CD166 promotes cancer stem cell-like phenotype via the EGFR/ERK1/2 pathway in the nasopharyngeal carcinoma cell line CNE-2R.

AIMS: The present study aimed to investigate the role and underlying mechanisms of CD166 in cancer stem cell-like (CSCs) phenotype of the radioresistant nasopharyngeal carcinoma cell CNE-2R. MAIN METHODS: Established CD166-shRNA- CNE-2R cell line by lentivirus-mediated silencing CD166. Then, CSC-related genes mRNAs and proteins, and EGFR/ERK1/2 signaling pathway were detected using RT-PCR and western blot. Sphere formation assay was performed to evaluate the sphere formation capacity in CD166-shRNA- CNE-2R cells. The tumorigenesis ability in vivo was examined in nude mice mode. KEY FINDINGS: Downregulation of CD166 inhibited the expression of the CSC-related genes, pEGFR and pERK in vitro and vivo. The capacity to form spheres and tumorigenesis was significantly decreased in CD166-shRNA cells. Furthermore, EGF-stimulated CD166-shRNA cells exhibited an increase in CSC-like traits by activating EGFR/ERK1/2 signaling. SIGNIFICANCE: CD166 induced CSCs formation by activating the EGFR/ERK1/2 signaling pathway in nasopharyngeal carcinoma, which may serve as a critical molecular target for NPC therapeutic strategies.

A novel HDGF-ALCAM axis promotes the metastasis of Ewing sarcoma via regulating the GTPases signaling pathway.

Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

Hic-5 regulates Src-induced invadopodia rosette formation and organization.

Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.

Fibrillar force generation by fibroblasts depends on formin.

Fibroblasts in the extra-cellular matrix (ECM) often adopt a predominantly one-dimensional fibrillar geometry by virtue of their adhesion to the fibrils in the ECM. How much forces such fibrillar fibroblasts exert and how they respond to the extended stiffness of their micro-environment comprising of other ECM components and cells are not clear. We use fibroblasts adherent on fibronectin lines micropatterned onto soft polyacrylamide gels as an in vitro experimental model that maintains fibrillar cell morphology while still letting the cell mechanically interact with a continuous micro-environment of specified stiffness. We find that the exerted traction, quantified as the strain energy or the maximum exerted traction stress, is not a function of cell length. Both the strain energy and the maximum traction stress exerted by fibrillar cells are similar for low (13 kPa) or high (45 kPa) micro-environmental stiffness. Furthermore, we find that fibrillar fibroblasts exhibit prominent linear actin structures. Accordingly, inhibition of the formin family of nucleators strongly decreases the exerted traction forces. Interestingly, fibrillar cell migration is, however, not affected under formin inhibition. Our results suggest that fibrillar cell migration in such soft microenvironments is not dependent on high cellular force exertion in the absence of other topological constraints.

Reporter Analyses Reveal Redundant Enhancers that Confer Robustness on Cis-Regulatory Mechanisms.

Reporter analyses of Hox1 and Brachyury (Bra) genes have revealed examples of redundant enhancers that provide regulatory robustness. Retinoic acid (RA) activates through an RA-response element the transcription of Hox1 in the nerve cord of the ascidian Ciona intestinalis. We also found a weak RA-independent neural enhancer within the second intron of Hox1. The Hox1 gene in the larvacean Oikopleura dioica is also expressed in the nerve cord. The O. dioica genome, however, does not contain the RA receptor-encoding gene, and the expression of Hox1 has become independent of RA. We have found that the upstream sequence of the O. dioica Hox1 was able to activate reporter gene expression in the nerve cord of the C. intestinalis embryo, suggesting that an RA-independent regulatory system in the nerve cord might be common in larvaceans and ascidians. This RA-independent redundant regulatory system may have facilitated the Oikopleura ancestor losing RA signaling without an apparent impact on Hox1 expression domains. On the other hand, vertebrate Bra is expressed in the ventral mesoderm and notochord, whereas its ascidian ortholog is exclusively expressed in the notochord. Fibroblast growth factor (FGF) induces Bra in the ventral mesoderm in vertebrates, whereas it induces Bra in the notochord in ascidians. Disruption of the FGF signal does not completely silence Bra expression in ascidians, suggesting that FGF-dependent and independent enhancers might comprise a redundant regulatory system in ascidians. The existence of redundant enhancers, therefore, provides regulatory robustness that may facilitate the acquisition of new expression domains.

