Tbxt alleviates senescence and apoptosis of nucleus pulposus cells through Atg7-mediated autophagy activation during intervertebral disk degeneration.

Intervertebral disk degeneration (IDD) is a significant cause of low back pain, characterized by excessive senescence and apoptosis of nucleus pulposus cells (NPCs). However, the precise mechanisms behind this senescence and apoptosis remain unclear. This study aimed to investigate the role of T-box transcription factor T (Tbxt) in IDD both in vitro and in vivo, using a hydrogen peroxide (H2O2)-induced NPCs senescence and apoptosis model, as well as a rat acupuncture IDD model. First, the expression of p16 and cleaved-caspase 3 significantly increased in degenerated human NPCs, accompanied by a decrease in Tbxt expression. Knockdown of Tbxt exacerbated senescence and apoptosis in the H2O2-induced NPCs degeneration model. Conversely, upregulation of Tbxt alleviated these effects induced by H2O2. Mechanistically, bioinformatic analysis revealed that the direct downstream target genes of Tbxt were highly enriched in autophagy-related pathways, and overexpression of Tbxt significantly activated autophagy in NPCs. Moreover, the administration of the autophagy inhibitor, 3-methyladenine, impeded the impact of Tbxt on the processes of senescence and apoptosis in NPCs. Further investigation revealed that Tbxt enhances autophagy by facilitating the transcription of ATG7 through its interaction with a specific motif within the promoter region. In conclusion, this study suggests that Tbxt mitigates H2O2-induced senescence and apoptosis of NPCs by activating ATG7-mediated autophagy.NEW & NOTEWORTHY This study investigates the role of Tbxt in IDD. The results demonstrate that knockdown of Tbxt exacerbates H2O2-induced senescence and apoptosis in NPCs and IDD, whereas upregulation of Tbxt significantly protects against IDD both in vivo and in vitro. Mechanistically, in the nucleus, Tbxt enhances the transcription of ATG7, leading to increased expression of ATG7 protein levels. This, in turn, promotes elevated autophagy levels, ultimately alleviating IDD.

Downregulated FGFR3 Expression Inhibits In Vitro Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells of Mice with TBXT Gene Mutation.

We studied the effect of fibroblast growth factor receptor 3 (FGFR3) inhibitor BGJ-398 on the differentiation of bone marrow mesenchymal stem cells (BM MSC) into osteoblasts in wild type (wt) mice and in animals with mutation in TBXT gene (mt) and possible differences in the pluripotency of these cells. Cytology tests showed that the cultured BM MSC could differentiate into osteoblasts and adipocytes. The effect of different BGJ-398 concentrations on the expression of FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 were studied by quantitative reverse transcription PCR. The expression of RUNX2 protein was evaluated by Western blotting. BM MSC of mt and wt mice did not differ in pluripotency and expressed the same membrane marker antigens. BGJ-398 inhibitor reduced the expression of FGFR3 and RUNX2. In BM MSC from mt and wt mice have similar gene expression (and its changing) in FGFR3, RUNX2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7, and SMAD8 genes. Thus, our experiments confirmed the effect of decreased expression of FGFR3 on osteogenic differentiation of BM MSC from wt and mt mice. However, BM MSC from mt and wt mice did not differ in pluripotency and are an adequate model for laboratory research.

Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T-ARMS-PCR assay.

BACKGROUND: Chordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T-box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells. METHODS: Herein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T-ARMS-PCR. The results were subsequently validated by Sanger sequencing. RESULTS: The genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p-value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p-value <0.05). DNA sequencing verified the T-ARMS-PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost-effective T-ARMS-PCR assay. CONCLUSIONS: Our results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T-ARMS-PCR assay to determine the genotype of rs2305089.

Transcription Factors with Targeting Potential in Gliomas.

