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Ewing sarcomas (ES) are aggressive pediatric cancers of bone and soft tissues characterized by in frame chromosomal translocations giving rise to chimeric transcription factors, such as EWS-FLI1. An emerging strategy to block EWS-FLI1 activity is represented by the small molecule YK-4-279, which binds to EWS-FLI1 and alters its transcriptional activity. The specific effectors of the anti-oncogenic activity of YK-4-279 are still largely unknown. Herein, by performing a high-throughput screening we identify the lncRNA HULC (Highly Upregulated in Liver Cancer) as a prominent target of YK-4-279 activity in ES cells. High levels of HULC correlate with ES aggressiveness, whereas HULC depletion reduces ES cell growth. Mechanistically, we find that HULC promotes the expression of TWIST1 oncogene by sponging miR-186. Downregulation of HULC upon treatment with YK-4-279 reduces the expression of TWIST1 by unleashing miR-186 and favoring its binding to TWIST1 transcripts. Notably, high levels of miR-186 and low levels of TWIST1 correlate with better prognosis in ES patients. Our results disclose a novel oncogenic regulatory circuit mediated by HULC lncRNA that is disrupted by the small molecule YK-4-279, with promising therapeutic implications for ES treatment. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) and Twist1-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination are well established, but the involvement of long noncoding RNAs (lncRNAs) in Twist1-mediated signaling remains largely unknown. Methods: RT-qPCR and western blotting were conducted to detect the expression levels of lncRNA JPX and Twist1 in lung cancer cell lines and tissues. The impact of JPX on Twist1 expression, cell growth, invasion, apoptosis, and in vivo tumor growth were investigated in lung cancer cells by western blotting, rescue experiments, colony formation assay, flow cytometry, and xenograft animal experiment. Results: We observed that lncRNA JPX was upregulated in lung cancer metastatic tissues and was closely correlated with tumor size and an advanced stage. Functionally, JPX promoted lung cancer cell proliferation in vitro and facilitated lung tumor growth in vivo. Additionally, JPX upregulated Twist1 by competitively sponging miR-33a-5p and subsequently induced EMT and lung cancer cell invasion. Interestingly, JPX and Twist1 were coordinately upregulated in lung cancer tissues and cells. Mechanically, the JPX/miR-33a-5p/Twist1 axis participated in EMT progression by activating Wnt/β-catenin signaling. Conclusions: These findings suggest that lncRNA JPX, a mediator of Twist1 signaling, could predispose lung cancer cells to metastasis and may serve as a potential target for targeted therapy. [ABSTRACT FROM AUTHOR]

Introduction: Prostate cancer is one of the most common cancers and the second major cause of mortality in men. Different researches have shown that the overexpression of a gene called twist1 leads to initiation of metastasis process in this cancer. TWIST1 protein triggers this process through stimulating the transition pathway of cells from epithelial to mesenchymal tissue. Materials and Methods: In this study, four oligonucleotides of antisense RNA have been designed for twist1 gene, and its anti-metastatic effect was examined in two cell lines PC3 and LNCaP. The antisense oligonucleotides (ASOs) were designed as single strands with a length of 20 nucleotides and chosen from ASOs suggested by Soligo program. The ASOs were synthesized in phosphorothioated form. MTT assay was used for evaluating the ASOs cytotoxicity effect on PC3 and LNCaP cell lines. The cell lines were transfected with 500 nmol of antisense oligonucleotides using cationic polymer turbofect and incubated for 48 hours, and then, their invasive ability were measured by CytoSelect™ Cell Invasion Assay Kit. Results: The anti-invasive effect of ASOs in LNCaP and PC3 had a significant difference. This effect was more significant in LNCaP cell line as compared to PC3. The most anti- invasive effect was observed in LNCaP cell line (50%). Conclusion: According to the results, Antisense oligonucleotides were effective in decreasing the invasion ability of two cell lines PC3 and LNCaP and therfore can be considered as a good candidate for preventing the prostate cancer metastasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution. [Display omitted] • Mutually dependent binding of TWIST1 and homeodomain TFs in embryonic mesenchyme • TF co-binding drives enhancer accessibility and shared transcriptional regulation • Weak TF-TF contacts guided by DNA mediate the selectivity of cooperating partners • TWIST1, partners, and bound targets enriched for face-shape-associated SNPs Epigenomic, biochemical, structural, and human phenotypic analyses of transcription factors that regulate a composite DNA motif in the embryonic face and limb mesenchyme reveal how DNA-guided cooperative binding gives rise to specificity among members of large TF families. This cooperativity promotes the integration of cellular and positional identity programs and contributes to the evolution and individual variation of human facial shape. [ABSTRACT FROM AUTHOR]

[Display omitted] • Epigenetic modification of TWIST1 mediates foam cell formation and enhances atherosclerotic plaque vulnerability during hypertension. It is accepted that hypertension is a major, independent risk factor for atherosclerotic cardiovascular ischemic events, which are mainly attributed to the formation of unstable, vulnerable atherosclerotic lesions. But the mechanisms by which hypertension aggravates atherosclerosis (AS) through increased macrophage recruitment are unknown. It has been reported that TWIST1 can regulate the shear stress of blood flow in endothelial cells to promote the development of atherosclerosis, but the function of TWIST1 in macrophage recruitment during hypertension remains undefined. Here, the roles of TWIST1 in macrophage activation during N w -nitro-l-arginine-methyl ester (L-NAME; NO-synthase (NOS) inhibitor)-induced hypertension were investigated in ApoE−/− mice fed a high-fat diet (HFD) and RAW264.7 cells treated with oxidized low-density lipoprotein(ox-LDL). Oil Red O staining and hematoxylin and eosin staining were adopted to analyze atherosclerotic lesions and plaque instability. Chromatin immunoprecipitation (ChIP)-PCR was used to explore whether Lysine-specific histone demethylase 1A (LSD1/KDM1A) and Variegated suppressor 3–9 homolog 1 (SUV39H1) could regulate histone modification of the TWIST1 promoter. We reported that L-NAME increased the expression of TWIST1 in the aortic tissues of ApoE−/− mice fed a high-fat diet (HFD) and RAW264.7 cells treated with ox-LDL. TWIST1 accelerated the development of an unstable atherosclerotic phenotype by promoting macrophage activation, inflammatory factor secretion, macrophage polarization, and lipid phagocytosis. Moreover, we found that H3K9me2 and H3K9me3 in the TWIST1 promoter could be coregulated by LSD1 and SUV39H1, and this process was modulated by CK2α. Taken together, these results revealed that TWIST1 in macrophages is a critical factor that mediates foam cell formation and enhances atherosclerotic plaque vulnerability during hypertension, and targeting TWIST1 may be a promising new therapeutic approach for delaying the progression of AS in hypertension. [ABSTRACT FROM AUTHOR]

Abstract 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.

