Brachyury Expression Research Articles

Intradural chordoma of the Meckel's cave: A challenging differential diagnosis

Chordomas are midline tumors that arise from embryonic remnants of the notochord and are considered to be malignant tumors because of their tendency to invade and destroy the involved bone. Cases of intradural chordomas without bone involvement have been rarely described with a predilection for prepontine location. The absence of bony invasion renders the complete excision of these tumors more feasible and is related to their better prognosis in comparison to conventional chordomas. Herein we report the first intradural chordoma arising in the Meckel's cave. The intradural location of the lesion, outside midline structures, in the absence of bone infiltration, made the differential diagnosis versus other meningeal lesions such as chordoid meningioma challenging. The intense and strong immunohistochemical expression of pan-cytokeratins, S100, cytokeratin-19 and of the notochordal marker brachyury allowed differential diagnosis toward other tumors showing chordoid morphology. The expression of brachyury, which had not been previously analyzed in intradural chordoma, definitely links the histogenesis of this neoplasia to the notochord, similar to that of conventional chordoma. We also show that, different from conventional chordoma, intradural chordoma does not express the metallo-proteinases (MMPs) -2 and -9, which may account for its indolent biological behavior.

The Effect of Human and Mouse Fibroblast Feeder Cells on Cardiac Differentiation of Human Pluripotent Stem Cells

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) are commonly used as feeder cells to maintain the pluripotent state of stem cells. The aim of the present study was to evaluate the effect of MEF and hFF feeders on the cardiac differentiation. Two human embryonic and two induced pluripotent stem cell lines were cultured on MEF and hFF before cardiac differentiation. The expression of Brachyury T was higher in cell lines cultured on MEF, than if cultured on hFF, suggesting enhanced mesoderm formation. However, significant positive influence of MEF feeders on cardiac differentiation was only seen with one cell line. Further, the ability of hFF to maintain pluripotency of stem cells originally cultured on MEF was quite poor. In conclusion, the cells behaved differently whether cultured on hFF or MEF feeders. However, the influence of the feeder cells on differentiation was less than the difference observed between the cell lines.

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0–2, A plus BMP-4 (4) during days 2–4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4–6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6–8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24+ cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.

Abstract P015: Hypoxia Regulates Embryonic Cardiac Myocyte Proliferation and Differentiation

Mammalian cardiac myocytes (CM) proliferate readily during development but exit the cell cycle perinatally. The molecular mechanism for this is poorly understood could lead to new approaches for cardiac regeneration. We employed a novel bioinformatics approach to identify transcription factors that may control perinatal cell-cycle arrest and identified hypoxia inducible factor 1α (Hif1α) as a potential regulator of CM proliferation and differentiation. To test our hypothesis, e12.5d embryonic heart cells were cultured in atmospheric (17%) or hypoxic (3%) [O2] and then assessed for proliferation over 4 days. Total cell numbers were significantly increased (∼2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2] after 4 days. Hypoxia increased numbers of cTn+ cells labeled with p-Histone-H3 indicating an increase in CM proliferation. Carboxyfluorescein succinimidyl ester staining indicated that the majority (70%) of cells cultured in 3% [O2] divided 2–3 times while the majority (73%) of cells in 17% [O2] divided 0–1 times after 3 days of culture. As expected, hypoxia stabilized the Hif1α protein and induced the expression of Vegf. Messenger RNA for the G1-S phase cyclin dependent kinase Ccnd2 was increased (>2 fold) in hypoxic vs. atmospheric cultured cells indicating that hypoxia my increase proliferation by regulating the expression of cell cycle machinery. Furthermore, early mesodermal transcription factors Brachyury and Mesp1 and the CM progenitor marker Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2] cultured cells suggesting that hypoxia positively regulates early mesodermal and CM progenitor cell differentiation. To validate this hypothesis, we differentiated mouse embryonic stem cells in atmospheric and hypoxic [O2] and observed similar increases in the expression of Brachyury, Mesp1 and Nkx2.5 in hypoxic differentiated cells. Transient culture (2 d) of embryoid bodies (EBs) in hypoxia increased the frequency of beating EBs and the % of cTn+ cells compared to cells differentiated in atmospheric O2. Taken together these data suggest that the hypoxic environment in which CMs develop may serve as a key signal that regulates mesodermal progenitor marker expression and CM progenitor cell differentiation and proliferation.

Brachyury and Related Tbx Proteins Interact with the Mixl1 Homeodomain Protein and Negatively Regulate Mixl1 Transcriptional Activity

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.