Gliomas portray a large and heterogeneous group of CNS tumors, encompassing a wide range of low- to high-grade tumors, as defined by histological and molecular characteristics. The identification of signature mutations and other molecular abnormalities has largely impacted tumor classification, diagnosis, and therapy. Transcription factors (TFs) are master regulators of gene expression programs, which ultimately shape cell fate and homeostasis. A variety of TFs have been detected to be aberrantly expressed in brain tumors, being highly implicated in critical pathological aspects and progression of gliomas. Herein, we describe a selection of oncogenic (GLI-1/2/3, E2F1-8, STAT3, and HIF-1/2) and tumor suppressor (NFI-A/B, TBXT, MYT1, and MYT1L) TFs that are deregulated in gliomas and are subsequently associated with tumor development, progression, and migratory potential. We further discuss the current targeting options against these TFs, including chemical (Bortezomib) and natural (Plumbagin) compounds, small molecules, and inhibitors, and address their potential implications in glioma therapy.

The T-box transcription factor brachyury behaves as a tumor suppressor in gliomas.

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[Chordoma: is there a molecular basis for diagnosis and treatment?] / Chordome: Gibt es eine molekulargenetische Grundlage für Diagnostik und Therapie?

Chordomas are malignant bone tumours with a reported annual incidence of 0.08 per 100,000 cases. They show a notochordal differentiation and are characterised by their nuclear expression of brachyury (TBXT). Chordomas are localised in the axial skeleton, where they occur from the clivus to the sacrococcygeal region. They are slow growing, locally destructive tumours, and are often not diagnosed until they have reached an advanced stage. Putative precursor-lesions are benign notochordal cell lesions, which are microscopically small and intraosseous. Different histological chordoma subtypes exist, which differ in their prognosis. To date, there are no known recurrent genetic drivers for this disease. Brachyury seems to play a key role in the pathogenesis of chordoma, though the detailed mechanism still needs to be elucidated. Surgical en bloc resection with negative margins is the only curative treatment for this disease. High-dose irradiation, particularly with protons and carbon ions, is a therapeutic alternative in cases of inoperable tumours. Currently, there is no approved medical treatment for chordoma. Clinical trials exploring additional therapeutic modalities are ongoing.

A mutation in TBXT causes congenital vertebral malformations in humans and mice.

T-box transcription factor T (TBXT; T) is required for mesodermal formation and axial skeletal development. Although it has been extensively studied in various model organisms, human congenital vertebral malformations (CVMs) involving T are not well established. Here, we report a family with 15 CVM patients distributed across 4 generations. All affected individuals carry a heterozygous mutation, T c.596A>G (p.Q199R), which is not found in unaffected family members, indicating co-segregation of the genotype and phenotype. In vitro assays show that T p.Q199R increases the nucleocytoplasmic ratio and enhances its DNA-binding affinity, but reduces its transcriptional activity compared to the wild-type. To determine the pathogenicity of this mutation in vivo, we generated a Q199R knock-in mouse model that recapitulates the human CVM phenotype. Most heterozygous Q199R mice show subtle kinked or shortened tails, while homozygous mice exhibit tail filaments and severe vertebral deformities. Overall, we show that the Q199R mutation in T causes CVM in humans and mice, providing previously unreported evidence supporting the function of T in the genetic etiology of human CVM.

Exploring the formation mechanism of short-tailed phenotypes in animals using mutant mice with the TBXT gene c.G334T developed by CRISPR/Cas9.