Background: In head and neck squamous cell carcinoma (HNSCC), invasiveness and metastasis of cancer cells contribute to make this insidious disease a major cause of mortality. Epithelial-mesenchymal transition (EMT) is generally considered to be the major mechanism of cancer metastasis. We previously demonstrated the crucial role of the EMT regulator Twist1 in HNSCC. A growing body of evidence suggests that microRNAs play essential roles in cancer progression and metastasis. The aim of this study was to investigate the role of microRNA in Twist1-mediated cancer metastasis. Materials and methods: The HNSCC cell lines FaDu, SAS, OECM1 and CAL-27, were used in this study. Quantitative RT-PCR for microRNAs and Western blot were performed on four HNSCC cell lines to study the molecular mechanisms involved. Results: Expression of the miR-29 family, including miR-29a, b, and c were increased in Twist1-overexpressing HNSCC cells. Furthermore, we discovered that SIN3A, a co-repressor of another EMT regulator Snail, is a target of miR-29s. With our results, we demonstrated that Twist1 modifies the function of gene expression through microRNA machinery. Conclusion: We herein have delineated the regulatory mechanism of the Twist1-miR29-SIN3A axis in HNSCC.

Abstract Background MEIS1 (Myeloid ecotropic viral integration site 1), as a homeobox (HOX) transcription factor, has a dual function in different types of cancer. Although numerous roles are proposed for MEIS1 in differentiation, stem cell function, gastrointestinal development and tumorigenesis, the involved molecular mechanisms are poor understood. Our aim in this study was to elucidate the functional correlation between MEIS1, as regulator of differentiation process, and the involved genes in cell differentiation in human esophageal squamous carcinoma (ESC) cell line KYSE‐30. Methods The KYSE‐30 cells were transduced using recombinant retroviral particles containing specific shRNA sequence against MEIS1 to knockdown MEIS1 gene expression. Following RNA extraction and cDNA synthesis, mRNA expression of MEIS1 and the selected genes including TWIST1, EGF, CDX2, and KRT4 was examined using relative comparative real‐time PCR. Results Retroviral transduction caused a significant underexpression of MEIS1 in GFP‐hMEIS1 compared to control GFP cells approximately 5.5‐fold. While knockdown of MEIS1 expression caused a significant decrease in EGF and TWIST1 mRNA expression, nearly ‐8‐ and ‐12‐fold respectively, it caused a significant increase in mRNA expression of differentiation markers including KRT4 and CDX2, approximately 34‐ and 1.14‐fold, correspondingly. Conclusion MEIS1 gene silencing in KYSE‐30 cells increased expression of epithelial markers and decreased expression of epithelial‐mesenchymal transition (EMT) marker TWIST1. It may highlight the role of MEIS1 in differentiation process of KYSE‐30 cells. These results may confirm that MEIS1 silencing promotes differentiation and decreases EMT capability of ESC cell line KYSE‐30.

Objective: To investigate DNA methylation of specific tumor suppressor genes in endometrial hyperplasia compared to normal endometrial tissue. File and methodology: To search for epigenetic events, methylation-specific multiplex ligation-dependent probe amplification was employed to compare the methylation status of 40 tissue samples with atypical endometrial hyperplasia, 40 tissue samples with endometrial hyperplasia without atypia, and 40 control tissue samples with a normal endometrium., Results and Conclusion: Differences in DNA methylation among the groups were found in TWIST1, GATA4, MUS81, and NTRK1 genes (TWIST1: atypical hyperplasia 67.5%, benign hyperplasia 2.5%, normal endometrium 22.5%; P < 0.00001; GATA4: atypical hyperplasia 95%, benign hyperplasia 65%, normal endometrium 22.5%; P < 0.00001; MUS81: atypical hyperplasia 57.5%, benign hyperplasia 22.5%, normal endometrium 5%; P < 0.00001; NTRK1: atypical hyperplasia 65%, benign hyperplasia 27.5%, normal endometrium 10%; P < 0.00001). Higher methylation rates were observed for the tumor suppressor genes of TWIST1, GATA4, MUS81, and NTRK1 in samples with atypical endometrial hyperplasia compared to samples with normal endometrial tissue, and higher methylation rates were found in samples with atypical endometrial hyperplasia compared to samples of benign endometrial hyperplasia. DNA methylation of TWIST1, GATA4, MUS81, and NTRK1 is involved in the pathogenesis of atypical endometrial hyperplasia.

Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Luteolin, a common dietary flavonoid found in plants, has been shown to have anti-cancer properties. However, its exact mechanisms of action in non-small cell lung cancer (NSCLC) are still not fully understood, particularly its role in regulating broader genomic networks and specific gene targets. In this study, we aimed to elucidate the role of microRNAs (miRNAs) in NSCLC treated with luteolin, using A549 cells as a model system., Materials and Methods: miRNA profiling was conducted on luteolin-treated A549 cells using Exiqon microarrays, with validation of selected miRNAs by qRT-PCR. Bioinformatic analysis identified the regulatory roles of miRNAs in biological processes and pathways following luteolin treatment. Computational algorithms were employed to identify potential target genes. A549 cells were transfected with miR-106a-5p mimic and inhibitor or their corresponding controls. The expression levels of 2 genes, twist basic helix-loop-helix transcription factor 1 (TWIST1) and matrix metallopeptidase 2 (MMP2), and cell migration were assessed., Results: miRNA profiling identified 341 miRNAs, with 18 exhibiting significantly altered expression ( P  < 0.05). Subsequent qRT-PCR analysis confirmed altered expression of 6 selected miRNAs. KEGG and GO analyses revealed significant alterations in pathways and biological processes crucial for tumor biology. TWIST1 and MMP2, which both contain conserved miR-106a-5p binding sites, exhibited an inverse correlation with the expression levels of miR-106a-5p. Dual-luciferase reporter assays confirmed TWIST1 and MMP2 as direct targets of miR-106a-5p. Luteolin treatment led to a reduction in A549 cell migration, and this reduction was further amplified by the overexpression of miR-106a-5p., Conclusion: Luteolin inhibits A549 cell migration by modulating the miRNA landscape, shedding light on its mechanisms and laying the foundation for miRNA-based therapeutic approaches for NSCLC., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Uveal melanoma (UM) is the most common primary intraocular tumor. Hepatic metastasis is the major and direct death-related reason in UM patients. Given that cancer stem-like cells (CSCs) are roots of metastasis, targeting CSCs may be a promising strategy to overcome hepatic metastasis in UM. Salinomycin, which has been identified as a selective inhibitor of CSCs in multiple types of cancer, may be an attractive agent against CSCs thereby restrain hepatic metastasis in UM. The objective of the study is to explore the antitumor activity of salinomycin against UM and clarify its underlying mechanism. Methods: UM cells were treated with salinomycin, and its effects on cell proliferation, apoptosis, migration, invasion, CSCs population, and the related signal transduction pathways were determined. The in vivo antitumor activity of salinomycin was evaluated in the NOD/SCID UM xenograft model and intrasplenic transplantation liver metastasis mouse model. Results: We found that salinomycin remarkably obviated growth and survival in UM cell lines and in a UM xenograft mouse model. Meanwhile, salinomycin significantly eliminated CSCs and efficiently hampered hepatic metastasis in UM liver metastasis mouse model. Mechanistically, Twist1 was fundamental for the salinomycin-enabled CSCs elimination and migration/invasion blockage in UM cells. Conclusions: Our findings suggest that targeting UM CSCs by salinomycin is a promising therapeutic strategy to hamper hepatic metastasis in UM. These results provide the first pre-clinical evidence for further testing of salinomycin for its antitumor efficacy in UM patients with hepatic metastasis. [ABSTRACT FROM AUTHOR]

Background: Substantive studies have described the ectopic microRNAs as a determinant of the pathogenesis of endometrial cancer (EC). miR-214-3p has been reported to be significantly downregulated in EC tissues, and its overexpression has been shown to inhibit the proliferation, migration, and invasion of EC cells. Our study sought to explore the molecular mechanism underlying the inhibitory effect of miR-214-3p on metastasis of EC cells. Methods: The expressions of miR-214-3p and TWIST1 in EC tissues and cells were detected by quantitative real-time PCR. Cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) were measured by transwell and Western blot analyses, respectively. The interaction between miR-214-3p and TWIST1 was confirmed by luciferase reporter assay. Xenograft tumor assay was performed to verify the role and underlying mechanism of miR-214-3p in EC in vivo. Results: miR-214-3p was downregulated and TWIST1 was upregulated in EC tissues and cells. miR-214-3p was negatively correlated with TWIST1 expression in EC tissues. Overexpression of miR-214-3p suppressed migration, invasion, and EMT in EC cells. TWIST1 was identified as a target of miR-214-3p in EC cells, and its overexpression significantly restored the inhibitory effects of miR-214-3p on cell migration, invasion, and EMT while its knockdown remarkably abolished miR-214-3p inhibitor-mediated promotion of progression of EC cells. Additionally, addition of miR-214-3p inhibited tumor growth by regulating EMT in vivo. Conclusion: miR-214-3p suppressed the EMT and metastasis of EC cells by targeting TWIST1, providing a novel biomarker for treatment of EC. [ABSTRACT FROM AUTHOR]

Deregulation of miR-186 and Twist1 has been identified to be involved in the progression of multiple cancers. However, the detailed molecular mechanisms underlying miR-186-involved cholangiocarcinoma (CCA) are still unknown. In this study, we found that miR-186 was downregulated in CCA tissues and cell lines, and negatively correlated with the expression of Twist1 protein. In vitro assays demonstrated that miR-186 mimics repressed cell proliferation, in vivo tumor formation, and caused cell cycle arrest. miR-186 mimics also inhibited the migration and invasion of CCLP1 and SG-231 cells. Mechanistically, the 3'-untranslated region (3'-UTR) of Twist1 mRNA is a direct target of miR-186. Further, miR-186 inhibited the expressions of Twist1, N-cadherin, vimentin, and matrix metallopeptidase 9 (MMP9) proteins, whereas it increased the expression of E-cadherin in CCLP1 and SG-231 cells. Silencing of Twist1 expression enhanced the inhibitory effects of miR- 186 on the proliferation, migration, and invasion of CCLP1 and SG-231 cells. In conclusion, miR-186 inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) through targeting Twist1 in human CCA. Thus, miR-186/Twist1 axis may benefit the development of therapies for CCA. [ABSTRACT FROM AUTHOR]

Pterygium as a complex disease shares common features with other malignant cells in its onset recurrence and especially epithelial–mesenchymal transition (EMT) transition. Although using different approaches including conjunctival autografts, amniotic membrane, radiotherapy, mitomycin C (MMC) has shown promising insights in the inhibition of pterygium recurrence, it needs to be investigated in more details in molecular pathways to present adjuvant target therapy. In this study, we aimed to evaluate the expression of and then illustrate the role of signaling pathways on EMT in pterygium. Using real‐time polymerase chain reaction, the twist‐related protein 1 (TWIST1) expression was compared in primary pterygium and normal conjunctiva. This study assessed the mRNA expression, as well as the association between the clinicopathological indices and the gene expression level. The expression level of TWIST1 was overexpressed in 36% of our cohort (n = 76). There was a significant positive correlation between recurrence with grade T, grade V and a significant negative correlation with growth activity. Our vast literature review on different signaling pathways in pterygium showed that EMT has centralization role in recurrence of this disease. Our data confirmed that EMT is important in the recurrence of pterygium samples and different signaling pathways end up activating the EMT markers. It is suggested to evaluate the environmental factors and their correlation with molecular markers to select favorable treatment for this kind of diseases. [ABSTRACT FROM AUTHOR]

