TWIST1 Research Articles

Research Article A targeted NGS approach to identify a c.352C>G variant in the TWIST1 gene in an Albanian family with Saethre–Chotzen syndrome

A targeted next generation sequencing (NGS) approach analysing contemporaneously 20 different genes mainly involved in craniosynostosis was adopted to molecularly diagnose the family of a 2-years old girl affected by Saethre–Chotzen syndrome, a syndromic form of craniosynostosis. The identified pathogenic variant in the TWIST1 gene lies in a conserved residue (Arg118) that shifts from a positively charged amino acid to a non-polar amino acid and segregates in all the examined affected familial members, although with variable phenotypic expression. An accurate molecular diagnosis is of obvious value for the clinical management of individuals with isolated or syndromic craniosynostosis to define their medical needs, the recurrence risk and allow the identification of available therapeutic opportunity. Given the high number of associated genes, an NGS approach is the election choice to increase the yield of genetic diagnosis, leading to an expansion of the genotypic and phenotypic spectrum of these rare syndromes

LGR5 Is a Gastric Cancer Stem Cell Marker Associated with Stemness and the EMT Signature Genes NANOG, NANOGP8, PRRX1, TWIST1, and BMI1.

BackgroundAccumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear.MethodsqPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. Tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness in vivo. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations.ResultsThe expression of LGR5 was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of LGR5 was also positively associated with stemness regulators (NANOG, OCT4, SOX2, and AICDA) and EMT inducers (PRRX1, TWIST1, and BMI1). In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the NANOG expression primarily originates from the retrogene NANOGP8. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. LGR5 over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. Tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results.ConclusionThe LGR5-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which LGR5 is closely associated with stemness and EMT core genes, and NANOG expression is mainly contributed by the retrogene NANOGP8. Sphere cells are the best starting materials for the characterization of CSCs.

Chordoma and its embryonic determinants

Background. Chordomas are rare malignant tumors occurring along the axial skeleton that originate from notochordal remnants. It is hypothesized that embryonic factors are involved in their aetiology. In the present study, four such factors are of interest: EVX1, which mediates posterior patterning of the embryo and is found in prostate. Wnt3a, localized to the placenta, is involved in different proliferation signaling pathways. Twist is a mediator of the epithelial-mesenchymal transition (EMT) and is expressed in the neuroblastoma cell line SHSY 5Y. Finally, brachyury is a crucial EMT mediator and involved in cell cycle regulation. Research question. The research question is tripartite. (I) It is the aim to obtain positive control tissue for validation of embryonic primers for the EVX1, Wnt3a and Twist genes. (II) Second, expression of brachyury mRNA in cultured chordoma cells will be assessed in order to validate the cell line. (III) Last, it will be investigated whether fibroblast conditioned cell culturing medium exerts a positive effect on chordoma proliferation in vitro. Materials and methods. (I) Placenta and prostate samples were obtained from patients in the Maastricht University Hospital. mRNA was isolated from prostate, placenta and SHSY 5Y cells and cDNA of the genes of interest was generated after which PCR and gel electrophoresis was employed to yield amplicon bands. (II) Chordoma cells were cultured and brachyury mRNA expression was assessed by PCR assay. (III) Cultured chordoma cells were grown in enriched DMEM or in enriched DMEM containing 33% fibroblast conditioned medium and proliferation was assessed. Results. (I) Both EVX1 and Twist are positive in prostate and SHSY 5Y respectively. Wnt3a is not positive in placenta. (II) Brachyury expression is either strongly downregulated or not expressed in two slowly growing chordoma cell cultures. (III) First data indicate enriched DMEM containing 33% fibroblast conditioned medium does not stimulate chordoma proliferation in vitro as compared to enriched DMEM without fibroblast conditioned medium.

Platelets Inhibit Migration of Canine Osteosarcoma Cells

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition.

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation.

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.