With the change in diet structure, individuals prefer to consume mutton with less fat. However, sheep tail has a lot of fat. We identified a breed of low-fat short-tailed sheep (i.e., Hulunbuir short-tailed sheep). It is necessary to develop an animal model that can promote research on the potential mechanisms of the short-tail phenotype in sheep, which results from the TBXT gene c.G334T mutation. To create animal models, we selected mice as experimental animals. Mouse embryos lacking the TBXT protein, which crucially regulates mouse embryonic development, cannot develop normally. We utilized CRISPR/Cas9 gene editing technology to generate site-specific mutation (c.G334T) in the TBXT gene of mice, and found that the mouse TBXT mutation (c.G334T) leads to a short-tail phenotype. Furthermore, we investigated the interaction between TBXT and Wnt signaling pathways. The expressions of TBXT, Axin2, Dkk1, Wnt3, Wnt3a, and Wnt5a were discovered to be significantly different between mutant embryos and wild embryos by obtaining mouse embryos at various developmental stages and examining the expression relationship between the TBXT and Wnt signaling pathway-related components in all of these embryos. Therefore, as a transcription factor, TBXT regulates the expression of the aforementioned Wnt signaling pathway components by forming a regulatory network for the normal development of mouse embryos. This study enriches the research on the functional role of the TBXT in the development of mouse embryos and the mechanism by which the short-tailed phenotype in sheep develops.

Mitochondrial genes modulate the phenotypic expression of congenital scoliosis syndrome caused by mutations in the TBXT gene.

BACKGROUND: Congenital scoliosis (CS) is a spinal disorder caused by genetic-congenital vertebral malformations and may be associated with other congenital defects or may occur alone. It is genetically heterogeneous and numerous genes contributing to this disease have been identified. In addition, CS has a wide range of phenotypic and genotypic variability, which has been explained by the intervention of genetic factors like modifiers and environment genes. The aim of the present study was to determine the possible cause of CS in a Tunisian patient and to examine the association between mtDNA mutations and mtDNA content and CS. METHODS: Here we performed Whole-Exome Sequencing (WES) in a patient presenting clinical features suggestive of severe congenital scoliosis syndrome. Direct sequencing of the whole mitochondrial DNA (mtDNA) was also performed in addition to copy number quantification in the blood of the indexed case. In silico prediction tools, 3D modeling and molecular docking approaches were used. RESULTS: The WES revealed the homozygous missense mutation c.512A > G (p.H171R) in the TBXT gene. Bioinformatic analysis demonstrated that the p.H171R variant was highly deleterious and caused the TBXT structure instability. Molecular docking revealed that the p.H171R mutation disrupted the monomer stability which seemed to be crucial for maintaining the stability of the homodimer and consequently to the destabilization of the homodimer-DNA complex. On the other hand, we hypothesized that mtDNA can be a modifier factor, so, the screening of the whole mtDNA showed a novel heteroplasmic m.10150T > A (p.M31K) variation in the MT-ND3 gene. Further, qPCR analyses of the patient's blood excluded mtDNA depletion. Bioinformatic investigation revealed that the p.M31K mutation in the ND3 protein was highly deleterious and may cause the ND3 protein structure destabilization and could disturb the interaction between complex I subunits. CONCLUSION: We described the possible role of mtDNA genetics on the pathogenesis of congenital scoliosis by hypothesizing that the presence of the homozygous variant in TBXT accounts for the CS phenotype in our patient and the MT-ND3 gene may act as a modifier gene.

Neuromesodermal specification during head-to-tail body axis formation.

The anterior-to-posterior (head-to-tail) body axis is extraordinarily diverse among vertebrates but conserved within species. Body axis development requires a population of axial progenitors that resides at the posterior of the embryo to sustain elongation and is then eliminated once axis extension is complete. These progenitors occupy distinct domains in the posterior (tail-end) of the embryo and contribute to various lineages along the body axis. The subset of axial progenitors with neuromesodermal competency will generate both the neural tube (the precursor of the spinal cord), and the trunk and tail somites (producing the musculoskeleton) during embryo development. These axial progenitors are called Neuromesodermal Competent cells (NMCs) and Neuromesodermal Progenitors (NMPs). NMCs/NMPs have recently attracted interest beyond the field of developmental biology due to their clinical potential. In the mouse, the maintenance of neuromesodermal competency relies on a fine balance between a trio of known signals: Wnt/ß-catenin, FGF signalling activity and suppression of retinoic acid signalling. These signals regulate the relative expression levels of the mesodermal transcription factor Brachyury and the neural transcription factor Sox2, permitting the maintenance of progenitor identity when co-expressed, and either mesoderm or neural lineage commitment when the balance is tilted towards either Brachyury or Sox2, respectively. Despite important advances in understanding key genes and cellular behaviours involved in these fate decisions, how the balance between mesodermal and neural fates is achieved remains largely unknown. In this chapter, we provide an overview of signalling and gene regulatory networks in NMCs/NMPs. We discuss mutant phenotypes associated with axial defects, hinting at the potential significant role of lesser studied proteins in the maintenance and differentiation of the progenitors that fuel axial elongation.