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

Astrocytomas are one of the most vascularized types tumor in human cancers. Micro-vascular proliferation is an important factor for the classification of astrocytoma. Vasculogenic mimicry (VM) is a novel tumor vascular model that develops independently of endothelial cells, and serves an important role in astrocytoma. Twist family bHLH transcription factor 1 (Twist1) is able to regulate the formation of VM; thus in the present study, the expression and importance of Twist1 was studied in astrocytoma tissues. The present study confirmed that the expression of Twist1 was associated with the grade of astrocytoma. Twist1 promotes the formation of VM and the development of astrocytomas, and may also regulate the formation of VM via vascular endothelial-cadherin and matrix metalloproteinase-9. [ABSTRACT FROM AUTHOR]

Although epithelial-to-mesenchymal transition (EMT) has been described in the development of complete hydatidiform moles and the invasion of the maternal decidua by trophoblasts during normal human placentation, its implication in gestational trophoblastic neoplasm (GTN) without villi is totally unknown. We studied the immunoexpression of EMT transcription factors (TWIST1, ZEB1, ZEB2), E-cadherin, and vimentin in 18 trophoblastic tumors and pseudo-tumors. Weak nuclear TWIST1 immunostaining was seen in 5% to 10% of all trophoblastic cells, without ZEB1 and ZEB2 nuclear staining. Trophoblastic cells did not express vimentin, and the expression of E-cadherin was maintained in all cases, indicating the absence of EMT features in GTN. [ABSTRACT FROM AUTHOR]

Objective: Craniosynostosis (CS) associated genes (FGFR1-3, TWIST1, MSX2, POR, FREM1 and RAB23) were investigated in order to determine the mutation rates and establish an effective flow chart for molecular genetic diagnosis for syndromic (SCS) and non-syndromic craniosynostosis (NSCS). Material and Method: A total of 40 cases, including six prenatal cases, with normal karyotypes, and 34 parents were investigated in the Medical Genetics Department of Istanbul Medical Faculty. The clinical diagnosis was NSCS in 9, Pfeiffer in 9 (PS), Crouzon in 6 (CRS), Apert in 5 (AS), Saethre-Chotzen in 7 (SaCS) and Muenke/ Saethre Chotzen in 4 (MUS/SaCS) of the cases. According to the clinical diagnosis, the hot spot mutation sites of genes/exons were screened initially and the whole gene and other genes were progressively examined by Sanger sequencing. The Multiplex Ligation-Depended Probe Amplification (MLPA) technique was applied to detect deletions/duplications. Results: Molecular results were achieved in 50% of cases by sequencing and in 2.5% by MLPA. Molecular diagnosis rate was 64.5% in SCSs and 11.1% in NSCSs. Conclusion: Molecular diagnosis rate was higher in the SCS than in the NSCS group. Including exons 12 and 13 to target exons (3, 5, 11, 14-17) of FGFR2 gene increases the mutation rate by 33% in the second step of the molecular investigation in PS cases. Genetic counseling with the families following molecular diagnosis is important. Our results supported the fact that CS cases with un-identified pathogenic variants in the first and second steps of the algorithmic chart, should be followed by clinical exome and high resolution microarray techniques. [ABSTRACT FROM AUTHOR]

The dysregulation of microRNA (miRNA) expression has been highlighted in a variety of human malignant conditions with reports implicating a critical role in the process of tumor growth. The role of miR-539 in pancreatic cancer (PC) is yet to be fully elucidated, hence the aim of the current study was to investigate the effect of miR-539 expression in relation to a cohort of 52 PC specimens. The application of a real-time quantitative polymerase chain reaction (qRT-PCR) revealed a significantly down-regulated miR-539 level, which was accompanied by an increased TWIST1 expression in PC when compared with the controls. The in vitro experiment results demonstrated that the endogenic mimic of miR-539 significantly suppressed the growth of the xenograft tumors in PANC-1 cells, when compared to the delivery of the control miRNA and blank control. Meanwhile, the key epithelial-mesenchymal transition (EMT) inducer, TWIST1 was verified as a direct target gene of miR-539 through the application of a luciferase reporter assay. In conclusion, the results of the current study present evidence emphasizing the significance of the interactions between miR-539 and TWIST1 in the development of and progression of PC, highlighting its potential as a therapeutic target in the treatment of PC patients. [ABSTRACT FROM AUTHOR]

The T790M mutation is recognized as a typical mechanism of acquired resistance to first generation of epithermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib in non-small cell lung cancer (NSCLC) patients who are commonly treated by third generation of EGFR-TKI AZD9291 (osimertinib). However, the therapeutic strategy for overcoming acquired resistance to EGFR-TKIs in NSCLC patients without T790M remains to be definitively determined. In the present study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing NSCLC cell line H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11 months. The cytotoxic effects of gefitinib or AZD9291 in vitro were evaluated via the half maximal inhibitory concentrations (IC50s) determined by the MTT assay. IC50 of gefitinib in H1650GR (50.0 ± 3.0 µM) significantly increased compared with H1650 (31.0 ± 1.0 µM) (p < 0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3 ± 0.9 µM) significantly increased compared with H1975 (5.5 ± 0.6 µM) (p < 0.05). However, IC50 of AZD9291 on H1650GR (8.5 ± 0.5 µM) did not increase compared with H1650 (9.7 ± 0.7 µM). On the other hand, IC50 of AZD9291 on gefitinib-resistant A549 (A549GR established in our previous study) (12.7 ± 0.8 µM) was significantly increased compared with A549 (7.0 ± 1.0 µM) (p < 0.05). AZD9291 induced caspase 3/7 activation in A549, H1650, and H1650GR, but not in A549GR. Western blot analyses showed that p-Akt played a key role in determining the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291. Additionally, increased expression of Twist1 was observed in all cells with acquired EGFR-TKI resistance and knockdown of Twist1 by shRNA was found to significantly enhance the sensitivity of A549GR to gefitinib or AZD9291 via reversing epithelial–mesenchymal transition and downregulating p-Akt, but not of H1975AR to AZD9291. The enhanced cytotoxic effect of AZD9291 on A549GR by Twist1 knockdown in vitro was further validated by in vivo studies which showed that Twist1 knockdown could lead to significantly delayed tumor growth of A549GR xenograft with increased sensitivity to AZD9291 treatment in nude mice without any observed side toxic effects. In summary, our study demonstrated that the mechanisms of acquired resistance in different NSCLC cell lines treated by even the same EGFR-TKI might be quite different, which provide a rationale for adopting different therapeutic strategies for those NSCLC patients with acquired EGFR-TKI resistance based on different status of heterogeneous mutations. [ABSTRACT FROM AUTHOR]