Silencing Twist gene leads to mesenchymal-to-epithelial transition and loss of stemlike properties in breast cancer

Objective To observe the effect of silencing Twist gene on mesenchymal-to-epithelial transition (MET) and stem-like properties in breast cancer. Methods The small interfering RNA (siRNA) vectors targeting Twist gene were transfected into MDA-MB-231 cells. The mRNA and protein expression levels of Twist and MET-associated genes (Slug, Snail and Vimentin) were detected by using real-time quantitative polymerase chain reaction(Real-time PCR) and Western blotting. Cell morphology was observed under microscope. The proportion of CD44+ CD24- cells was measured by flow cytometry. In vitro self-renewal capacity was evaluated by sphere formation assay. Tumor-initiating ability was measured by limited dilution assays. Results Compared to the control group, the Twist mRNA (0.24±0.04, P<0.01) and protein expression in siRNA group was significantly decreased. Cells with Twist silencing underwent MET reversal, showing an epithelial-like appearance, decreased mRNA expression of mesenchymal markers, including Slug (0.25±0.05, P<0.01), Snail (0.34±0.03, P<0.01) and Vimentin (0.44±0.04, P<0.01), increased mRNA expression of epithelial marker, E-cadherin (58.71±1.27, P<0.01), down-regulated protein levels of Slug, Snail and Vimentin, and up-regulated protein level of E-cadherin. The proportion of CD44+ CD24- cells in the si-Twist group and control group was (63.30±0.80)% and (86.50±0.70)% respectively (P<0.01). The sphere number in the si-Twist and control groups was (46.67±2.52) and (109.67±2.52) respectively (P<0.01). Twist silencing led to the decrease of about 6-folds in tumor-initiating ability in nude mice compared to the control (P<0.01). Conclusion Silencing Twist gene leads to MET and loss of self-renewal capacity and tumor-initiating ability in breast cancer. Key words: Breast cancer; Twist; Small interfering RNA; Mesenchymal-to-epithelial transition; Tumor stem cells

Genetic research on five children with craniostenosis

Objective To conduct clinical molecular diagnosis for Chinese Han nationality children with craniostenosis in order to find the related pathogenic genes. Methods Five children with craniostenosis admitted to the Department of Neurosurgery, Shanghai Children’s Medical Center of Shanghai Jiao Tong University conducted whole-exome sequencing (WES) on peripheral blood nucleated cell genes using the high-throughput WES between February 2014 and January 2015. The obtained data were used conduct bioinformatic information analysis, and Sanger sequencing was used to validate the gene related mutation found by WES. Results The exon 1 of the regulator of epithelial-mesenchymal transition TWIST1 of 2 children had a heterozygous missense mutation respectively: c. 528C>G, p. Ser176Arg和c.487C>T, p.Leu163Phe; the other 3 children had a fibroblast growth factor receptor 2 (FGFR2) gene heterozygous missense mutation respectively. Two of them were located in the exon 7 (c.755C>G, p. Ser252Trp and c. 833G>T, p. Cys278Phe); and 1 was located in the exon 8 (c.1 012G>C, p. Gly338Arg). Conclusions With the aid of high-throughput DNA sequencing technology and combined with clinical features, 2 children were diagnosed as Saethre-Chotzen syndrome caused by TWIST1 mutation; 2 were diagnosed as Crouzon syndrome caused by FGFR2 mutation, and 1 was diagnosed as Apert syndrome caused by FGFR2 mutation. Genetic studies contribute to diagnose the children with craniostenosis whose clinical feature is not significant, and provide the basis for the diagnosis of craniostenosis and typing. Key words: Receptor, fibroblast growth factor, type 2; Exome; Sequence analysis, DNA; Point mutation; TWIST1; Craniosynostoses

Tyrosine kinase receptor c-ros-oncogene 1 mediates TWIST-1 regulation of human mesenchymal stem cell lineage commitment