DNA methylation, combined with RNA sequencing, provide novel insight into molecular classification of chordomas and their microenvironment.

Chordomas are rare tumors of notochord remnants, occurring mainly in the sacrum and skull base. Despite of their unusually slow growth, chordomas are highly invasive and the involvement of adjacent critical structures causes treatment challenges. Due to the low incidence, the molecular pathogenesis of this entity remains largely unknown. This study aimed to investigate DNA methylation abnormalities and their impact on gene expression profiles in skull base chordomas. 32 tumor and 4 normal nucleus pulposus samples were subjected to DNA methylation and gene expression profiling with methylation microarrays and RNA sequencing. Genome-wide DNA methylation analysis revealed two distinct clusters for chordoma (termed subtypes C and I) with different patterns of aberrant DNA methylation. C Chordomas were characterized by general hypomethylation with hypermethylation of CpG islands, while I chordomas were generally hypermethylated. These differences were reflected by distinct distribution of differentially methylated probes (DMPs). Differentially methylated regions (DMRs) were identified, indicating aberrant methylation in known tumor-related genes in booth chordoma subtypes and regions encoding small RNAs in subtype C chordomas. Correlation between methylation and expression was observed in a minority of genes. Upregulation of TBXT in chordomas appeared to be related to lower methylation of tumor-specific DMR in gene promoter. Gene expression-based clusters of tumor samples did not overlap with DNA methylation-based subtypes. Nevertheless, they differ in transcriptomic profile that shows immune infiltration in I chordomas and up-regulation of cell cycle in C chordomas. Immune enrichment in chordomas I was confirmed with 3 independent deconvolution methods and immunohistochemistry. Copy number analysis showed higher chromosomal instability in C chordomas. Nine out of eight had deletion of CDKN2A/B loci and downregulation of genes encoded in related chromosomal band. No significant difference in patients' survival was observed between tumor subtypes, however, shorter survival was observed in patients with higher number of copy number alterations.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion.

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial-mesenchymal transition (EMT) inducer, by directly binding to TBXT 3'-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.

Multiple malignant tumors in a patient with familial chordoma, a case report.

BACKGROUND: Chordoma is a rare bone tumor that is typically resistant to chemotherapy and is associated with genetic abnormalities of the T-box transcription factor T (TBXT) gene, which encodes the transcription factor brachyury. Brachyury is felt to be a major contributor to the development of chordomas. CASE PRESENTATION: We describe a 67-year-old woman who developed an undifferentiated pleomorphic sarcoma in her thigh. Despite treatment with standard chemotherapy regimens, she had a rapidly progressive course of disease with pulmonary metastases and passed away 8 months from diagnosis with pulmonary complications. Her medical history was remarkable in that she had a spheno-occipital chordoma at age 39 and later developed multiple other tumors throughout her life including Hodgkin lymphoma and squamous cell carcinoma and basal cell carcinoma of the skin. She had a family history of chordoma and her family underwent extensive genetic study in the past and were found to have a duplication of the TBXT gene. CONCLUSIONS: Brachyury has been found to associate with tumor progression, treatment resistance, and metastasis in various epithelial cancers, and it might play roles in tumorigenesis and aggressiveness in this patient with multiple rare tumors and germ line duplication of the TBXT gene. Targeting this molecule may be useful for some malignancies.

