Brachyury Expression Research Articles

In-Vivo Nucleus Pulposus-Specific Regulation of Adult Murine Intervertebral Disc Degeneration via Wnt/Beta-Catenin Signaling

B-Catenin, transcription factor of Wnt signaling, is promoted in patients with intervertebral disc (IVD) degeneration, but Wnt signaling decreases with aging. We hypothesize that IVD degeneration is associated with decreased Wnt signaling despite more b-Catenin. Chronic compression of tail IVDs of young-adult and aged Wnt-reporter (TOPGAL) animals initiated an age-related cascade of degenerative-like changes, which included reduced Wnt ligand expression and Wnt signaling in nucleus pulposus cells, despite elevation of b-Catenin protein and gene expression. To determine the effect of upregulated and downregulated Wnt signaling in adult discs, b-Catenin in the nucleus pulposus was stabilized (Shh-CreErT2/b-Cateninfl(Ex3)/fl(Ex3), cACT) or knocked out (Shh-CreErT2/b-Cateninfl/fl, cKO). cACT discs had promoted expression of Wnt-targets and -ligands, brachyury, extracellular matrix production and 34% greater compressive stiffness than WT (b-Cateninfl(Ex3)/fl(Ex3)) discs, but 50% less tensile stiffness. By contrast, knockout reversed the cACT phenotype: less protein expression of b-catenin in the nucleus pulposus, less expression of brachyury, heightened expression of extracellular matrix breakdown and 46% less compressive stiffness than wild-type (b-Cateninfl/fl,WT) discs. These data suggest that intervertebral disc degeneration is associated with loss of Wnt signaling and that the concomitant increase in b-catenin is a regenerative response, potentially offering a therapeutic approach to degeneration.

Abstract 2150: Rapid and efficient evaluation of drug sensitivity in a diverse panel of chordoma xenograft models

Abstract Background: Chordoma is a rare cancer found in the base of the skull and the spine that develops from residual embryonic notochord cells. Currently, no systemic therapies are approved for treatment of the disease, but emerging understanding of chordoma biology and tumor dependencies has revealed a number of potential therapeutic targets. To date, validation of novel targets and preclinical evaluation of corresponding therapies has been hampered by scarcity of preclinical models as well the high cost of starting in-vivo experiments. To facilitate more rapid and efficient evaluation of new therapeutic approaches, the Chordoma Foundation has developed a centralized Drug Screening Program (DSP) operated through a partnership with South Texas Accelerated Research Therapeutics (START). Methods: Patients undergoing surgical resection are consented through the Chordoma Foundation's IRB approved biobanking protocol. Tumor tissue is shipped to START, engrafted in immunodeficient mice, and expanded. Resulting patient-derived xenograft (PDX) models are validated by confirmation of histology and expression of brachyury, a protein highly expressed in chordoma tumors. Additional PDX models and cell line derived xenograft (CDX) models are acquired from academic institutions and established at START for use in screening experiments. Drugs are nominated by academic and industry investigators and prioritized by the Chordoma Foundation on the basis of molecular rationale and existing in vitro data. Selected drugs are evaluated for efficacy in the panel of PDX and CDX models. Data is provided back to the nominating investigator, and, when permitted, is made public through an online data repository. Results: A diverse collection of seven validated PDX and CDX models has been developed and is available for in-vivo drug screening experiments. These models represent different clinical subtypes of chordoma, including five derived from conventional chordomas and two derived from poorly differentiated tumors, the latter being an aggressive subtype of chordoma typically found in pediatric patients and young adults. To date, twenty-four drugs have been tested as single agents or in combination in various models. A range of activities have been observed, which may be dependent on molecular alterations present in the individual models. Inhibitors of EGFR and CDK4/6 demonstrated the greatest activity across models. This data has supported the initiation of two clinical trials specific for chordoma patients testing the targeted agents afatinib and palbociclib. Conclusion: The Chordoma Foundation's DSP serves as a core facility for the chordoma research community, providing investigators with a rapid and low-cost means of evaluating novel targets and therapies. It overcomes a key barrier to chordoma drug development and may provide a template that can be employed to other cancer types. Citation Format: Patricia Cogswell, Michael Wick, Melissa Rundle, Amanda Mangold, Josh Sommer, Joan Levy. Rapid and efficient evaluation of drug sensitivity in a diverse panel of chordoma xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2150.