The TWIST-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor important in mediating skeletal and head mesodermal tissue development. Bone marrow-derived mesenchymal stem/stromal cells (BMSC), express high levels of TWIST-1, which is down regulated during ex vivo expansion. Cultured BMSC over-expressing TWIST-1 display decreased capacity for osteogenic differentiation and an enhanced capacity to undergo adipogenesis, suggesting that TWIST-1 is a mediator of lineage commitment. However, little is known regarding the mechanism(s) by which TWIST-1 mediates cell fate determination. In this study, microarray analysis was used to identify a novel downstream TWIST-1 target, tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1), which was down regulated in TWIST-1 over-expressing BMSC. Chromatin immunoprecipitation analysis showed that TWIST-1 directly bound to two E-box binding sites on the proximal C-ROS-1 promoter. Knock-down of C-ROS-1 in human BMSC and cranial bone cells resulted in a decreased capacity for osteogenic differentiation in vitro. Conversely, suppression of C-ROS-1 in BMSC resulted in an enhanced capacity to undergo adipogenesis. Furthermore, reduced C-ROS-1 levels led to activation of different components of the PI3K/AKT/mTORC1 signalling pathway during osteogenic and adipogenic differentiation. Collectively, these data suggest that C-ROS-1 is involved in BMSC fate switching between osteogenesis and adipogenesis, mediated via PI3K/AKT/mTORC1 signalling.

Differential expression pattern of Twist1 in mouse preimplantation embryos suggests its multiple roles during early development.

The purpose of the present study is to understand Twist-related protein 1 (Twist1) spatiotemporal expression patterns and functions during early embryo development. We performed whole-mount double immunofluorescence staining and reverse transcription (RT)-PCR analysis of the Twist1 protein and gene throughout the preimplantation development in mice. We determined that after compaction, the expression of Twist1 becomes developmentally differentiated and targeted in the inner cells of embryos. In blastocysts at E4.5, uniform staining of the inner cell mass was apparent, and it had been gradually translocated to the nucleus of hatched embryonic cells at E4.75. Furthermore, the effect of potential regulators of Twist on its expression level during blastocyst development was also sought. Accordingly, Twist1 expression appeared to be upregulated in both mRNA and protein level following culture of embryos in the presence of high glucose. Our study revealed the dynamic Twist localization within the early stage of embryo. The results are discussed in terms of potential roles of Twist1 in the processes of lineage segregation, hatching, and implantation in post-compaction embryos and in blastocysts.

The Urtica dioica extract enhances sensitivity of paclitaxel drug to MDA-MB-468 breast cancer cells.

Due to the chemo resistant nature of cancer cells and adverse effects of current therapies, researchers are looking for the most efficient therapeutic approach which has the lowest side effects and the highest toxicity on cancer cells. The aim of the present study was to investigate the synergic effect of Urtica dioica extract in combination with paclitaxel on cell death and invasion of human breast cancer MDA-MB-468 cell line. To determine the cytotoxic effects of Urtica dioica extract with paclitaxel, MTT assay was performed. The scratch test was exploited to assess the effects of Urtica dioica, Paclitaxel alone and combination on migration of cancer cells. The expression levels of snail-1, ZEB1, ZEB2, twist, Cdc2, cyclin B1 and Wee1 genes were quantified using qRT-PCR and western blot performed for snail-1expression. The effects of plant extract, Paclitaxel alone and combination on different phases of cell cycle was analyzed using flow cytometry. Results of MTT assay showed that Urtica dioica significantly destroyed cancer cells. Interestingly, Concurrent use of Urtica dioica extract with paclitaxel resulted in decreased IC50 dose of paclitaxel. Moreover, findings of scratch assay exhibited the inhibitory effects of Urtica dioica, Paclitaxel alone and combination on migration of MDA-MB-468 cell line. Our findings also demonstrated that the extract substantially decreased the Snail-1 and related gene expression. Ultimately, Cell cycle arrest occurred at G2/M phase post-treatment by deregulating Cdc2 and wee1. Our results demonstrated that the dichloromethane extract of Urtica dioica inhibit cell growth and migration. Also, Urtica dioica extract substantially increased sensitivity of breast cancer cells to paclitaxel. Therefore, it can be used as a potential candidate for treatment of breast cancer with paclitaxel.