Id1 Maintains Embryonic Stem Cell Self-Renewal by Up-Regulation of Nanog and Repression of Brachyury Expression

Understanding the mechanism by which embryonic stem (ES) cells self-renew is crucial for the realization of their therapeutic potential. Earlier, overexpression of Id proteins was shown to be sufficient to maintain mouse ES cells in a self-renewing state even in the absence of serum. Here, we use ES cells derived from Id deficient mice to investigate the requirement for Id proteins in maintaining ES cell self-renewal. We find that Id1(-/-) ES cells have a defect in self-renewal and a propensity to differentiate. We observe that chronic or acute loss of Id1 leads to a down-regulation of Nanog, a critical regulator of self-renewal. In addition, in the absence of Id1, ES cells express elevated levels of Brachyury, a marker of mesendoderm differentiation. We find that loss of both Nanog and Id1 is required for the up-regulation of Brachyury, and ectopic Nanog expression in Id1(-/-) ES cells rescues the self-renewal defect, indicating that Nanog is the major downstream target of Id1. These results identify Id1 as a critical factor in the maintenance of ES cell self-renewal and suggest a plausible mechanism for its control of lineage commitment.

Efficient Induction of CD34+ Progenitor Cells From Human Bone Marrow Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells with Defined Chemicals

Abstract 2347Induced pluripotent stem cells (iPSCs) hold great potential in clinical application with indefinite passages, pluripotency of forming three germ layers and immunological compatibility with patients. Human bone marrow mesenchymal stem cell-derived iPSCs (hBMMSC-iPSCs) may have more virtues than other types of cell–derived iPSCs do in diseases treatment for their wide application in clinic for many years. Efficient differentiation of induced pluripotent stem cells (iPSCs) to a specialized cell type is a crucial step for iPSC clinical application. CD34+ progenitor cells derived from hBMMSC-iPSCs by treatment with defined chemicals have not been reported. Here, base on the efficient generation of hBMMSC-iPSCs with cDNA encoding four transcription factors OCT4, SOX2, KLF4 and c-MYC, and the compounds of p53 siRNA, VPA and Vc, we proposed that CD34+ progenitor cells, retaining hematopoietic and endothelial cell potential, could be efficiently obtained from hBMMSC-iPSCs by treatments with chemicals exerting function on mesoderm and hematopoietic cells. The differentiated hBMMSC-iPSCs treated with the chemicals comprised nearly 20% proportion of CD34+ progenitor cells, about 20-fold increasing compared to the spontaneous differentiation hBMMSC-iPSCs and higher than that from human skin fibroblast-derived iPSCs (hFib-iPSCs) (nearly 15%) and human embryonic stem cell line H1 (nearly 17%) by flow cytometry analysis. Those cells retained the hematopoietic and endothelial cell potential as CD34+ progenitor cells derived from umbilical cord blood. After coculture with the first chemical cocktails including BMP4 and SCF for 5 days, a higher proportion of the mesoderm cells were induced from hBMMSC-iPSCs than that from the untreated groups (P<0.05) by analysis the expression of Brachyury and GATA2 with RT-PCR and immunochemistry analysis. The efficient mesoderm cell activity was then directed to CD34+ progenitor cells with the following chemicals as SCF and IL-3 for another 7–9 days by analysis of cellular phenotype and the expression of the hematopoietic transcription factors TAL-1 and GATA-2. Sorted by CD34 magnetic separation and exposed to semisolid media with the hematopoietic cytokines including EPO and G-CSF, CD34+ progenitor cells formed various hematopoietic colonies as BFU-E, CFU-E, CFU-G, CFU-GM, and CFU-GEMM. CFUs derived from hBMMSC-iPSC groups treated with the chemicals outnumbered those from hFib-iPSCs, the spontaneous differentiation hBMMSC-iPSCs and H1 groups. Hematopoietic cell lineages as erythroid, granulocyte, and megakaryocyte were identified with Wright-Giemsa staining. During the hematopoietic commitment of hBMMSC-iPSCs, the pluripotent gene Oct4 continued to be down-regulated and completely vanished at day 14 after the induction. However, the expression of the hematologic transcription factor GATA-2 experienced a dynamic process with up-regulation at the initial induction stage and then gradual down-regulation after CD34+ progenitor cell commitment to CFU forming. Endothelial cell potential of CD34+ progenitor cells was also verified by demonstration of the vascular tube-like structure formation and the expression of endothelial specific markers CD31 and VE-CADHERIN. The efficient induction of CD34+ progenitor cells from hBMMSC-iPSCs with defined chemicals, avoiding contamination from indentified factors with using mouse feeder lays or other poorly defined induction components, gives an opportunity for the generation of unlimited HLA matched transplantable cells, provides cell models for study the mechanism of haemopoiesis and related diseases, and therefore promotes iPSC clinical application. Disclosures:No relevant conflicts of interest to declare.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of β-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic β-catenin protein expression and axin2 gene expression, but β-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/β-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β-catenin signaling activity in early IVD degeneration may be a reflection of its dual role.