Investigating Evolutionarily Conserved Molecular Mechanisms Controlling Gene Expression in the Notochord.

Ascidian embryos have been employed as model systems for studies of developmental biology for well over a century, owing to their desirable blend of experimental advantages, which include their rapid development, traceable cell lineage, and evolutionarily conserved morphogenetic movements. Two decades ago, the development of a streamlined electroporation method drastically reduced the time and cost of transgenic experiments, and, along with the elucidation of the complete genomic sequences of several ascidian species, propelled these simple chordates to the forefront of the model organisms available for studies of regulation of gene expression. Numerous ascidian sequences with tissue-specific enhancer activity were isolated and rapidly characterized through systematic in vivo experiments that would require several weeks in most other model systems. These cis-regulatory sequences include a large collection of notochord enhancers, which have been used to visualize notochord development in vivo, to generate mutant phenotypes, and to knock down genes of interest. Moreover, their detailed characterization has allowed the reconstruction of different branches of the notochord gene regulatory network. This chapter describes how the use of transgenic techniques has rendered the ascidian Ciona a competitive model organism for studies of notochord development, evolution, and gene regulation.

ß-Catenin acts in a position-independent regeneration response in the simple eumetazoan Hydra.

Wnt/ß-Catenin signaling plays crucial roles in regenerative processes in eumetazoans. It also acts in regeneration and axial patterning in the simple freshwater polyp Hydra, whose morphallactic regenerative capacity is unparalleled in the animal kingdom. Previous studies have identified ß-catenin as an early response gene activated within the first 30min in Hydra head regeneration. Here, we have studied the role of ß-Catenin in more detail. First, we show that nuclear ß-Catenin signaling is required for head and foot regeneration. Loss of nuclear ß-Catenin function blocks head and foot regeneration. Transgenic Hydra tissue, in which ß-Catenin is over-expressed, regenerates more heads and feet. In addition, we have identified a set of putative ß-Catenin target genes by transcriptional profiling, and these genes exhibit distinct expression patterns in the hypostome, in the tentacles, or in an apical gradient in the body column. All of them are transcriptionally up-regulated in the tips of early head and foot regenerates. In foot regenerates, this is a transient response, and expression starts to disappear after 12-36h. ChIP experiments using an anti-HydraTcf antibody show Tcf binding at promoters of these targets. We propose that gene regulatory ß-Catenin activity in the pre-patterning phase is generally required as an early regeneration response. When regenerates are blocked with iCRT14, initial local transcriptional activation of ß-catenin and the target genes occurs, and all these genes remain upregulated at the site of both head and foot regeneration for the following 2-3 days. This indicates that the initial regulatory network is followed by position-specific programs that inactivate fractions of this network in order to proceed to differentiation of head or foot structures. brachyury1 (hybra1) has previously been described as early response gene in head and foot regeneration. The HyBra1 protein, however, appears in head regenerating tips not earlier than about twelve hours after decapitation, and HyBra1 translation does not occur in iCRT14-treated regenerates. Foot regenerates never show detectable levels of HyBra1 protein at all. These results suggest that translational control mechanisms may play a decisive role in the head- and foot-specific differentiation phase, and HyBra1 is an excellent candidate for such a key regulator of head specification.

ALCAM a novel biomarker in patients with type 2 diabetes mellitus complicated with diabetic nephropathy.