Abstract 1707: Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells

Abstract Background: EpCAMhigh CD44+colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a property of cancer stem cells. Epithelial-mesenchymal transition (EMT) is a possible process of acquisition of cancer stem cell (CSC)-like properties. However, it is unclear whether EMT can be induced in primary human CRC cells. Patients and Methods: We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing growth factors. For induction of EMT, TGF-beta was added into the culture medium. Otherwise, TWIST1 expression was enforced in the cells by using lentiviral transduction. Immunocytochemical analysis, flow cytometric analysis and single cell PCR analysis using 24-genes set containing embryonic stem cell (ES)-related and EMT-related genes were performed. PCR analysis was carried out by C1 single cell auto prep system. CD44- CRC cells with or without enforced expression of TWIST1 were injected into immunodeficient mice. Results: Fifteen out of 51 samples of cancer cells formed sphere (&amp;gt;50 μm in diameter) after one week culture. The sphere-forming ability was related with clinical stage (Stage 1 and 2;16.7%, Stage 3 and 4;41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in short term culture than in long-term culture, suggesting that differentiation occurred in sphere cells after long-term culture. Single cell PCR analysis revealed that sphere-forming cells were classified into 2 different populations on the basis of primary component analysis. Correlation analysis showed expression of TWIST1 and ES-related genes were correlated. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population, and EMT is involved in acquisition of CSC-like properties. TGF-beta increased the number of CD44+ cells in CD44- cells, and enhanced sphere-forming ability of CD44- cells. TGF-beta also induced expression of ES-related genes and TWIST1. Enforced expression of TWIST1 induced sphere-forming ability and tumorigenicity in CD44- cells. Conclusions: We established the culture system to observe the differentiation of CSCs and revealed that EMT might be involved in maintenance of CSCs. We firstly demonstrated that primary CD44- CRC cells undergo EMT and become CD44+ cells by TGF-beta treatment or enforced expression of TWIST1. Citation Format: Michitaka Nakano, Mamoru Tanaka, Taichi Isobe, Kohta Miyawaki, Yoshikane Kikushige, Hitoshi Kusaba, Shigeo Takaishi, Takashi Ueki, Eishi Baba, Koichi Akashi. Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1707.

Abstract 427: Integrated PDEF and Twist1 expression distinguishes lethal prostate cancer from an indolent disease