β-Catenin–dependent mechanotransduction dates back to the common ancestor of Cnidaria and Bilateria

Although the genetic regulation of cellular differentiation processes is well established, recent studies have revealed the role of mechanotransduction on a variety of biological processes, including regulation of gene expression. However, it remains unclear how universal and widespread mechanotransduction is in embryonic development of animals. Here, we investigate mechanosensitive gene expression during gastrulation of the starlet sea anemone Nematostella vectensis, a cnidarian model organism. We show that the blastoporal marker gene brachyury is down-regulated by blocking myosin II-dependent gastrulation movements. Brachyury expression can be restored by applying external mechanical force. Using CRISPR/Cas9 and morpholino antisense technology, we also show that mechanotransduction leading to brachyury expression is β-catenin dependent, similar to recent findings in fish and Drosophila [Brunet T, et al. (2013) Nat Commun 4:1-15]. Finally, we demonstrate that prolonged application of mechanical stress on the embryo leads to ectopic brachyury expression. Thus, our data indicate that β-catenin-dependent mechanotransduction is an ancient gene regulatory mechanism, which was present in the common ancestor of cnidarians and bilaterians, at least 600 million years ago.

Brachyury oncogene is a prognostic factor in high-risk testicular germ cell tumors.

The T-box transcription factor Brachyury has been considered a cancer-specific marker and a novel oncotarget in solid tumors. Brachyury overexpression has been described in various cancers, being associated with epithelial-mesenchymal transition, metastasis, and poor prognosis. However, its clinical association with testicular germ cell tumor is unknown. We analyzed the expression of Brachyury by immunohistochemistry in a series of well-characterized testicular germ cell tumor samples and at transcript level by in silico analysis. Additionally, we aimed to investigate the clinical significance of Brachyury in testicular germ cell tumor. Brachyury cytoplasm immunostaining was present in 89.6% (86/96) of cases with nuclear staining observed in 24% (23/96) of testicular germ cell tumor. Bioinformatics microarray expression analysis of two independent cohorts of testicular germ cell tumors showed similar results with increased levels of Brachyury in testicular germ cell tumors and metastasis compared with normal testis. Clinically, Brachyury nuclear staining was statistically associated with lower event-free survival (p=0.04) and overall survival (p=0.01) in intermediate/high-risk testicular germ cell tumors. Univariate analysis showed that Brachyury nuclear subcellular localization was a predictor of poor prognosis (p=0.02), while a tendency was observed by multivariate analysis (HR: 3.56, p=0.06). In conclusion, these results indicate that Brachyury plays an oncogenic role in testicular germ cell tumors and its subcellular localization in the nucleus may constitute a novel biomarker of poor prognosis and a putative oncotarget for intermediate/high-risk testicular germ cell tumor patients.

Loss of the Cyclin-Dependent Kinase Inhibitor 1 in the Context of Brachyury-Mediated Phenotypic Plasticity Drives Tumor Resistance to Immune Attack.

