between KiSS-1-positive expression and OS time for patients with CAC. Multivariate Cox analysis demonstrated that overexpression of MACC1, CD44, Twist1, and low expression of KiSS-1 and LNM and TNM stages were independent predictors of prognosis in patients with CAC. Conclusions The results in this study indicated that levels of expression of MACC1, CD44, Twist1, and KiSS-1 are related to the duration of OS in patients with CAC. MACC1, CD44, Twist1, and KiSS-1 may be suitable for use as biomarkers and therapeutic targets in CAC.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial-to-mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self-renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss-of-function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW-associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.

Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM) greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion, and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, overexpression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.

It is well established that hypoxia induces epithelial‑mesenchymal transition in vitro and in vivo. However, the role of hypoxia in endothelial‑mesenchymal transition (EndMT), an important process in the pathogenesis of pulmonary hypertension, is not well‑characterized. The present study demonstrated a significant downregulation of the endothelial marker CD31 and its co‑localization with a mesenchymal marker, α‑smooth muscle actin (α‑SMA), in the intimal layer of small pulmonary arteries of rats exposed to chronic hypoxia. These results suggest a possible role of hypoxia in inducing EndMT in vivo. Consistent with these observations, pulmonary microvascular endothelial cells (PMVECs) cultured under hypoxic conditions exhibited a significant decrease in CD31 expression, alongside a marked increase in the expression of α‑SMA and two other mesenchymal markers, collagen (Col) 1A1 and Col3A1. In addition, hypoxia promoted the proliferation and migration of α‑SMA‑expressing mesenchymal‑like cells, but not of PMVECs. Of note, knockdown of hypoxia‑inducible factor 1α (HIF‑1α) effectively inhibited hypoxic induction of α‑SMA, Col1A1 and the transcription factor Twist1, while rescuing hypoxic suppression of CD31; these results suggest that HIF‑1α is essential for hypoxia‑induced EndMT and that it serves as an upstream regulator of Twist1. Mechanistically, HIF‑1α was identified to bind to the promoter of the Twist1 gene, thus activating Twist1 transcription and regulating EndMT. Collectively, the present results indicate that the HIF‑1α/Twist1 pathway has a critical role in mediating the effect of hypoxia‑induced EndMT in pulmonary arterial remodeling.

Epithelial–mesenchymal transition (EMT) is a pathological process that occurs in a variety of diseases, including organ fibrosis. Twist1, a basic helix–loop–helix transcription factor, is involved in EMT and plays significant roles in various fibrotic diseases. Suppression of the EMT process represents a promising approach for the treatment of fibrotic diseases. In this review, we discuss the roles and the underlying molecular mechanisms of Twist1 in fibrotic diseases, including those affecting kidney, lung, skin, oral submucosa and other tissues. We aim at providing new insight into the pathogenesis of various fibrotic diseases and facilitating the development of novel diagnostic and therapeutic methods for their treatment.

Background/Aim: Currently, it has been proposed that combination of 5-fluorouracil (5FU) with inhibitors of the mitogen-activated protein kinases (MAPKs) signaling pathway might enhance the efficacy of 5FU-based chemotherapy in colon cancer. Our study aimed to investigate an impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU and selected MAPK inhibitors. Materials and Methods: The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-sh TWIST1). The statistical calculation was performed with analysis of variance or Dunnett's test for comparison to control group. Paired Student's t-test was performed when two groups were analyzed. Results: Suppression of TWIST1 reduced the proliferation rate of colon cancer cells and enhanced their sensitivity to 5FU and MAPKs inhibitors. The sensitivity of HT29 cells to examined compounds was more dependent on TWIST1 expression level compared to HCT116 cells. The most noticeable effect of TWIST1 suppression on sensitivity of both colon cancer cell lines to combined treatment of 5FU and the MAPKs inhibitors was observed for inhibitors of p38α/β and JNK1-3. We also noted that the suppression of TWIST1 significantly sensitized both cell lines to combined treatment of 5FU and Rac inhibitor. Conclusions: Our observations point to TWIST1 expression level as a marker of colon cancer sensitivity to combined treatment of 5FU and MAPKs inhibitors.