Abstract Introduction: Prostate cancer (PCa) is the third most commonly diagnosed cancer in the world. Advanced genomic studies have characterized it as a molecularly heterogeneous disease with a multiple clinical spectrum ranging from indolent to highly aggressive. Conventional therapies produce a high rate of cure for patients with localized PCa, but there is no effective treatment for metastatic PCa. These facts underline the urgent, yet unmet, need for identification and characterization of new targets that can help distinguish metastatic PCa from an indolent disease, and pave a way for novel mechanism based anti-metastasis therapies against PCa. Our previous studies have shown that loss of PDEF, a putative tumor suppressor, could lead to more invasive phenotype in PCa cell lines through promoting Epithelial to Mesenchymal transition (EMT). In this study, we evaluate the role of PDEF and Twist1 in PCa through NCBI omnibus and The Cancer Genome Atlas. We also evaluated the mechanism by which PDEF is regulated epigenetically. Methods: PDEF expression was monitored by immunohistochemistry using tissue microarray. Western blot was employed to measure the PDEF level in PCa cell lines. PDEF and Twist1 gene expression was measured by real time PCR. The PDEF promoter was cloned and methylation specific PCR was performed. PDEF methylation in clinical samples was extracted from TCGA database and PDEF/Twist1 microarray data was extracted from GSE16560 in NCBI gene expression omnibus with R language. GraphPad prism was used to generate figure and statistical analysis. P &amp;lt; 0.05 is considered significant Results: IHC staining of PDEF on PCa patient samples and in commonly used PCa cell lines suggested an inverse correlation between the level of PDEF and Twist1. Over-expression of PDEF in PC3 cells inhibits Twist1 expression. DNA Methylation status near the promoter region of PDEF was negatively associated with the expression levels of PDEF in established PCa cells. These results suggest that PDEF levels are regulated in part by promoter methylation. Using TCGA data we identified 12 methylation site and the methylation level of multiple site correlates with the increased of Gleason Score (GS). Analysis of gene expression data from GSE16560 revealed that low expression of PDEF and high expression of Twist1 were independently associated with poor survival in PCa patients. Integrated PDEF and Twist1 expression can distinguished a high-risk group of people for hormone refractory PCa along with short median survival time. Conclusions: Loss of PDEF combined with gain of Twist1 expression can serve as a potential biomarker set for distinguishing aggressive PCa from an indolent disease. PDEF expression is epigenetically regulated in part by promoter hyper-methylation. Acknowledgement: Carroll W. Feist Endowed Chair Funds. Citation Format: Fengtian Wang, Sweaty Koul, Parveen K. Jaiswal, Runhua Shi, Glenn Mills, Hari K. Koul. Integrated PDEF and Twist1 expression distinguishes lethal prostate cancer from an indolent disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 427.

Abstract 4486: Epigenetic vulnerabilities in ovarian cancer

Abstract Background: Histone acetylation regulates gene expression in cancer and can be exploited for therapeutic targeting. Histone deacetylase inhibitors (HDACi) increase histone acetylation, promoting transcription of genes involved in growth arrest and apoptosis and have been used as anti-cancer agents. BRCA mutations are the best characterized genetic alteration associated with ovarian cancer and can be targeted therapeutically by recently approved PARP inhibitors. We hypothesized that the epigenetic landscape of ovarian cancer is altered by the presence of BRCA mutations and can be manipulated by HDACi for therapeutic benefit. Materials and Methods: We utilized isogenic cell lines carrying mutated/functional BRCA 1 (UWB1.289; UWB1.289-BRCA), and respectively BRCA 2 genes (PE01/PEO4). We measured HDAC expression and activity by western blotting and an enzymatic assay and characterized globally chromatin marked by H3K9ac (active chromatin) and H3K27ac (poised chromatin) by using ChIP sequencing. To determine whether HDAC inhibitors increase susceptibility to PARP inhibitors, cellular proliferation was measured in ovarian cancer cells expressing mutated and wild type (WT) BRCA. Results: HDAC1 expression and activity were decreased in cell lines carrying defective BRCA proteins. ChIP sequencing identified chromatin regions differentially marked by H3K9ac (178 promoters in BRCA1-null compared to BRCA WT cells) and H3K27ac (131 promoters in BRCA WT compared to BRCA null cells) suggesting differences in gene transcription dependent on histone acetylation in BRCA mutated vs BRCA functional cancer cells. A subset of genes differentially marked by H3K9ac were validated at mRNA level by RT PCR confirming BRCA dependent regulation (TGM2, Wnt7a, DKK1, MET, FLI1, AXL, TG2, PMEPA1, TWIST2 upregulated) and (E2F2, NID2 downregulated) in BRCA1-null ovarian cancer cells. HDAC1 inhibition with trichostatin A (TSA) and entinostat (MS-275) increased H3K9ac and was associated with an increase in TGM2, FLI1, DKK1 and E2F2, and a decrease in TWIST2 gene expression in both BRCA1-wt and -null cell lines confirming epigenetic regulation of this subset of genes. Functionally, HDACi synergized with PARP inhibitors in BRCA null ovarian cancer cells (combination index 0.63), consistent with the altered gene expression changes induced by differences in chromatin marks. Conclusions: In conclusion, the chromatin landscape in BRCA null ovarian cancer cells is altered and can be targeted by HDAC inhibitors. Identifying pathways and specific genes altered by histone acetylation may enable future rational targeted combination therapies. Citation Format: Jessica A. Thomes Pepin, Horacio Cardenas, Cody Moore, Salvatore Condello, Jiang Guanglong, Liu Yunlong, Daniela Matei. Epigenetic vulnerabilities in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4486.