P63 does not regulate brachyury expression in human chordomas and osteosarcomas

P63 does not regulate brachyury expression in human chordomas and osteosarcomas

Abstract C53: Overexpression of Brachyury in human carcinoma cells drives the acquisition of resistance to anticancer therapeutics

Abstract The epithelial-mesenchymal transition (EMT) has been recognized as a process crucial to the progression of carcinomas, mediating the conversion of stationary epithelial tumor cells into mesenchymal-like, invasive tumor cells. Recent reports have also demonstrated a potential association between tumor EMT and the acquisition of resistance to cell death in carcinoma cells. We recently identified the T-box transcription factor Brachyury, a molecule prevalently expressed in human tumors but seldom found in normal adult tissues, as a novel driver of EMT in human carcinomas. Brachyury was demonstrated to induce the expression of molecules associated with the mesenchymal phenotype, tumor cell motility and invasiveness in vitro, as well as metastatic propensity in xenograft models. In the current study, we investigated whether deregulated expression of Brachyury in human lung tumor cells is also associated with acquisition of resistance to the conventional anti-cancer modalities, chemotherapy and radiation. Our results demonstrated that over-expression of Brachyury in epithelial lung tumor cells significantly improved their survival in response to treatment with various doses of Taxotere, Cisplatin, Vinorelbine, and combinations of Cisplatin plus Vinorelbine, as well as to various doses of gamma-radiation. Similarly, inhibition of Brachyury expression by using Brachyury-specific shRNA constructs in mesenchymal-like, human lung tumor cells resulted in enhanced susceptibility to the same cytotoxic agents. Analysis of clonal lines derived from single-cell isolates of human lung tumor cells expressing various amounts of Brachyury revealed an inverse relationship between Brachyury levels and tumor growth, and a positive correlation of Brachyury with the ability to resist treatment by Taxotere, Cisplatin, Vinorelbine, and Cisplatin plus Vinorelbine combinations. Detailed gene expression analysis demonstrated a positive association between Brachyury expression and markers of tumor stemness, such as the MDR1/ABCB1 transporter, and the self-renewal transcription factors Sox-2, Oct4, and Nanog. These results thus raise the possibility that in addition to being a mediator of EMT, Brachyury may also be a marker for human tumor cells bearing stem-like characteristics and resistance to conventional therapeutics. One approach that may overcome the therapeutic resistance of tumors undergoing Brachyury-mediated EMT is that of immune-mediated targeting – we have previously characterized the immunogenicity of the Brachyury protein and generated Brachyury-specific human T-cell lines that have the ability to lyse Brachyury-positive tumor cells. We hypothesize that the eradication of Brachyury-expressing tumor cells via Brachyury-based immunotherapeutic approaches could be efficient at eliminating tumor cells with metastatic propensity as well as at alleviating tumor resistance in response to conventional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C53.

Abstract A64: Brachyury-mediated epithelial-mesenchymal transition of human carcinoma cells is associated with an increased resistance to immune-mediated attack