The acquisition of mesenchymal features by carcinoma cells is now recognized as a driver of metastasis and tumor resistance to a range of anticancer therapeutics, including chemotherapy, radiation, and certain small-molecule targeted therapies. With the recent successful implementation of immunotherapies for the treatment of various types of cancer, there is growing interest in understanding whether an immunological approach could be effective at eradicating carcinoma cells bearing mesenchymal features. Recent studies, however, demonstrated that carcinoma cells that have acquired mesenchymal features may also exhibit decreased susceptibility to lysis mediated by immune effector cells, including antigen-specific CD8+ T cells, innate natural killer (NK), and lymphokine-activated killer (LAK) cells. Here, we investigated the mechanism involved in the immune resistance of carcinoma cells that express very high levels of the transcription factor brachyury, a molecule previously shown to drive the acquisition of mesenchymal features by carcinoma cells. Our results demonstrate that very high levels of brachyury expression drive the loss of the cyclin-dependent kinase inhibitor 1 (p21CIP1, p21), an event that results in decreased tumor susceptibility to immune-mediated lysis. We show here that reconstitution of p21 expression markedly increases the lysis of brachyury-high tumor cells mediated by antigen-specific CD8+ T cells, NK, and LAK cells, TNF-related apoptosis-inducing ligand, and chemotherapy. Several reports have now demonstrated a role for p21 loss in cancer as an inducer of the epithelial–mesenchymal transition. The results from the present study situate p21 as a central player in many of the aspects of the phenomenon of brachyury-mediated mesenchymalization of carcinomas, including resistance to chemotherapy and immune-mediated cytotoxicity. We also demonstrate here that the defects in tumor cell death described in association with very high levels of brachyury could be alleviated via the use of a WEE1 inhibitor. Several vaccine platforms targeting brachyury have been developed and are undergoing clinical evaluation. These studies provide further rationale for the use of WEE1 inhibition in combination with brachyury-based immunotherapeutic approaches.

Extra-axial chordoma: a clinicopathologic analysis of six cases.

Extra-axial chordoma is an exceedingly rare tumor, with only 28 cases reported in the literature to date. Axial and extra-axial chordoma exhibits complete morphologic and immunophenotypic (expression of brachyury) overlap. However, in consideration of the non-canonical presentation, extra-axial chordoma is under-recognized and often misdiagnosed, most often as extraskeletal myxoid chondrosarcoma or myoepithelioma. To increase our understanding of the clinicopathologic features of extra-axial chordoma, six cases have been retrieved from the files of the Istituto Ortopedico Rizzoli and of the General Hospital of Treviso. The clinicoradiologic, morphologic, and molecular features have been analyzed, and the follow-up was updated. Our series included four female and two male patients; their ages ranged from 20 to 67years (mean 45.8years). All patients presented with a single mass localized in four cases in the soft tissue (posterior arm, left leg, dorsal aspect of the foot, and popliteal fossa), and in two cases in the bone (radius and second metacarpal bone). Grossly, the neoplasm was lobulated, with a fleshy cut surface and a diameter ranging between 0.8 and 8cm (mean 3.4cm). Morphologically, all six cases showed an epithelioid cell proliferation organized in nests and cords demarcated by fibrous septa and set in an abundant extracellular myxoid matrix. Neoplastic cells featured hyperchromatic nuclei and abundant vacuolated cytoplasm. Immunohistochemically, all six cases were strongly positive for EMA, cytokeratin AE1/AE3, S100, and brachyury. INI1 nuclear expression was retained. Smooth muscle actin, calponin, p63, and GFAP were all negative. Fluorescent in situ hybridization (FISH) analysis did not reveal rearrangements involving NR4A3, FUS, and EWSR1 genes. At follow-up (mean 55months), all patients were alive without disease after local surgical treatment. One patient underwent thigh amputation following multiple local recurrences and inguinal node metastases treated with marginal resection. In conclusion, primary extra-axial chordoma is an extremely rare neoplasm with distinct morphological and immunohistochemical features. Immunomorphology and molecular analysis allow distinction from both extraskeletal myxoid chondrosarcoma and myoepithelioma. Complete surgical resection appears to be curative.