TWIST1 Polymorphisms Predict Survival in Patients with Metastatic Colorectal Cancer Receiving First-Line Bevacizumab plus Oxaliplatin-Based Chemotherapy.

The epithelial-mesenchymal transition (EMT) is an important mechanism of resistance to angiogenesis inhibition. The ability of EMT pathway genetic variants to predict the efficacy of antiangiogenic therapy is unknown. We analyzed associations between functional SNPs in EMT-related genes and outcomes in metastatic colorectal cancer (mCRC) patients undergoing first-line bevacizumab-based chemotherapy. A total of 220 mCRC patients were included in this study: 143 patients treated with first-line bevacizumab-based chemotherapy (bevacizumab cohort) and 77 patients treated with cetuximab-based chemotherapy (cetuximab cohort). SNPs in TWIST1 (rs2285682, rs2285681), ZEB1 (rs10826943, rs2839658), SNAIL (rs1543442, rs4647958), and E-cadherin (rs16260) genes were analyzed by PCR-based direct sequencing. Patients carrying a TWIST1 rs2285682 G allele had a significantly longer median progression-free survival (PFS) of 18.1 months and overall survival (OS) of 44.1 months compared with those with the T/T genotype, who had a median PFS of 13.3 months (HR, 0.57; P = 0.003) and OS of 29.2 months (HR, 0.53; P = 0.001) in the bevacizumab cohort. In multivariate analysis, associations between TWIST1 rs2285682 and PFS and OS remained significant. Among women, the G allele of TWIST1 rs2285682 (PFS HR, 0.39; P = 0.007; OS HR, 0.30; P = 0.001) and TWIST1 rs2285681 (PFS HR, 0.27; P < 0.001; OS HR, 0.25; P < 0.001) was associated with improved survival. No significant associations were found in the cetuximab cohort. Our findings suggest that TWIST1 polymorphisms are associated with survival in mCRC patients treated with first-line bevacizumab-based chemotherapy and may serve as clinically useful biomarkers for antiangiogenic therapy. Mol Cancer Ther; 15(6); 1405-11. ©2016 AACR.

Genetic Polymorphisms of TGFB1, TGFBR1, SNAI1 and TWIST1 Are Associated with Endometrial Cancer Susceptibility in Chinese Han Women.

Endometrial cancer (EC) is a complex disease involving multiple gene-gene and gene–environment interactions. TGF-β signaling plays pivotal roles in EC development. This study aimed to investigate whether the genetic polymorphisms of TGF-β signaling related genes TGFB1, TGFBR1, SNAI1 and TWIST1 contribute to EC susceptibility. Using the TaqMan Genotyping Assay, 19 tagging-SNPs of these four genes were genotyped in 516 EC cases and 707 controls among Chinese Han women. Logistic regression (LR) showed that the genetic variants of TGFB1 rs1800469, TGFBR1 rs6478974 and rs10733710, TWIST1 rs4721745 were associated with decreased EC risk, and these four loci showed a dose-dependent effect (Ptrend < 0.0001). Classification and regression tree (CART) demonstrated that women carrying both the genotypes of TGFBR1 rs6478974 TT and rs10512263 TC/CC had the highest risk of EC (aOR = 7.86, 95% CI = 3.42–18.07, P<0.0001). Multifactor dimensionality reduction (MDR) revealed that TGFB1 rs1800469 plus TGFBR1 rs6478974 was the best interactional model to detect EC risk. LR, CART and MDR all revealed that TGFBR1 rs6478974 was the most important protective locus for EC. In haplotype association study, TGFBR1 haplotype CACGA carrier showed the lowest EC risk among women with longer menarche-first full term pregnancy intervals (˃11 years) and BMI˂24 (aOR = 0.39, 95% CI = 0.17–0.90, P = 0.0275). These results suggest that polymorphisms in TGFB1, TGFBR1, SNAI1 and TWIST1 may modulate EC susceptibility, both separately and corporately.