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

Background/Aims: TNF-α and TGF-β associated epithelial-mesenchymal transition (EMT) occurs via NF-κB-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-α (10.0 ng/mL) and TGF-β (5.0 ng/mL) for 24h following TGF-β (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0μM). 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-κBp65 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-α after prolonged TGF-β treatment, in a concentration-dependent fashion. NF-κBp65 knockdown and ChR(10.0μM) cooperatively enhanced the inhibition of NF-κBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-κBp65 combined with ChR(10.0μM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-κBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-κBp65 knockdown and ChR. Conclusion: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-kB/Twist axis. [ABSTRACT FROM AUTHOR]

Intracranial aneurysms (IA) are weakened outpouchings of the arterial wall in the cerebrovasculature. Rupture of an IA often leads to devastating consequences. The early identification of IA patients is crucial for management of their condition. A genetic variant at rs10230207, located nearby the HDAC9, TWIST1, and FERD3L genes, is associated with IA. HDAC9 is a class IIa histone deacetylase that mediates vascular smooth muscle cell dysfunction. TWIST1 is a mechanosensitive transcription factor and its expression is reduced in unstable carotid atherosclerotic plaques. In this study, the expression of the HDAC9, TWIST1, and FERD3L genes was characterized and associated with the presence of the rs10230207 genetic variant. Allelic discrimination and gene expression analysis were performed using lymphoblasts from 85 population controls and 109 IA patients. Subjects that were heterozygous (GT) within rs10230207 were 4.32 times more likely to have an IA than those that were homozygous for the reference allele (GG; 95%CI 1.23 to 14.16). Subjects that were homozygous (TT) were 8.27 times more likely to have an IA than those that were GG (95%CI 2.45 to 27.85). While the presence of the risk allele was not associated with changes in FERD3L gene expression, the risk allele was associated with increased HDAC9 and decrease in TWIST1 mRNA expression. The significant inverse correlation between HDAC9 and TWIST1 gene expression suggests that changes in the expression of both of genes may contribute to the formation of IAs. [ABSTRACT FROM AUTHOR]

Abstract This study identified LIMK2 kinase as a disease-specific target in castration resistant prostate cancer (CRPC) pathogenesis, which is upregulated in response to androgen deprivation therapy, the current standard of treatment for prostate cancer. Surgical castration increases LIMK2 expression in mouse prostates due to increased hypoxia. Similarly, human clinical specimens showed highest LIMK2 levels in CRPC tissues compared to other stages, while minimal LIMK2 was observed in normal prostates. Most notably, inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice, underscoring its potential as a clinical target for CRPC. We also identified TWIST1 as a direct substrate of LIMK2, which uncovered the molecular mechanism of LIMK2-induced malignancy. TWIST1 is strongly associated with CRPC initiation, progression and poor prognosis. LIMK2 increases TWIST1 mRNA levels upon hypoxia; and stabilizes TWIST1 by direct phosphorylation. TWIST1 also stabilizes LIMK2 by inhibiting its ubiquitylation. Phosphorylation-dead TWIST1 acts as dominant negative and fully prevents EMT and tumor formation in vivo , thereby highlighting the significance of LIMK2-TWIST1 signaling axis in CRPC. As LIMK2 null mice are viable, targeting LIMK2 should have minimal collateral toxicity, thereby improving the overall survival of CRPC patients. Highlights • LIMK2 was identified as a disease-specific target in CRPC. • We show that LIMK2 is upregulated in castrated prostates due to increased hypoxia. • Inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice. • TWIST1 was identified a direct target of LIMK2. • LIMK2 inhibitor shows very high synergy with docetaxel. [ABSTRACT FROM AUTHOR]

Overexpression of long noncoding RNA (lncRNA) H19 has been observed in various cancers, which indicates that H19 exert important roles in the progression of carcinogenesis. MiR‐326 has been reported to play tumor suppressive roles in multiple tumors. Recently, the competing endogenous RNA (ceRNA) hypothesis has implied that lncRNAs might function as molecular sponges for microRNAs in various cancers. However, the roles of H19/miR‐326 in human hepatocellular carcinoma (HCC) still remain unclear. The aim of our study was to determine H19/miR‐326 expression in HCC cells and investigate their roles in HCC development. We found that H19 was significantly elevated and miR‐326 was decreased in HCC cells including Hep3B, HepG2, MHCC‐97L, SK‐hep1, Hun7, SMCC‐7721 compared with LO2 cells, respectively. In the subsequent experiments, we observed that inhibition of H19 can repress HCC cell growth, migration, and invasion in vitro. H19 downregulation can increase miR‐326 expression in HCC cells. Meanwhile, miR‐326 mimics can also inhibit HCC progression, whereas miR‐326 inhibitors exhibited a reverse phenomenon by modulating H19 expression. In addition, a negative association between H19 and miR‐326 was predicted and confirmed. Furthermore, the transcription factor TWIST1 has been recognized as a significant regulator in tumor progression. Here, by performing bioinformatics analysis, TWIST1 was identified as a downstream target of miR‐326. The findings of our study implied that lncRNA H19 can serve as a ceRNA to sponge miR‐326 and modulate TWIST1 levels in HCC pathogenesis. Taken these together, these findings indicated that H19/miR‐326/TWIST1 axis was involved in HCC development and can indicate a novel HCC target. We observed that H19 was elevated and miR‐326 was decreased in hepatocellular carcinoma (HCC) cells. TWIST1 was found to be upregulated in HCC cells and it can serve as a direct target of miR‐326. In conclusion, we indicated that H19/miR‐326/TWIST1 axis was involved in HCC and it can provide a new insight in to HCC pathogenesis. [ABSTRACT FROM AUTHOR]