Simulation of Embryonic Environments Produced by the Effect of Estradiol on Induced Pluripotent Stem Cells

Estradiol (E2) is one of the sex steroid hormones, whose various well-known functions include control of the female reproductive system, causing breast cancers, and inhibiting arterial sclerosis. In this study to investigate how E2 affected cell differentiation of induced pluripotent stem cells (iPSCs), we used different methods to culture hiPSCs, the undifferentiated state, longterm culture without passages (natural differentiation), embryoid bodies (EBs), and accelerated differentiation by activin, focusing on marker gene expression, including Brachyury, hepatocyte nuclear factor (HNF) 3β, estrogen receptor α, and other markers. When iPSCs began to differentiate by responding to E2, Brachyury expression decreased regardless of the amount of E2, cell line, or protocol. Other markers were up or down-regulated, but did not show any tendency except in the early stage of EBs. The definite timing that E2 had the strongest effect on the aspect of differentiation was the EB stage analogous to gastrulation, in which E2 repressed Brachyury expression and induced HNF3β at its restricted amount. These results suggest that the first target of E2 is the gastrulation stage.

Prognostic significance of expression of epithelial-mesenchymal transition driver brachyury in breast cancer and its association with subtype and characteristics.

Brachyury is a T-box transcription factor characterized as a driver of the epithelial-to-mesenchymal process, which is associated with poor prognosis of patients with breast cancer. The present study investigated expression of brachyury in breast cancer including primary tumor, metastatic and recurred tumor tissues, and the clinical significance and value of brachyury as a prognostic biomarker. This retrospective study included a series of 102 consecutive patients surgically resected between January 2005 and December 2011. Brachyury expression in tumor cell was evaluated using immunohistochemistry and scored as the immunoactivity. Of 102 patients, 62 primary tumors were positive for brachyury expression and 40 were negative. Multivariate analysis of disease-free survival (DFS) revealed brachyury expression, HER2 and lymphovascular invasion as independent prognostic factors [brachyury negative vs. positive hazard ratio (HR), 3.0; P=0.024; HER2 negative vs. positive HR, 4.9; P=0.003; lymphovascular invasion absent vs. present HR, 3.5; P=0.020]. These results were particularly observed in triple-negative breast cancer (TNBC), no recurrence or mortality occurred in brachyury negative group during the follow-up period, and therefore a significantly improved prognosis was demonstrated in these patients compared with the brachyury positive group [overall survival (OS), P=0.022; DFS, P=0.002]. Brachyury expression in metastatic lymph node/recurred tumors was not significantly associated with prognosis (OS, P=0.745; DFS, P=0.189). Therefore, Brachyury expression in primary tumor independently is a potential predictor of poor prognosis, particularly in TNBC, where it appears to serve a crucial function in recurrence and mortality. Brachyury vaccines under clinical trials are likely to be useful in patients with breast cancer.

Functional and evolutionary insights from the Ciona notochord transcriptome.

The notochord of the ascidian Ciona consists of only 40 cells, and is a longstanding model for studying organogenesis in a small, simple embryo. Here, we perform RNAseq on flow-sorted notochord cells from multiple stages to define a comprehensive Ciona notochord transcriptome. We identify 1364 genes with enriched expression and extensively validate the results by in situ hybridization. These genes are highly enriched for Gene Ontology terms related to the extracellular matrix, cell adhesion and cytoskeleton. Orthologs of 112 of the Ciona notochord genes have known notochord expression in vertebrates, more than twice as many as predicted by chance alone. This set of putative effector genes with notochord expression conserved from tunicates to vertebrates will be invaluable for testing hypotheses about notochord evolution. The full set of Ciona notochord genes provides a foundation for systems-level studies of notochord gene regulation and morphogenesis. We find only modest overlap between this set of notochord-enriched transcripts and the genes upregulated by ectopic expression of the key notochord transcription factor Brachyury, indicating that Brachyury is not a notochord master regulator gene as strictly defined.