Abstract 585: Endothelial Cells from Adipose Tissue of Obese Humans Display Mesenchymal Characteristics

The mechanisms underlying vascular dysfunction in adipose tissue (AT) in obesity are not clearly understood. Our hypothesis is that in response to pro-inflammatory cytokines (PIC) present in obese AT, endothelial cells (EC) can de-differentiate and acquire a mesenchymal-like phenotype (EndoMT) that leads to endothelial dysfunction. To test our hypothesis, we measured endothelial and mesenchymal markers of CD31 + CD34 + EC isolated from omental (OM) and subcutaneous (SC) AT of bariatric subjects (BAMVEC) using RT-PCR and western blot. Permeability and oxidative metabolism were determined by ECIS and Seahorse analyzer XF e 24, respectively. BAMVEC isolated from both OM and SC fat showed very low protein expression of vWF and VE-Cadherin (EC markers) and abundantly expressed αSMA and the EMT transcription factor twist-1. To determine effects of PIC on EndoMT, commercially available primary endothelial cells from AT (HAMVEC) were treated in vitro with PIC (2.5ng/mL TNFα, IFNγ and TGFβ) for 1, 3 or 6 days. We found progressive down-regulation by &gt;2-fold (p&lt;0.001) of the EC markers vWF, VE-Cadherin, and Occludin compared to controls. As early as 1 day of PIC treatment twist-1 (p&lt;0.001) and snail1 (p&lt;0.05) showed an increase by &gt;2-fold. Similarly, OM and SC BAMVEC expressed &gt;2-fold increase in the mesenchymal genes twist-1, FSP1, αSMA, and snail1 compared to untreated HAMVEC. Metabolically, BAMVEC had increased ATP production and maximal respiration compared to HAMVEC suggesting increased oxidative phosphorylation, a marker of mesenchymal-like cells. PIC stimulation of HAMVEC yielded significant increases in endothelial permeability and motility (p&lt;0.001). Notably, there were no significant differences in any of the markers between OM and SC BAMVEC. These results show that EC in obese AT exhibit a mesenchymal-like phenotype which may account for functional changes such as increased permeability and migration and are not depot specific. Using primary EC from human AT we showed that prolonged exposure to PIC induces a phenotype similar to CD31+CD34+ EC from obese AT. This supports the concept that AT inflammation can promote EC de-differentiation in vivo and our in vitro model is suitable for future studies to uncover the relevant mechanisms.

A rare case of acrocephaly: Saethre-Chotzen syndrome or Crouzon?

Abstract Acrocephaly is a common neonatal craniofacial malformation. Saethre-Chotzen syndrome (SCS) is one of the acrocephaly related syndromes less frequently described in the literature. A female newborn term was admitted to our Neonatal Unit to study craniofacial dysmorphia without family history of interest. Pregnancy, childbirth and the neonatal period were uneventful. She had exotropia, short anterior-posterior cranial diameter, flat occiput and wide normotensive anterior fontanelle (beginning at the nose root, continuing through the sagittal suture with the posterior fontanelle) without syndactyly. The scanner imaging confirmed an acrocephaly with fusion of bilateral coronal sutures. We initially suspected a cranyosinostosis due to a Crouzon syndrome or SCS. After differential diagnosis and genetic study the patient was diagnosed as having SCS due to a de novo TWIST1 gene mutation. The craniofacial dysmorphias were corrected early by neurosurgical with good results. This case shows a new example of the phenotypic and genotypic variability of these TWIST1 gene mutations.