// Michelle Faria 1 , Samaneh Karami 1 , Sergio Granados-Principal 2 , Prasenjit Dey 3, 4 , Akanksha Verma 5 , Dong S. Choi 6 , Olivier Elemento 5 , Tasneem Bawa-Khalfe 1 , Jenny C. Chang 6 , Anders M. Strom 1 and Jan-Ake Gustafsson 1, 7 1 University of Houston, Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, Science & Engineering Research Center, Houston, Texas, USA 2 Department of Medical Oncology, Hospital of Jaen, Jaen, Spain 3 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Institute for Computational Biomedicine, Department of Physiology and Biophysics Weill Cornell Medicine, New York, NY, USA 6 Methodist Cancer Center, Houston Methodist Hospital, Houston, Texas, USA 7 Department of BioSciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Correspondence to: Anders M. Strom, email: amstrom@uh.edu Keywords: slug; twist1; SOX2; CD133; c-Myc Received: July 22, 2017 Accepted: November 05, 2017 Published: January 10, 2018 ABSTRACT Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that two of the variants of ERβ, namely ERβ2 and ERβ5, control aggressiveness of TNBC by regulating hypoxic signaling through stabilization of HIF-1α. RNA-seq of patient derived xenografts (PDX) from TNBC shows expression of ERβ2, ERβ4 and ERβ5 variants in more than half of the samples. Furthermore, expression of ERβ4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to tumorsphere formation caused transformation and development of tumorspheres. By contrast, ERβ1, ERβ2 or ERβ5 were unable to support tumorsphere formation. We have previously shown that all variants except ERβ1 stabilize HIF-1α but only ERβ4 appears to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERβ4. We propose that ERβ variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer.

Background/Aims: Non-small-cell lung carcinoma (NSCLC) is the leading cause of cancer death, with tumor metastasis being mainly responsible for lung cancer-associated mortality. Our previous studies have found that tubulin polymerization promoting protein family member 3 (TPPP3) acted as a potential oncogene in NSCLC. Little is known about the function of TPPP3 in tumor metastasis. Methods: RT-qPCR and IHC were used to investigate the expression of TPPP3 in NSCLC tissues. CCK8 assay and transwell assay were used to measure proliferation and migration of NSCLC cells in vitro and xenograft model was performed to assess the tumor growth and metastasis in vivo. Results: In the present study, upregulation of TPPP3 was found to correlate with an increased metastasis capability of NSCLC. Ectopic expression of TPPP3 significantly enhanced cell proliferation in vitro and promoted tumor growth in vivo. Furthermore, overexpression of TPPP3 remarkably promoted NSCLC cell migration and invasion along with the upregulation of Twist1 both in vitro and in vivo. Further investigations showed that activation of STAT3 was required for TPPP3-mediated upregulation of Twist1, cell migration and invasion. A strong positive correlation between TPPP3 and Twist1 expression was identified in NSCLC tissues. Patients with low TPPP3 or low Twist1 in NSCLC tissues had a better prognosis with longer overall survival (OS) and disease-free survival (DFS). Conclusion: Overall, this study demonstrates that TPPP3 promotes the metastasis of NSCLC through the STAT3/Twist1 pathway.

Background The transcription factor TWIST1 plays an important role in the epithelial–mesenchymal transition (EMT) process and in the migration, invasion and metastasis of cancer cells. OCT4, which is a homeobox transcription factor, has an important role in the self-renewal potential of cancer cells. Our aim here is to elucidate impact of ectopic expression of TWIST1 on OCT4 gene expression in esophageal squamous cell carcinoma (ESCC). Methods The ESCC line was KYSE30. GP293T cells were transfected with purf-IRES-GFP and pGP plasmids to produce recombinant viral particles. A semi-confluent KYSE30 culture was transduced with the prepared retroviral particles. mRNA extraction and cDNA synthesis were performed from normal KYSE30 cells and those ectopically expressing TWIST1. Expressional analysis of TWIST1 and OCT4 were performed with relative comparative real-time PCR. Results Ectopic expression of TWIST1 in KYSE30 cells was related to its significant overexpression: nearly nine-fold higher in GFP-hTWIST1 KYSE-30 cells than in control GFP cells. This induced expression of TWIST1 caused significant upregulation of OCT4 in GFP-hTWIST1 KYSE-30 cells: nearly eight-fold higher. In silico analysis predicted the correlation of TWIST1 and OCT4 through ETS2. Conclusions Overexpressed TWIST1 can be correlated with upregulation of the cancer stem cell marker OCT4 and the protein may play critical regulatory role in OCT4 gene expression. Since OCT4 is involved in the self-renewal process, the results may suggest a new linkage between TWIST1 and OCT4 in the cell biology of ESCC, highlighting the probable role of TWIST1 in inducing self-renewal.

Twist1 is a master regulator of epithelial mesenchymal transition and carcinoma metastasis. Twist1 has also been associated with increased malignancy of human glioma. However, the impact of inhibiting Twist1 on tumorigenicity has not been characterized in glioma models in the context of different oncogenic transformation paradigms. Here we used an orthotopic mouse glioma model of transplanted transformed neural progenitor cells (NPCs) to demonstrate the effects of Twist1 loss of function on tumorigenicity. Decreased tumorigenicity was observed after shRNA mediated Twist knockdown in HPV E6/7 Ha-RasV12 transformed NPCs and Cre mediated Twist1 deletion in Twist1 fl/fl NPCs transformed by p53 knockdown and Ha-RasV12 expression. By contrast, Twist1 deletion had no effect on tumorigenicity of NPCs transformed by co-expression of Akt and Ha-RasV12. We demonstrated a dramatic off-target effect of Twist1 deletion with constitutive Cre expression, which was completely reversed when Twist1 deletion was achieved by transient administration of recombinant Cre protein. Together these findings demonstrate that the function of Twist1 in these models is highly dependent on specific oncogenic contexts of NPC transformation. Therefore, the driver mutational context in which Twist1 functions may need to be taken into account when evaluating mechanisms of action and developing therapeutic approaches to target Twist1 in human gliomas.