Emerging evidence has illustrated the vital roles of long noncoding RNAs (lncRNAs) in human cancers. However, the role of lncRNAs in non‐small cell lung cancer (NSCLC) is still elusive and poorly understood. In the current study, our team conducted extensive experiments to identify the role of long intergenic nonprotein coding (LINC01296) on NSCLC tumorigenesis. The results illustrated that the elevated LINC01296 expression in NSCLC tissue specimens and cell lines were closely correlated with the poor prognosis of patients with NSCLC. Functional studies revealed that LINC01296 knockdown silenced by small interfering RNAs inhibited proliferation, accelerated apoptosis in vitro, and impaired tumor growth in vivo. Mechanical studies showed that INC01296 harbored miR‐598, acting as a microRNA "sponge." Besides, miR‐598 targeted the 3′‐UTR of Twist1. Interestingly, transcription factor Twist1 could bind with the promoter of INC01296 and activate its transcriptional level. In summary, we conclude that INC01296/miR‐598/Twist1 constitutes a positive feedback loop to promote the tumorigenesis of NSCLC, providing a novel insight and a valuable therapeutic strategy. We conclude that INC01296/miR‐598/Twist1 constitute a positive feedback loop to promote the tumorigenesis of non‐small cell lung cancer (NSCLC). [ABSTRACT FROM AUTHOR]

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ), a novel synthesized combretastatin A-4
(CA-4) analogue, is identified as a microtubule inhibitor and has been shown to exert anticancer activity in breast cancer cells. Here, we found that SQ reversed epidermal growth factor (EGF)-induced motility and invasion in breast cancer cell lines by the in vitro Wound healing and Transwell assay. Further studies showed that SQ treatment resulted in inhibitory alteration of EGF-stimulated epithelial-to-mesenchymal transition (EMT) and MMP-2 activity. What is more, SQ significantly inhibited the EGF-induced mouse double minute 2- (MDM2) expression and transcription factor Twist1 expression. In addition, compared with the control cells, MDM2 overexpression up-regulated Twist1 expression and dramatically promoted cell migration and invasion, MDM2 under-expression also down-regulated Twist1 expression and suppressed cell motility and invasion. Taken together, our findings suggest that the inhibitory effects of SQ on migration and invasion were related to the suppression of MDM2 and Twist1 signal axis. [ABSTRACT FROM AUTHOR]

Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expression through epigenetic modifications of H3K4me3 and H3K27ac at the TWIST1 proximal promoter. We also identified that the FZD7‐TWIST1 axis regulates the expression of BCL2, a gene that controls apoptosis. Identification of this FZD7‐TWIST1‐BCL2 pathway reaffirms the mechanism of anoikis resistance in OC. We subsequently showed that the FZD7‐TWIST1 axis can be targeted by using a small molecule inhibitor of porcupine, an enzyme essential for secretion and functional activation of Wnts. In conclusion, our results identified that the FZD7‐TWIST1 axis is important for tumorigenesis and anoikis resistance, and therapeutic inhibition results in cell death in OCs. Frizzled family receptor 7 (FZD7) expression maintains the mesenchymal phenotype and anchorage independence via epigenetic regulation of TWIST1. Knockdown of FZD7 results in inhibition of TWIST1 with consequent downregulation of epithelial to mesenchymal transition and BCL2 leading to anoikis and loss of tumorigenesis. Our results demonstrate that the FZD7‐TWIST1 axis is important for tumorigenesis and anoikis in ovarian cancer. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate the roles of miR-15a-3p in ovarian cancer cell growth and metastasis. PATIENTS AND METHODS: A key role of miR- 15a-3p was identified via gene profiling and bioinformatics analysis. The impact of miR-15a- 3p on ovarian cancer cell growth, migration and invasion was measured by 3-(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), wound-healing and transwell invasion assays. Bioinformatics and luciferase reporter assays were applied to identify that twist family BHLH transcription factor 1 (Twist1) was the target gene of miR-15a-3p. The miR-15a-3p level and the expression of Twist1 were detected using quantitative Real-time polymerase chain reaction (qRT-PCR) assay. The expressions of N-cadherin and E-cadherin were measured by immunofluorescence staining. Small interfering RNA targeting Twist1 and pCDNA3.1 containing Twist1 were applied to decrease and increase the expression of Twist1, respectively. RESULTS: miR-15a-3p was markedly down-regulated in ovarian cancer. Exogenous up-regulation of miR-15a-3p inhibited the growth, colony formation, migration and invasion of ovarian cancer cell in vitro. Furthermore, a xenograft model indicated that miR-15a-3p inhibited tumour growth and the metastatic potential of ovarian cancer cell in vivo. We found that Twist1 was the direct target of miR-15a-3p in ovarian cancer and that its expression was negatively correlated with the level of miR-15a-3p in ovarian cancer tissues. Up-regulation of miR-15a- 3p rescued the inhibitory impact of miR-15a-3p on ovarian cancer cell growth, migration and invasion. Finally, down-regulation of Twist1 mimicked the suppressive effects of miR-15a-3p on ovarian cancer cell. CONCLUSIONS: We demonstrated that miR-15a-3p is down-regulated in ovarian cancer. Up-regulation of miR-15a-3p restrains the growth and metastasis of ovarian cancer cell by regulating Twist1. [ABSTRACT FROM AUTHOR]

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 EMTassociated 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. [ABSTRACT FROM AUTHOR]

Abstract Cancer-associated fibroblasts (CAFs) play critical roles in tumor progression. However, the role and mechanism underlying CAFs in esophageal cancer (EC) remain unclear. In this study, primary CAFs and normal esophageal fibroblasts (NOFs) were isolated and characterized by immunofluorescence, qRT-PCR and western blot. Clinical significance of twist1 in CAFs were evaluated by immunohistochemistry assay. Conditioned medium (CM) was collected from CAFs to evaluate the influence on epithelial-mesenchymal transition (EMT) of EC cells. EC cells were mixed with CAFs and subcutaneously injected into nude mice to assess the in vivo tumor growth. As the result, twist1 was overexpressed in CAFs compared with NOFs and exhibited adverse prognostic significance. In CAFs, twist1 promoted the expression and secretion of CXCL12. In EC cells, activated CXCL12/CXCR4 signaling promoted the EMT process through ERK/AKT - twist1 - MMP1/E-cadherin pathway. In addition, knockdown of twist1 in CAFs also suppressed in vivo tumor growth. In conclusion, our results revealed a dual role of twist1 in CAFs and EC cells to promote the EMT process. Highlights • Twist1 promoted the expression and secretion of CXCL12 in CAFs of EC. • CXCL12 secreted by CAFs promoted EMT of EC cells. • CXCL12 increased the expression of twist1 via AKT and ERK signaling in EC cells. • Knockdown of twist1 in CAFs suppressed in vivo tumorigenesis of EC cells. [ABSTRACT FROM AUTHOR]