Abstract The epithelial-mesenchymal transition (EMT) has recently been recognized as a relevant process during the progression of carcinomas. Epithelial tumor cells undergoing EMT have been shown to acquire a mesenchymal-like phenotype, cell motility, and invasiveness, in vivo metastatic propensity and stem cell-like properties, including the ability to resist cell death initiated by traditional cancer treatment modalities such as chemotherapy and radiation. The development of therapeutic interventions aimed at interfering with EMT is therefore emerging as a rational approach for the prevention of cancer metastasis and alleviation of therapeutic resistance. Promising results recently obtained in preclinical and clinical studies with cancer vaccines may lead to their use for the treatment of various types of cancer; the concept of utilizing a vaccine to specifically target essential regulators of EMT is yet to be exploited in the field of tumor immunology. Recently, we have demonstrated that Brachyury, a T-box transcription factor essential for embryonic development, plays a central role in the induction of EMT in human carcinoma cells. In contrast to other drivers of EMT, Brachyury is highly expressed in various types of human tumors, while its expression in most normal adult tissues is undetectable. Moreover, Brachyury-specific T cells capable of lysing human tumor cell lines in an antigen-specific, HLA-restricted manner can be expanded from the peripheral blood of cancer patients, making Brachyury an attractive target for a vaccination strategy designed to specifically target cells undergoing an EMT. In light of the documented resistance of mesenchymal-like tumor cells to chemotherapy and radiation, we have thoroughly investigated the susceptibility of tumor cells undergoing EMT to immune-mediated attack. Utilizing several epithelial human tumor cell lines stably transfected to express various levels of Brachyury, we have observed that tumor cells with low to moderate amounts of Brachyury can be efficiently lysed by various immune-mediated mechanisms, while very high levels of Brachyury expression correlate with increased resistance to lysis mediated by natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and antigen-specific CD8+ T cells. It has been suggested that EMT is a reversible process, whereby cells can undergo a mesenchymal-epithelial transition (MET). By stably inhibiting the expression of Brachyury in various mesenchymal-like human tumor cell lines, we have also demonstrated that induction of MET restores susceptibility of tumor cells to immune-mediated attack. Currently, the mechanisms responsible for the acquisition of increased resistance to immune-mediated killing by cells undergoing an EMT are being investigated. The results from these studies will be fundamental in designing strategies aimed at rendering metastatic carcinoma cells more susceptible not only to chemotherapy and radiation, but also to immunotherapeutic interventions against the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A64.

Gastrulation in rabbit blastocysts depends on insulin and insulin-like-growth-factor 1

Insulin and insulin-like-growth-factor 1 (IGF1) are components of the uterine secretions. As potent growth factors they influence early embryo development. The underlying molecular mechanisms are largely unknown. Here we report on the effects of insulin and IGF1 on early gastrulation in rabbit blastocysts. We have studied blastocysts grown in vivo in metabolically healthy rabbits, in rabbits with type 1 diabetes and in vitro in the presence or absence of insulin or IGF1. Embryonic disc morphology and expression of Brachyury, Wnt3a and Wnt4 were analysed by qPCR and IHC. Pre-gastrulated blastocysts (stage 0/1) cultured with insulin or IGF1 showed a significantly higher capacity to form the posterior mesoderm and primitive streak (stage 2 and 3) than blastocysts cultured without growth factors. In gastrulating blastocysts the levels of the mesoderm-specific transcription factor Brachyury and the Wnt signalling molecules Wnt3a and Wnt4 showed a stage-specific expression pattern with Brachyury transcripts increasing from stage 0/1 to 3. Wnt4 protein was found spread over the whole embryoblast. Insulin induced Wnt3a, Wnt4 and Brachyury expression in a temporal- and stage-specific pattern. Only blastocysts cultured with insulin reached the Wnt3a, Wnt4 and Brachyury expression levels of stage 2 in vivo blastocysts, indicating that insulin is required for Wnt3a, Wnt4 and Brachyury expression during gastrulation. Insulin-induced Wnt3a and Wnt4 expression preceded Brachyury. Wnt3a-induced expression could be depleted by MEK1 inhibition (PD98059). Involvement of insulin in embryonic Wnt3a expression was further shown in vivo with Wnt3a expression being notably down regulated in stage 2 blastocysts from rabbits with type 1 diabetes. Blastocysts grown in diabetic rabbits are retarded in development, a finding which supports our current results that insulin is highly likely required for early mesoderm formation in rabbit blastocysts by inducing a distinct spatiotemporal expression profile of Wnt3a, Wnt4 and Brachyury.

Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target

Chordoma is a malignant bone neoplasm hypothesized to arise from notochordal remnants along the length of the neuraxis. Recent genomic investigation of chordomas has identified T (Brachyury) gene duplication as a major susceptibility mutation in familial chordomas. Brachyury plays a vital role during embryonic development of the notochord and has recently been shown to regulate epithelial-to-mesenchymal transition in epithelial-derived cancers. However, current understanding of the role of this transcription factor in chordoma is limited due to the lack of availability of a fully characterized chordoma cell line expressing Brachyury. Thus, the objective of this study was to establish the first fully characterized primary chordoma cell line expressing gain of the T gene locus that readily recapitulates the original parental tumor phenotype in vitro and in vivo. Using an intraoperatively obtained tumor sample from a 61-year-old woman with primary sacral chordoma, a chordoma cell line (JHC7, or Johns Hopkins Chordoma Line 7) was established. Molecular characterization of the primary tumor and cell line was conducted using standard immunostaining and Western blotting. Chromosomal aberrations and genomic amplification of the T gene in this cell line were determined. Using this cell line, a xenograft model was established and the histopathological analysis of the tumor was performed. Silencing of Brachyury and changes in gene expression were assessed. The authors report, for the first time, the successful establishment of a chordoma cell line (JHC7) from a patient with pathologically confirmed sacral chordoma. This cell line readily forms tumors in immunodeficient mice that recapitulate the parental tumor phenotype with conserved histological features consistent with the parental tumor. Furthermore, it is demonstrated for the first time that silencing of Brachyury using short hairpin RNA renders the morphology of chordoma cells to a more differentiated-like state and leads to complete growth arrest and senescence with an inability to be passaged serially in vitro. This report represents the first xenograft model of a sacral chordoma line described in the literature and the first cell line established with stable Brachyury expression. The authors propose that Brachyury is an attractive therapeutic target in chordoma and that JHC7 will serve as a clinically relevant model for the study of this disease.

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27Kip1 in hESC lead to a G1 phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27Kip1 protein occupies the Twist1 gene promoter and manipulation of p27Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.

Abstract 2418: Establishment and characterization of a primary chordoma xenograft

Abstract Chordomas are slow growing, malignant cancers believed to originate from notochord remnants. Chordoma accounts for 1% of cancers of the central nervous system with an annual incidence of 0.08 cases per 100,000 individuals. There are 3 different histologic subtypes- classic chordoma, chondroid chordoma, and dedifferentiated chordoma. Chordomas are characterized by the presence of physaliphorous cells with vacuolated cytoplasm and high expression of the transcription factor T Brachury. Management of chordoma is primarily surgical – chemotherapy and radiation therapy have limited effect. The development of effective treatments for this tumor is hampered by the lack of adequate animal models. We report here the establishment of the first primary xenograft model of chordoma. Tumor excised from a patient diagnosed with classic chordoma was implanted adjacent to the right proximal tibia in a NOD/SCID/IL2Rγ (null) mouse. The resulting tumor has been serially transplanted at least 10 times with no diminution in growth rate over time. Histologically, the xenograft does not appear to be a classic chordoma; interestingly, the patient from whom the xenograft was derived relapsed with a dedifferentiated morphology. To confirm the identity of the tumor xenograft, T brachyury expression was demonstrated by immunohistochemistry and PCR. We have begun to characterize this xenograft by gene expression profiling and comparative genomic hybridization. Expression profiling results were verified by RT-PCR. Cells isolated from xenografts were analyzed by the Aldefluor assay to assess the levels of expression of aldehyde dehydrogenase activity. A cell line was generated from this xenograft as well and is being characterized. In summary, we report here the establishment of the first primary chordoma xenograft. Initial molecular characterization of the xenograft and a cell line derived from this tumor is ongoing. These tools will shed valuable light on the pathophysiology of this rare tumor and allow the preclinical development of novel treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2011-2418

Abstract SY24-02: Development of recombinant vaccines for the prevention and therapy of human carcinomas

Abstract SY24-02: Development of recombinant vaccines for the prevention and therapy of human carcinomas

Nodal signal is required for morphogenetic movements of epiblast layer in the pre‐streak chick blastoderm

During axis formation in amniotes, posterior and lateral epiblast cells in the area pellucida undergo a counter-rotating movement along the midline to form primitive streak (Polonaise movements). Using chick blastoderms, we investigated the signaling involved in this cellular movement in epithelial-epiblast. In cultured posterior blastoderm explants from stage X to XI embryos, either Lefty1 or Cerberus-S inhibited initial migration of the explants on chamber slides. In vivo analysis showed that inhibition of Nodal signaling by Lefty1 affected the movement of DiI-marked epiblast cells prior to the formation of primitive streak. In Lefty1-treated embryos without a primitive streak, Brachyury expression showed a patchy distribution. However, SU5402 did not affect the movement of DiI-marked epiblast cells. Multi-cellular rosette, which is thought to be involved in epithelial morphogenesis, was found predominantly in the posterior half of the epiblast, and Lefty1 inhibited the formation of rosettes. Three-dimensional reconstruction showed two types of rosette, one with a protruding cell, the other with a ventral hollow. Our results suggest that Nodal signaling may have a pivotal role in the morphogenetic movements of epithelial epiblast including Polonaise movements and formation of multi-cellular rosette.