Background: Metastatic castration-resistant prostate cancer (mCRPC) is one of the main contributors to the death of prostate cancer patients. To date, the detailed molecular mechanisms underlying mCRPC are unclear. Given the crucial role of epithelial–mesenchymal transition (EMT) in cancer metastasis, we aimed to analyse the expression and function of Transforming growth factor-beta (TGF-β) signal-associated protein named Sox5 in mCRPC. Methods: The protein expression levels were analysed by western blot, immunohistochemistry and immunofluorescence. Luciferase reporter assays and chromatin immunoprecipitation were employed to validate the target of Sox5. The effect of Smad3/Sox5/Twist1 on PCa progression was investigated in vitro and in vivo. Results: Here, we found that TGF-β-induced EMT was accompanied by increased Sox5 expression. Interestingly, knockdown of Sox5 expression attenuated EMT induced by TGF-β signalling. Furthermore, we demonstrated that Smad3 could bind to the promoter of Sox5 and regulate its expression. Mechanistically, Sox5 could bind to Twist1 promoter and active Twist1, which initiated EMT. Importantly, knockdown of Sox5 in prostate cancer cells resulted in less of the mesenchymal phenotype and cell migration ability. Furthermore, targeting Sox5 could inhibit prostate cancer progression in a xenograft mouse model. In clinic, patients with high Sox5 expression were more likely to suffer from metastases, and high Sox5 expression also has a lower progression-free survival and cancer specific-survival in clinic database. Conclusions: Therefore, we propose a new mechanism in which Smad3/Sox5/Twist1 promotes EMT and contributes to PCa progression.

Metformin had exerted important inhibitory effects in multiple cancers. However, the correlation between metformin and hepatocellular carcinoma (HCC) metastasis, and the relevant mechanisms are still unclear. By quantitative proteomics analysis technique, we found metformin could suppress FGF signalling significantly. In FGF signalling basic fibroblast growth factor (bFGF) is a crucial member, it initially binds to its receptors, the complex of bFGF and receptors activate FGF signallings, and promote many cancers progressions. When treating HCC cell lines HepG2 and Huh7 with bFGF, we observed the cells exhibited epithelial mesenchymal transition (EMT) and these cells metastasis potential was enhanced dramaticlly. However, when treating with metformin and bFGF together, EMT and metastasis induced by bFGF could be inhibited in these cells. Furthermore, bFGF could activate AKT/GSK-3β signalling, sequentially decrease the interaction between GSK-3β and Twist1 and decrease ubiquitination of Twist1 leading to Twist1 degradation reducing. While metformin could repress the bFGF-mediated activation in AKT/GSK-3β signalling, inhibition on interaction between GSK-3β and Twist1, enhancement of Twist1 stability. Taken together, our findings suggested that metformin had prominent negative effects on bFGF-induced EMT and metastasis in HCC cells.

Background Twist1 is involved in tumor initiation and progression, which especially contributes to tumor invasion and metastasis. Wogonoside is the main in-vivo metabolite of wogonin, and it is also a natural product with potential treatment effects against cancer. Methods In this study, we investigated the in-vitro anti-invasion and in-vivo anti-metastasis effects of wogonoside on breast cancer cells and uncovered its underlying mechanism. Results The results showed that wogonoside could suppress the growth and metastasis of breast tumor in the orthotopic model of MDA-MB-231 cells. We found that wogonoside could reduce the overexpression of TNF-α, TRAF2 and TRAF4 in later stage of tumor, and improved tumor microenvironment. Therefore, TNF-α was utilized to induce metastases of breast cancer cell in vitro. Wogonoside could inhibit invasion and migration in TNF-α-induced MDA-MB-231, MDA-MB-435, and BT-474 cells. Mechanically, wogonoside inactivated NF-κB signaling through decreasing the protein expression of TRAF2/4, which further inhibited Twist1 expression. Consequently, wogonoside could down-regulate MMP-9, MMP-2, vimentin and CD44v6 expression in TNF-α-induced MDA-MB-231 and MDA-MB-435 cells. Then, these findings were proved in TNF-α + TGF-β1-induced MCF7 cells. Conclusions Wogonoside might be a potential therapeutic agent for the treatment of tumor metastasis in breast cancer.