Knockdown of PSCA induces EMT and decreases metastatic potentials of the human prostate cancer DU145 cells.

BackgroundProstate stem cell antigen (PSCA) expression has been shown to correlate with prostatic carcinogenesis and prostate cancer (PCa) progression. The underlying mechanisms for these processes are currently unknown. Epithelial to mesenchymal transition (EMT) has been associated with the invasiveness and the distant metastasis of PCa. In this study, we investigated the effects of knocking down the PSCA on the cell migration, the invasiveness, and the EMT of the PCa cell line DU145 in vitro and in vivo.MethodsFour target sequences of the small hairpin RNA for PSCA were designed, and the best effect knockdown sequence shRNA#1 was screened to construct the stable transfected DU145 cell line (DU145 shRNA#1), the scramble sequence was also designed to construct the stable transfected DU145 cell line(DU145 scramble). Cell migration and invasion were studied using Transwell assay. Quantitative RT-PCR, Western blot (WB) were used to quantify PSCA, E-cadherin, β-catenin, Vimentin, Fibronectin expression in DU145, DU145 scramble, DU145 shRNA#1 in vitro and in vivo. RT-PCR, immunofluorescent staining were used to quantify PSCA, E-cadherin, and Vimentin expression in vitro. EMT-related genes Snail, Slug, and Twist, were quantified by quantitative RT-PCR in vitro.ResultsThe constructed stable knockdown of the PSCA in the DU145 cell had a silencing effect up to 90.5 %. DU145 shRNA#1 became scattered from the tightly packed colonies. It was associated with decreased cell migration and invasion. There was also an increased Vimentin and Fibronectin expression, an inhibited E-cadherin and β-catenin expression at both the mRNA and the protein levels when compared to the DU145 and the DU145 scramble in vitro and vivo. Furthermore, with the exception of the Snail, the expression of EMT-related Slug and Twist genes were upregulated.ConclusionsOur data indicated that knockdown of PSCA induced EMT and reduced metastatic potentials of the DU145 cells, suggesting that PSCA played an important role in prostatic carcinogenesis and progression.

A Familial Case of Saethre-Chotzen Syndrome in Japan

Saethre-Chotzen syndrome is extremely rare in Japan. We experienced a familial case of Saethre-Chotzen syndrome in 4 individuals of 3 generations. In all 4 individuals, a mutation in the TWIST gene was observed by a gene test. Some of these patients underwent surgical correction of brachycephaly and blepharoptosis with good results.

Inhibition of the STAT3 signaling pathway contributes to apigenin-mediated anti-metastatic effect in melanoma.

Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in human melanoma, and promotes melanoma metastasis. The dietary flavonoid apigenin is a bioactive compound that possesses low toxicity and exerts anti-metastatic activity in melanoma. However, the anti-metastasis mechanism of apigenin has not been fully elucidated. In the present study, we showed that apigenin suppressed murine melanoma B16F10 cell lung metastasis in mice, and inhibited cell migration and invasion in human and murine melanoma cells. Further study indicated that apigenin effectively suppressed STAT3 phosphorylation, decreased STAT3 nuclear localization and inhibited STAT3 transcriptional activity. Apigenin also down-regulated STAT3 target genes MMP-2, MMP-9, VEGF and Twist1, which are involved in cell migration and invasion. More importantly, overexpression of STAT3 or Twist1 partially reversed apigenin-impaired cell migration and invasion. Our data not only reveal a novel anti-metastasis mechanism of apigenin but also support the notion that STAT3 is an attractive and promising target for melanoma treatment.