Smek promotes histone deacetylation to suppress transcription of Wnt target gene brachyury in pluripotent embryonic stem cells

In embryonic stem cells (ESCs), Wnt-responsive development-related genes are silenced to maintain pluripotency and their expression is activated during differentiation. Acetylation of histones by histone acetyltransferases stimulates transcription, whereas deacetylation of histones by HDACs is correlated with transcriptional repression. Although Wnt-mediated gene transcription has been intimately linked to the acetylation or deacetylation of histones, how Wnt signaling regulates this type of histone modification is poorly understood. Here, we report that Smek, a regulatory subunit of protein phosphatase 4 (PP4) complex, plays an important role in histone deacetylation and silencing of the Wnt-responsive gene, brachyury, in ESCs. Smek mediates recruitment of PP4c and HDAC1 to the Tcf/Lef binding site of the brachyury promoter and inhibits brachyury expression in ESCs. Activation of Wnt signaling during differentiation causes disruption of the Smek/PP4c/HDAC1 complex, resulting in an increase in histones H3 and H4 acetylation at the brachyury gene locus. These results suggest that the Smek-containing PP4 complex represses transcription of Wnt-responsive development-related genes through histone deacetylation, and that this complex is essential for ESC pluripotency maintenance.

Three-dimensional immunohistochemical characterization of lineage commitment by localization of T and FOXA2 in porcine peri-implantation embryos

Gene expression during gastrulation in porcine embryos has been sparsely studied, but there are indications that species-specific patterns exist. Here, we investigated the three-dimensional (3D) expression of the T-box transcription factor Brachyury (T) and the forkhead box transcription factor FOXA2 by immunocytochemistry in porcine peri-gastrulation embryos. The first T(+) cells were detected in posterior epiblast of ovoid blastocysts. Later T(+) FOXA2(-) cells were found in the posterior primitive streak (PS) and nascent mesoderm, T(+) FOXA2(+) cells in the anterior PS, probably identifying the organizer region, and T(-) FOXA2(+) cells anterior to this region. In embryos with a neural groove, T and FOXA2 were co-expressed in the node and notochord, FOXA2 was expressed in the floor plate and posteriorly T was expressed in the streak. In all developmental stages, FOXA2 was expressed in the entire hypoblast/definitive endoderm. We conclude that the expression pattern of T and FOXA2 is largely conserved between pig and mouse.

Specificity of brachyury in the distinction of chordoma from clear cell renal cell carcinoma and germ cell tumors: a study of 305 cases

Brachyury is recognized as a specific marker for notochord-derived tissues and neoplasms, and has become a defining immunohistochemical feature of chordoma. The main differential diagnostic consideration for chordoma is chondrosarcoma, which is known to lack brachyury expression. However, within the spectrum of genitourinary neoplasia, metastatic germ cell tumors and clear cell renal cell carcinoma may also be close morphological mimics of chordoma, particularly given the increasing prevalence of small tissue samples from image-guided biopsies. Although immunoreactivity for brachyury has been reported in a few germ cell tumors, a thorough characterization of staining by specific subtype has not been performed in a large series. Additionally, brachyury expression in clear cell renal cell carcinoma has not been well studied. In this study, immunohistochemical expression with the brachyury antibody was evaluated in 111 germ cell tumors, 30 non-neoplastic and neoplastic (non-germ cell) testicular tissues, and 184 metastatic clear cell renal cell carcinomas using tissue microarray technology. In addition, immunoreactivity for PAX-8 and SALL-4 was evaluated in 12 chordomas on whole section. No nuclear brachyury expression was identified in any of the 101 germ cell tumors within the tissue microarray (including choriocarcinoma (1), embryonal carcinoma (20), intratubular germ cell neoplasia unclassified (2), seminoma (64), spermatocytic seminoma (1), teratoma (5) and yolk sac tumor (8)), in any of the 30 non-neoplastic and neoplastic (non-germ cell) testicular tissues, or in any of the 10 whole-section seminomas. All 184 metastatic clear cell renal cell carcinomas were also non-reactive for brachyury. All 12 chordomas showed strong nuclear immunoreactivity for brachyury, but no expression of SALL-4. In all, 1 of 12 chordoma cases showed patchy, 1+ nuclear immunoreactivity for PAX-8. This study confirms the specificity of brachyury for chordoma in the differential diagnostic distinction from the potential genitourinary mimics, germ cell tumors and metastatic clear cell renal cell carcinoma.