Establishment and genomic characterization of the new chordoma cell line Chor-IN-1

Chordomas are rare, slowly growing tumors with high medical need, arising in the axial skeleton from notochord remnants. The transcription factor “brachyury” represents a distinctive molecular marker and a key oncogenic driver of chordomas. Tyrosine kinase receptors are also expressed, but so far kinase inhibitors have not shown clear clinical efficacy in chordoma patients. The need for effective therapies is extremely high, but the paucity of established chordoma cell lines has limited preclinical research. Here we describe the isolation of the new Chor-IN-1 cell line from a recurrent sacral chordoma and its characterization as compared to other chordoma cell lines. Chor-IN-1 displays genomic identity to the tumor of origin and has morphological features, growth characteristics and chromosomal abnormalities typical of chordoma, with expression of brachyury and other relevant biomarkers. Chor-IN-1 gene variants, copy number alterations and kinome gene expression were analyzed in comparison to other four chordoma cell lines, generating large scale DNA and mRNA genomic data that can be exploited for the identification of novel pharmacological targets and candidate predictive biomarkers of drug sensitivity in chordoma. The establishment of this new, well characterized chordoma cell line provides a useful tool for the identification of drugs active in chordoma.

Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma.

OBJECTIVE Chordoma is a slow-growing but clinically malignant tumor, and the prognosis remains poor in many cases. There is a strong impetus to develop more effective targeted molecular therapies. On this basis, the authors investigated the potential of Brachyury, a transcription factor involved in notochord development, as a candidate molecular target for the treatment of chordoma. METHODS Brachyury gene copy number and expression levels were evaluated by quantitative polymerase chain reaction in 27 chordoma samples, and the transcriptomes of Brachyury high-expression tumors (n = 4) and Brachyury low-expression tumors (n = 4) were analyzed. A chordoma cell line (U-CH2) was used to investigate the signaling pathways that regulate Brachyury expression. RESULTS All chordoma specimens expressed Brachyury, and expression levels varied widely. Patients with higher Brachyury expression had significantly shorter progression-free survival (5 months, n = 11) than those with lower expression (13 months, n = 16) (p = 0.03). Somatic copy number gain was confirmed in 12 of 27 (44%) cases, and copy number was positively correlated with Brachyury expression (R = 0.61, p < 0.001). Expression of PI3K/Akt pathway genes was upregulated in Brachyury high-expression tumors, and suppression of PI3K signaling led to reduced Brachyury expression and inhibition of cell growth in the U-CH2 chordoma cell line. CONCLUSIONS Activation of the PI3K/Akt pathway and Brachyury copy number gain are strongly associated with Brachyury overexpression, which appears to be a key event in chordoma growth regulation. These findings suggest that targeting Brachyury and PI3K/Akt signaling may be an effective new approach for treating chordoma.

Two-Element Transcriptional Regulation in the Canonical Wnt Pathway

The canonical Wnt pathway regulates numerous fundamental processes throughout development and adult physiology and is often disrupted in diseases [1-4]. Signal in the pathway is transduced by β-catenin, which in complex with Tcf/Lef regulates transcription. Despite the many processes that the Wnt pathway governs, β-catenin acts primarily on a single cis element in the DNA, the Wnt-responsive element (WRE), at times potentiated by a nearby Helper site. In this study, working with Xenopus, mouse, and human systems, we identified a cis element, distinct from WRE, upon which β-catenin and Tcf act. The element is 11bp long, hundreds of bases apart from the WRE, and exhibits a suppressive effect. In Xenopus patterning, loss of the 11-bp negative regulatory elements (11-bp NREs) broadened dorsal expression of siamois. In mouse embryonic stem cells, genomic deletion of the 11-bp NREs in the promoter elevated Brachyury expression. This reveals a previously unappreciated mechanism within the Wnt pathway, where gene response is not only driven by WREs but also tuned by 11-bp NREs. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), we found evidence for the NREs binding to β-catenin and Tcf-suggesting a dual action by β-catenin as a signal and a feedforward sensor. Analyzing β-catenin ChIP sequencing in human cells, we found the 11-bp NREs co-localizing with the WRE in 45%-71% of the peaks, suggesting a widespread role for the mechanism. This study presents an example of a more complex cis regulation by a signaling pathway, where a signal is processed through two distinct cis elements in a gene circuitry.