The aim of the present study was to identify the relation between Tenascin‐C (TNC) and Twist1 and determine their clinical significance in gastric cancer (GC). We analyzed the expression of TNC and Twist1 in 159 GC samples and in 91 non‐tumor samples using immunohistochemistry. In this study, TNC expression in stromal fibroblasts of GC was remarkably higher than non‐tumor gastric lesions. The expression of TNC in GC stromal fibroblasts was significantly associated with pT stage, lymph node metastasis, distant metastasis. Twist1 expression in stromal fibroblasts of GC was remarkably higher than non‐tumor gastric lesions. Twist1 expression in the stromal fibroblasts of GC was associated with tumor size, lymph node metastasis, and clinical stage. Furthermore, TNC expression levels in GC stromal fibroblasts were positively associated with Twist1. The simultaneous expression of TNC and Twist1 was significantly higher in stromal fibroblasts of GC than in noncancerous tissues. The simultaneous expression of TNC and Twist1 in GC stromal fibroblasts was positively associated with tumor location, pT stage, lymph node metastasis and clinical stage. Moreover, patients with co‐expression of TNC and Twist1 had a poorer prognosis than either TNC or Twist1 positive in GC. Our study revealed that the simultaneous expression of TNC and Twist1 indicated the poorer prognosis of GC. Co‐targeting TNC and Twist1 confer significant clinical advantage, which offers a novel therapeutic target in GC. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is a leading cause of global cancer-related death. The incidence and mortality rates of gastric cancer in China are second and third ranked in all forms of malignant tumors. Krüppel-like factor11 (KLF11) is a member of the KLF family, and previous studies have shown it significantly influences epithelial ovarian, pancreatic and liver cancer proliferation, differentiation and apoptosis. However, the expression and some biological functions of KLF11 in GC are still unclear. We therefore collected and analyzed the mRNA and protein expressions of KLF11 in 59 paired gastric cancer tissues and matched healthy gastric tissue samples. We then investigated the KLF 11 biological functions and potential mechanisms in BGC823 and HGC27 gastric cancer cell lines. Analysis of KLF11 in gastric cancer specimens confirmed up-regulation compared to adjacent healthy gastric tissues, and similar results were evident in the GC cell lines. Ectopic expression of KLF11 was significantly associated with GC cell invasion and migration. KLF11 functions were most effective in Twist1 expression and knockdown, and also in KLF11 up-regulation which was accompanied by corresponding change in Twist1 expression; but these effects were inhibited when KLF11 was silenced by the small interfering RNA (siRNA). The relative Twist1 promoter region activity increased gradually with increasing KLF11 plasma, and KLF11 therefore has a critical role in regulating gastric cancer migration and invasion by increasing Twist1 expression. Finally, the results of this study should improve understanding of the KLF11 and EMT regulating network and KLF11's use as a potential therapeutic target in gastric cancer. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study aimed to characterize the effect of Twist2 on epithelial-to-mesenchymal transition (EMT) and the invasive potential of colorectal cancer (CRC) cells and to explore the mechanisms underlying the regulative effect of Twist1 and Twist2 on matrix metalloproteinase 2 (MMP2) expression in CRC. PATIENTS AND METHODS: Data mining was performed in colorectal cancer cohort (COADREAD) in the Cancer Genome Atlas (TCGA-COADREAD). CRC LoVo and HCT116 cells were used as in vitro cell models. RESULTS: CRC tumors with lymphatic invasion (N = 102) had a significantly higher expression of TWIST1 (p = 0.01) and TWIST2 (p = 0.02) than the lymphatic invasion negative cases (N = 228). TWIST2 overexpression enhanced EMT and the invasive potential of the CRC LoVo and HCT116 cells, while TWIST2 knockdown reversed the EMT process and weakened the invasive potential of the cells. TWIST1 and TWIST2 were co-upregulated with MMP2 and MMP9 in COADREAD cohort. TWIST1 or TWIST2 overexpression significantly elevated nuclear β-catenin accumulation, which is a known signaling pathway elevating MMP2 and MMP9 expression. More importantly, we found that both Twist1 and Twist2 could transcriptionally activate MMP2 via directly binding to its promoter. However, this mechanism was not observed in the MMP9 promoter. CONCLUSIONS: TWIST1/2 is associated with lymphatic invasion in CRC. TWIST2 upregulation enhances EMT and the invasive potential of CRC cells. TWIST1/2 can enhance the Wnt/β-catenin signaling pathway in CRC cells. In addition, Twist1/2 can bind to the MMP2 promoter and promote its transcription. [ABSTRACT FROM AUTHOR]

The TWIST family is a group of highly conserved basic helix–loop–helix transcription factors. In humans, TWIST1 haploinsufficiency causes Saethre–Chotzen syndrome, which is characterized by craniosynostosis. Heterozygous localized TWIST1 and TWIST2 basic domain substitutions exert antimorphic effects to cause Sweeney–Cox syndrome, Barber–Say syndrome, and ablepharon‐macrostomia syndrome, respectively. Sweeney–Cox syndrome, Barber–Say syndrome, and ablepharon‐macrostomia syndrome share the facial features of ablepharon, hypertelorism, underdevelopment of the eyelids, and cheek pads adjacent to the corners of the mouth. Existence of phenotypic overlap between Saethre–Chotzen syndrome and Sweeney–Cox syndrome remains unknown. Herein, we document a male infant with the distinctive facial features of ablepharon, hypertelorism, cheek pads adjacent to the corners of the mouth, and bilateral coronal suture craniosynostosis who had a de novo heterozygous mutation in the basic domain of TWIST1, that is, c.351C>G p.Glu117Asp. The pathogenicity of this variant was supported by in silico and in vivo evidence. Our review showed that Sweeney–Cox syndrome appears to share many characteristics with Barber–Say syndrome and ablepharon‐macrostomia syndrome except for craniosynostosis, which is a cardinal feature of Saethre–Chotzen syndrome. An amino acid substitution from Glu117 to Asp, both of which are the sole members of negatively charged amino acids, resulted in a prototypic Sweeney–Cox syndrome phenotype. This suggests that any amino acid substitutions at Glu117 would likely lead to the Sweeney–Cox syndrome phenotype or lethality. The present observation suggests that a localized TWIST1 basic domain substitution, that is, p.Glu117Asp, in TWIST1 may exert a mild antimorphic effect similar to that of haploinsufficiency, leading to craniosynostosis and ablepharon. [ABSTRACT FROM AUTHOR]