BACKGROUND & AIM: Activated leukocyte cell adhesion molecule (ALCAM/CD166) functions analogue to the receptor of advanced glycation end products, which has been implicated in the development of diabetic nephropathy (DN). We investigated the expression of ALCAM and its ligand S100B in patients with DN. METHODS: A total of 34 non-diabetic patients, 29 patients with type 2 diabetes and normal albuminuria and 107 patients with type 2 diabetes complicated with DN were assessed for serum concentration of soluble ALCAM (sALCAM) by ELISA. Expression of ALCAM and S100B in kidney histology from patients with DN was determined by immunohistochemistry. Cell expression of ALCAM and S100B was analyzed through confocal immunofluorescence microscopy. RESULTS: Serum concentration of sALCAM was increased in diabetic patients with DN compared to non-diabetic (59.85±14.99ng/ml vs. 126.88±66.45ng/ml, P<0.0001). Moreover sALCAM correlated positively with HbA1c (R=0.31, P<0.0001), as well as with the stages of chronic kidney disease and negatively correlated with eGFR (R=-0.20, P<0.05). In diabetic patients with normal albuminuria sALCAM was increased compared to patients with DN (126.88±66.45ng/ml vs. 197.50±37.17ng/ml, P<0.0001). In diabetic patients, ALCAM expression was significantly upregulated in both the glomeruli and tubules (P<0.001). ALCAM expression in the glomeruli correlated with presence of sclerosis (R=0.25, P<0.001) and localized mainly in the podocytes supporting the hypothesis that membrane bound ALCAM drives diabetic nephropathy and thus explaining sALCAM decrease in diabetic patients with DN. The expression of S100B was increased significantly in the glomeruli of diabetic patients (P<0.001), but not in the tubules. S100B was as well localized in the podocytes. CONCLUSIONS: This study identifies for the first time ALCAM as a potential mediator in the late complications of diabetes in the kidney.

Regulation of brachyury by fibroblast growth factor receptor 1 in lung cancer.

Recent evidence suggests that T-box transcription factor brachyury plays an important role in lung cancer development and progression. However, the mechanisms underlying brachyury-driven cellular processes remain unclear. Here we found that fibroblast growth factor receptor 1/mitogen-activated protein kinase (FGFR1/MAPK) signaling regulated brachyury in lung cancer. Analysis of FGFR1-4 and brachyury expression in human lung tumor tissue and cell lines found that only expression of FGFR1 was positively correlated with brachyury expression. Specific knockdown of FGFR1 by siRNA suppressed brachyury expression and epithelial-mesenchymal transition (EMT) (upregulation of E-cadherin and ß-catenin and downregulation of Snail and fibronectin), whereas forced overexpression of FGFR1 induced brachyury expression and promoted EMT in lung cancer cells. Activation of fibroblast growth factor (FGF)/FGFR1 signaling promoted phosphorylated MAPK extracellular signal-regulated kinase (ERK) 1/2 translocation from cytoplasm to nucleus, upregulated brachyury expression, and increased cell growth and invasion. In addition, human lung cancer cells with higher brachyury expression were more sensitive to inhibitors targeting FGFR1/MAPK pathway. These findings suggest that FGFR1/MAPK may be important for brachyury activation in lung cancer, and this pathway may be an appealing therapeutic target for a subset of brachyury-driven lung cancer.

Deep Sequencing Reveals a Novel miR-22 Regulatory Network with Therapeutic Potential in Rhabdomyosarcoma.