Abstract 26: Tcf7l1 Intrinsically Contributes to Cardiac Lineage Establishment

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins are critical downstream effectors of the canonical Wnt pathway. Tcf7l1 is the most abundant among the four TCF/LEF proteins and acts as a key regulator of self-renewal in ESCs. It negatively modulates the expression level of pluripotent genes, and prepares the epiblast for transition to lineage specification. Tcf7l1-/- embryos show a range of defects related to axis misinduction. Some severely affected embryos lack the heart and somites. Because of defective axis induction, whether Tcf7l1 intrinsically contributes to cardiac development is unknown. Earlier work in our lab established that TCF/LEF proteins cooperate with Oct4 in driving the transcription of the cardiac mesoderm factor Mesp1, but the responsible TCF/LEF protein is not identified. Here, we have built ESC systems to conditionally manipulate Tcf7l1 expression in a Tcf7l1-/- background. A combination of tTA/tet-O-Tcf7l1 transgenes allows us to temporally “knockout” Tcf7l1 with doxycycline, while a combination of rtTA/tet-O-Tcf7l1 transgenes allows us to temporally activate Tcf7l1 expression with doxycycline. We found that “knockout” of Tcf7l1 on day 4 during ESC differentiation caused decreased expression in cardiac genes including Tbx5, Nkx2-5, and α-MHC, without affecting the expression of Goosecoid, Eomes, and Brachyury, markers of mesendoderm development. Tcf7l1-/- ESCs showed slower progression toward mesoderm formation, but temporally activated Tcf7l1 induced cardiac gene expression after mesoderm was formed. Tcf7l1 ChIP-PCR demonstrated that Tcf7l1 bound to cis-regulatory regions of core cardiac transcription factor genes. Our data suggest that Tcf7l1 intrinsically contributes to cardiac lineage development through activating Mesp1 and downstream cardiac transcription factors.

Abstract 1533: Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype

Abstract BACKGROUND: Brachyury is a mesoderm specification transcription factor involved in notochord development and overexpressed in a variety of cancers, including chordoma. High levels of brachyury protein expression in cancers is associated with a poor prognosis in part due to its role in mediating epithelial-mesenchymal transition (EMT). TG02 is a multikinase inhibitor that targets transcriptional regulation of cyclin-dependent kinases (CDKs). Because chordoma is characterized by a relative paucity of genomic mutations and largely driven by the super-enhancer activity of brachyury expression, we investigated the downstream effect of a transcriptional inhibitor, TG02, in chordomas. METHODS: Established chordoma cell lines, UCH-1 and UM-Chor1, were exposed to increasing concentrations of TG02 to determine effect on brachyury protein expression. qPCR was used to analyze brachyury mRNA expression as well as the mesenchymal proteins, vimentin and fibronectin, typically associated with brachyury expression in tumor cells. Cell migration was evaluated using wound healing assay. CellTiterGlo Luminescence Assay was used to quantify cell viability. RESULTS: TG02 down-regulated protein expression of brachyury in a dose-dependent manner. Attenuation of brachyury expression was seen within 4 hours and persisted for 5 days upon single exposure to a clinically relevant concentration of TG02. Brachyury downregulation did not affect cell count or viability, and was associated with a quiescent phenotype, including impaired migration. Expression of vimentin and fibronectin, both associated with EMT, was also suppressed within 4 hours of TG02 treatment and persisted for 24 hours. CONCLUSIONS: Inhibition of brachyury expression and its downstream signaling is associated with quiescent behavior of chordoma cells. Pharmacologically induced transcriptional targeting of brachyury using TG02, a potent CDK9 inhibitor, represents a potential therapeutic strategy. Citation Format: Ashlee N. Seldomridge, Jinkyu Jung, Heather M. Sonnemann, Amber Giles, Kristan Meetze, Mark R. Gilbert, Claudia M. Palena, Deric M. Park. Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1533. doi:10.1158/1538-7445.AM2017-1533

Expression of epithelial-mesenchymal transition driver brachyury and status of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in predicting treatment responses to neoadjuvant chemotherapy of breast cancer.