During metastasis, tumor cells invade through the basement membrane and intravasate into blood vessels and then extravasate into distant organs to establish metastases. Here, we report a critical role of a transmembrane serine protease fibroblast activation protein (FAP) in tumor metastasis. Expression of FAP and TWIST1, a metastasis driver, is significantly correlated in several types of human carcinomas, and FAP is required for TWIST1-induced breast cancer metastasis to the lung. Mechanistically, FAP is localized at invadopodia and required for invadopodia-mediated extracellular matrix degradation independent of its proteolytic activity. Live cell imaging shows that association of invadopodia precursors with FAP at the cell membrane promotes the stabilization and growth of invadopodia precursors into mature invadopodia. Together, our study identified FAP as a functional target of TWIST1 in driving tumor metastasis via promoting invadopodia-mediated matrix degradation and uncovered a proteolytic activity-independent role of FAP in stabilizing invadopodia precursors for maturation. [Display omitted] • FAP promotes breast tumor metastasis • FAP expression is significantly correlated with TWIST1 in various human cancers • FAP is required for matrix degradation independent of its proteolytic activity • FAP localizes at invadopodia and promotes invadopodia precursor stabilization Bukhari et al. report a functional role of the transmembrane serine protease FAP in tumor metastasis. FAP is required for invadopodia-mediated matrix degradation independent of its proteolytic activity. FAP localizes at invadopodia and promotes invadopodia precursor stabilization. These results indicate a structural role of FAP at invadopodia to promote tumor metastasis. [ABSTRACT FROM AUTHOR]

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation. [Display omitted] • ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to regulate gene expression. • We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. • ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. • ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. • Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). • Treatment with sublethal doses of the HDAC inhibitor vorinostat that targets the Sin3a HDAC complex via ING proteins promoted breast cancer cell migration, which increased further upon ING1 knockout. • This suggests that use of HDAC inhibitors as adjuvant therapies in tumours that are prone to metastasize may not be optimal and requires further investigation. [ABSTRACT FROM AUTHOR]

Abstract Background Current evidence suggests that endometrial-derived stem cells, spilled in the peritoneal cavity via retrograde menstruation, are key players in the establishment of endometriotic lesions. The aim of this study was to determine the presence and distribution of the stemness-related factors OCT4, SOX15, TWIST1 and DCAMLK1 in women with and without endometriosis. Methods Immunohistochemical analysis was used to determine stromal and epithelial expression of OCT4, SOX15, TWIST1 and DCAMLK1 in endometriosis patient (EP) endometrium (n = 69) and endometriotic tissue (n = 90) and in control endometrium (n = 50). Quantitative Real-Time PCR of OCT4, SOX15 TWIST1 and DCAMLK1 was performed in paired samples of EP endometrium and endometriotic tissue. Co-immunofluorescence staining was performed for OCT4 and SOX15. For statistical analyses we used unpaired t-test, Fisher combination test and Spearman test. For paired analyses, paired t-test and McNemar test were used. Results We detected a significant correlation between the expression of the established stem cell marker OCT4 and the stemness-related markers SOX15 (p

Abstract Background DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear. Methods We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism. Results OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial–mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration. Conclusions Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1+ epithelium displayed extensive intercellular junctions, and Twist1– luminal epithelial cells could still adhere to disseminating Twist1+ cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1+ cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate.

The cranial neural crest and the cranial mesoderm are the source of tissues from which the bone and cartilage of the skull, face and jaws are constructed. The development of the cranial mesoderm is not well studied, which is inconsistent with its importance in craniofacial morphogenesis as a source of precursor tissue of the chondrocranium, muscles, vasculature and connective tissues, mechanical support for tissue morphogenesis, and the signaling activity that mediate interactions with the cranial neural crest. Phenotypic analysis of conditional knockout mouse mutants, complemented by the transcriptome analysis of differentially enriched genes in the cranial mesoderm and cranial neural crest, have identified signaling pathways that may mediate cross-talk between the two tissues. In the cranial mesenchyme, Bmp4 is expressed in the mesoderm cells while its signaling activity could impact on both the mesoderm and the neural crest cells. In contrast, Fgf8 is predominantly expressed in the cranial neural crest cells and it influences skeletal development and myogenesis in the cranial mesoderm. WNT signaling, which emanates from the cranial neural crest cells, interacts with BMP and FGF signaling in monitoring the switch between tissue progenitor expansion and differentiation. The transcription factor Twist1, a critical molecular regulator of many aspects of craniofacial development, coordinates the activity of the above pathways in cranial mesoderm and cranial neural crest tissue compartments.

In addition to their stem/progenitor properties, mesenchymal stem cells (MSCs) also exhibit potent effector (angiogenic, antiinflammatory, immuno-modulatory) functions that are largely paracrine in nature. It is widely believed that effector functions underlie most of the therapeutic potential of MSCs and are independent of their stem/progenitor properties. Here we demonstrate that stem/progenitor and effector functions are coordinately regulated at the cellular level by the transcription factor Twist1 and specified within populations according to a hierarchical model. We further show that manipulation of Twist1 levels by genetic approaches or by exposure to widely used culture supplements including fibroblast growth factor 2 (Ffg2) and interferon gamma (IFN-gamma) alters MSC efficacy in cell-based and in vivo assays in a predictable manner. Thus, by mechanistically linking stem/progenitor and effector functions our studies provide a unifying framework in the form of an MSC hierarchy that models the functional complexity of populations. Using this framework, we developed a CLinical Indications Prediction (CLIP) scale that predicts how donor-to-donor heterogeneity and culture conditions impact the therapeutic efficacy of MSC populations for different disease indications.