Genetic variants of TBX6 and TBXT identified in patients with congenital scoliosis in Southern China.

Congenital scoliosis (CS) is a spinal deformity present at birth due to underlying congenital vertebral malformation (CVM) that occurs during embryonic development. Hemivertebrae is the most common anomaly that causes CS. Recently, compound heterozygosity in TBX6 has been identified in Northern Chinese, Japanese, and European CS patient cohorts, which explains about 7%-10% of the affected population. In this report, we recruited 67 CS patients characterized with hemivertebrae in the Southern Chinese population and investigated the TBX6 variant and risk haplotype. We found that two patients with hemivertebrae in the thoracic spine and one patient with hemivertebrae in the lumbar spine carry the previously defined pathogenic TBX6 compound heterozygous variants. In addition, whole exome sequencing of patients with CS and their family members identified a de novo missense mutation (c.G47T: p.R16L) in another member of the T-box family, TBXT. This rare mutation compromised the binding of TBXT to its target sequence, leading to reduced transcriptional activity, and exhibited dominant-negative effect on wild-type TBXT. Our findings further highlight the importance of T-box family genes in the development of congenital scoliosis.

Brachyury Is Associated with Glioma Differentiation and Response to Temozolomide.

Glioblastomas (GBMs) are the most aggressive tumor type of the central nervous system, mainly due to their high invasiveness and innate resistance to radiotherapy and chemotherapy, with temozolomide (TMZ) being the current standard therapy. Recently, brachyury was described as a novel tumor suppressor gene in gliomas, and its loss was associated with increased gliomagenesis. Here, we aimed to explore the role of brachyury as a suppressor of glioma invasion, stem cell features, and resistance to TMZ. Using gene-edited glioma cells to overexpress brachyury, we found that brachyury-positive cells exhibit reduced invasive and migratory capabilities and stem cell features. Importantly, these brachyury-expressing cells have increased expression of differentiation markers, which corroborates the results from human glioma samples and in vivo tumors. Glioma cells treated with retinoic acid increased the differentiation status with concomitant increased expression of brachyury. We then selected TMZ-resistant (SNB-19) and TMZ-responsive (A172 and U373) cell lines to evaluate the role of brachyury in the response to TMZ treatment. We observed that both exogenous and endogenous brachyury activation, through overexpression and retinoic acid treatment, are associated with TMZ sensitization in glioma-resistant cell lines. In this study, we demonstrate that brachyury expression can impair aggressive glioma features associated with treatment resistance. Finally, we provide the first evidence that brachyury can be a potential therapeutic target in GBM patients who do not respond to conventional chemotherapeutic drugs.

Active receptor tyrosine kinases, but not Brachyury, are sufficient to trigger chordoma in zebrafish.

The aberrant activation of developmental processes triggers diverse cancer types. Chordoma is a rare, aggressive tumor arising from transformed notochord remnants. Several potentially oncogenic factors have been found to be deregulated in chordoma, yet causation remains uncertain. In particular, sustained expression of TBXT - encoding the notochord regulator protein brachyury - is hypothesized as a key driver of chordoma, yet experimental evidence is absent. Here, we employ a zebrafish chordoma model to identify the notochord-transforming potential of implicated genes in vivo We find that Brachyury, including a form with augmented transcriptional activity, is insufficient to initiate notochord hyperplasia. In contrast, the chordoma-implicated receptor tyrosine kinases (RTKs) EGFR and Kdr/VEGFR2 are sufficient to transform notochord cells. Aberrant activation of RTK/Ras signaling attenuates processes required for notochord differentiation, including the unfolded protein response and endoplasmic reticulum stress pathways. Our results provide the first in vivo evidence against a tumor-initiating potential of Brachyury in the notochord, and imply activated RTK signaling as a possible initiating event in chordoma. Furthermore, our work points at modulating endoplasmic reticulum and protein stress pathways as possible therapeutic avenues against chordoma.

Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis.

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.