Brachyury has been characterized as a driver of epithelial-mesenchymal transition process which is regarded as an important mechanism of cancer cell invasion and metastatic progression. The status of tumor-infiltrating lymphocytes has been proposed to predict response to neoadjuvant chemotherapy in breast cancer. We investigated the clinical significance and value of tumor-infiltrating lymphocytes and brachyury as biomarkers to predict treatment responses to neoadjuvant chemotherapy in breast cancer. We also examined the correlation of the Neo-Bioscore with tumor-infiltrating lymphocytes and brachyury to indirectly predict long-term outcome. This retrospective study included a series of 44 consecutive patients treated between January 2011 and December 2015. All patient samples were obtained using core needle biopsy before neoadjuvant chemotherapy. The relationship of expression of Brachyury and tumor-infiltrating lymphocyte subsets (CD8+, forkhead box protein 3 tumor-infiltrating lymphocytes) with clinicopathological factors was assessed to identify its predictive role with respect to tumor response to neoadjuvant chemotherapy and the outcome. Of 44 patients, 6 showed no response, 31 had partial response, and 7 demonstrated pathological complete response. Forkhead box protein 3 was significantly higher in the response group than in the no response group (no response = 2.6, partial response = 7.0, complete response = 9.7, p = 0.020). Brachyury expression was inversely associated with response to neoadjuvant chemotherapy, but the difference was not statistically significant ( p = 0.62). We also observed a significant association between forkhead box protein 3 ( p = 0.001) and the Neo-Bioscore, while only a marginal difference was observed with CD8+ expression ( p = 0.074). This study demonstrated that forkhead box protein 3 expression has value as the only independent marker that predicts a good response to neoadjuvant chemotherapy and that it is related with a good prognosis according to the Neo-Bioscore. Brachyury was significantly associated with estrogen receptor positive and human epidermal growth factor receptor 2 negative status; further study would be needed to clarify how it affects treatment prognosis.

Brachyury, a driver of epithelial mesenchymal transition, as an independent prognostic factor in high-grade testicular germ cell tumors.

e16039 Background: The T-box transcription factor Brachyury has been considered a cancer-specific marker and a novel oncotarget in solid tumors. Brachyury overexpression has been described in various cancers, being associated with epithelial-mesenchymal transition, metastasis and poor prognosis. However, its clinical association with testicular germ cell tumor (TGCT) is unknown. Methods: We analyzed the expression of Brachyury both at protein and transcript levels in a series of 96 TGCT samples and by in silico analysis, respectively. Additionally, we investigated the clinical significance of Brachyury in TGCT. Results: Brachyury protein showed to be over-represented in 89.6% (86/96) of TGCT cases with nuclear staining in 24% (23/96) of them. Microarray expression analysis in two independent cohorts of TGCTs showed similar results with increased levels of Brachyury in TGCTs and metastasis compared with normal testis. Clinically, Brachyury nuclear staining was statistically associated with event-free survival (p = 0.04) and overall survival (p = 0.01) in intermediate/high-risk TGCTs. Multivariate analysis showed that Brachyury nuclear subcellular localization was an independent predictor of poor prognosis (HR: 3.56, p = 0.06). Conclusions: These results indicate that Brachyury plays an oncogenic role in TGCTs and its subcellular localization in the nucleus is a potential novel biomarker of poor prognosis and an oncotarget for intermediate/high-risk TGCTs treatment.

Mechanism of arsenite toxicity in embryonic stem cells.