Current therapeutic options for the pediatric cancer rhabdomyosarcoma have not improved significantly, especially for metastatic rhabdomyosarcoma. In the current work, we performed a deep miRNA profiling of the three major human rhabdomyosarcoma subtypes, along with cell lines and normal muscle, to identify novel molecular circuits with therapeutic potential. The signature we determined could discriminate rhabdomyosarcoma from muscle, revealing a subset of muscle-enriched miRNA (myomiR), including miR-22, which was strongly underexpressed in tumors. miR-22 was physiologically induced during normal myogenic differentiation and was transcriptionally regulated by MyoD, confirming its identity as a myomiR. Once introduced into rhabdomyosarcoma cells, miR-22 decreased cell proliferation, anchorage-independent growth, invasiveness, and promoted apoptosis. Moreover, restoring miR-22 expression blocked tumor growth and prevented tumor dissemination in vivo Gene expression profiling analysis of miR-22-expressing cells suggested TACC1 and RAB5B as possible direct miR-22 targets. Accordingly, loss- and gain-of-function experiments defined the biological relevance of these genes in rhabdomyosarcoma pathogenesis. Finally, we demonstrated the ability of miR-22 to intercept and overcome the intrinsic resistance to MEK inhibition based on ERBB3 upregulation. Overall, our results identified a novel miR-22 regulatory network with critical therapeutic implications in rhabdomyosarcoma. Cancer Res; 76(20); 6095-106. ©2016 AACR.

Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila.

The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.

The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes.

Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.

The potential role of Brachyury in inducing epithelial-to-mesenchymal transition (EMT) and HIF-1α expression in breast cancer cells.

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1α (HIF-1α) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury on tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling.

Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity.

During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics.

Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting.

Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.

A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.

During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.

Charting Brachyury-mediated developmental pathways during early mouse embryogenesis.

To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator--Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies.

The T-box transcription factor Brachyury, a molecule frequently detected in human cancers but seldom found in normal adult tissue, has recently been characterized as a driver of the epithelial-to-mesenchymal switch of human carcinomas. In the current investigation, we present data demonstrating that in two different human lung carcinoma models expression of Brachyury strongly correlates with increased in vitro resistance to cytotoxic therapies, such as chemotherapy and radiation. We also demonstrate that chemotherapy treatment in vitro selects for tumor cells with high levels of Brachyury and that the degree of resistance to therapy correlates with the level of Brachyury expression. In vitro and in vivo, human lung carcinoma cells with higher levels of Brachyury divide at slower rates than those with lower levels of Brachyury, a phenomenon associated with marked downregulation of cyclin D1, phosphorylated Rb and CDKN1A (p21). Chromatin immunoprecipitation and luciferase reporter assays revealed that Brachyury binds to a half T-box consensus site located within the promoter region of the p21 gene, indicating a potential mechanism for the observed therapeutic resistance associated with Brachyury expression. Finally, we demonstrate that in vivo treatment of tumor xenografts with chemotherapy results in the selective growth of resistant tumors characterized by high levels of Brachyury expression. Altogether, these results suggest that Brachyury expression may attenuate cell cycle progression, enabling tumor cells to become less susceptible to chemotherapy and radiation in human carcinomas.

An intact brachyury function is necessary to prevent spurious axial development in Xenopus laevis.

We have previously shown that the member of the HES family hairy2 induces the ectopic expression of dorsal markers when it is overexpressed in the ventral side of Xenopus embryos. Intriguingly, hairy2 represses the mesoderm transcription factor brachyury (bra) throughout its domain in the marginal zone. Here we show that in early gastrula, bra and hairy2 are expressed in complementary domains. Overexpression of bra repressed hairy2. Interference of bra function with a dominant-negative construct expanded the hairy2 domain and, like hairy2 overexpression, promoted ectopic expression of dorsal axial markers in the ventral side and induced secondary axes without head and notochord. Hairy2 depletion rescued the ectopic dorsal development induced by interference of bra function. We concluded that an intact bra function is necessary to exclude hairy2 expression from the non-organiser field, to impede the ectopic specification of dorsal axial fates and the appearance of incomplete secondary axes. This evidence supports a previously unrecognised role for bra in maintaining the dorsal fates inhibited in the ventral marginal zone, preventing the appearance of trunk duplications.