Environmental arsenite exposure has been linked to cancer as well as other diseases, presenting an important and serious public health problem. Toxicity of inorganic arsenite (iAs) has been investigated using animal models and cell culture, yet its developmental effects are poorly understood. This study investigated the molecular mechanism of iAs toxicity to ascertain insight into development and differentiation processes using mouse embryonic stem cells (ESCs). The results showed that iAs exposure affected morphology and integrity of ESC colonies as well as inhibited cell growth in a concentration-dependent manner, excluding concentrations <1 μM iAs which stimulated ESC growth. ESCs self-renewal and pluripotency was also affected as evident from the downregulation of transcription circuitry, Oct4, Nanog, Sox2 and Klf4 resulting in non-specific differentiation. ESCs exposed to iAs randomly differentiated into three germ layers, mesoderm, endoderm and ectoderm, as judged by transcriptional expression of Brachyury, Gata4 and FGF2, as well as translational expression of BRACHYURY, GATA4 and TUJ1 respectively. The differentiated cells represented osteogenic, chondrogenic, myogenic and neurogenic lineages as evident from upregulation of Col1, Sox9, Col2, Myog, Notch, Nes and Nef. Although iAs caused slight apoptosis with a concomitant increase in ROS levels, the exposed ESCs had significant Bcl2 expression, which could be involved in the protection against apoptosis. Further analysis revealed upregulation of Jun and P38 in ESCs with an increase in iAs concentration. These observations indicated that iAs stress caused random differentiation of ESCs via JNK/P38 pathways. These findings suggest that iAs exposure may cause teratogenicity during early fetal development. Copyright © 2017 John Wiley & Sons, Ltd.

Gastrulation in an annual killifish: Molecular and cellular events during germ layer formation in Austrolebias.

Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.

Inhibition of Histone Methyltransferase, Histone Deacetylase, and β-Catenin Synergistically Enhance the Cardiac Potential of Bone Marrow Cells.

Previously, we reported that treatment with the G9a histone methyltransferase inhibitor BIX01294 causes bone marrow mesenchymal stem cells (MSCs) to exhibit a cardiocompetent phenotype, as indicated by the induction of the precardiac markers Mesp1 and brachyury. Here, we report that combining the histone deacetylase inhibitor trichostatin A (TSA) with BIX01294 synergistically enhances MSC cardiogenesis. Although TSA by itself had no effect on cardiac gene expression, coaddition of TSA to MSC cultures enhanced BIX01294-induced levels of Mesp1 and brachyury expression 5.6- and 7.2-fold. Moreover, MSCs exposed to the cardiogenic stimulus Wnt11 generated 2.6- to 5.6-fold higher levels of the cardiomyocyte markers GATA4, Nkx2.5, and myocardin when pretreated with TSA in addition to BIX01294. MSC cultures also showed a corresponding increase in the prevalence of sarcomeric protein-positive cells when treated with these small molecule inhibitors. These results correlated with data showing synergism between (1) TSA and BIX01294 in promoting acetylation of lysine 27 on histone H3 and (2) BIX01294 and Wnt11 in decreasing β-catenin accumulation in MSCs. The implications of these findings are discussed in light of observations in the early embryo on the importance of β-catenin signaling and histone modifications for cardiomyocyte differentiation and heart development.

Bmp signaling maintains a mesoderm progenitor cell state in the mouse tailbud

Caudal somites are generated from a pool of progenitor cells located in the tailbud region. These progenitor cells form the presomitic mesoderm that gradually differentiates into somites under the action of the segmentation clock. The signals responsible for tailbud mesoderm progenitor pool maintenance during axial elongation are still elusive. Here, we show that Bmp signaling is sufficient to activate the entire mesoderm progenitor gene signature in primary cultures of caudal mesoderm cells. Bmp signaling acts through the key regulatory genes brachyury (T) and Nkx1-2 and contributes to the activation of several other regulators of the mesoderm progenitor gene network. In the absence of Bmp signaling, tailbud mesoderm progenitor cells acquire aberrant gene expression signatures of the heart, blood, muscle and skeletal embryonic lineages. Treatment of embryos with the Bmp inhibitor noggin confirmed the requirement for Bmp signaling for normal T expression and the prevention of abnormal lineage marker activation. Together, these results identify Bmp signaling as a non-cell-autonomous signal necessary for mesoderm progenitor cell homeostasis.