The roles and regulation of TBX3 in development and disease

“Malignant cells form tumours, generate a tumour-associated neo vasculature (angiogenesis) which supplies the tumour with nutrients and oxygen and removes metabolic wastes, and they break away from the primary tumour and metastasise and invade distant organs (Hanahan and Weinberg, 2011).”

“The tumour suppressor role of TBX3—During oncogenesis, tumour suppressor genes are frequently silenced by methylation (Jones and Baylin, 2002).”

“TBX3 in cancer stem cells—Cancer stem cells (CSCs) are a small subpopulation of tumour cells which exhibit capabilities such as self-renewal, differentiation and tumorigenicity (Clarke et al., 2006).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“…M anuscript A uthor M anuscript A uthor M anuscript senescence and apoptosis (Berkovich et al., 2003; Brugarolas et al., 1995; Inoue et al., 1999; Pomerantz et al., 1998). p16INK4a, like other members of the Ink4 family, functions by blocking the kinase active sites of cyclin-dependent kinases…”

“Fgf signalling is regulated by, among other pathways, Bmp4 and Shh and when Fgf signalling is disrupted, branching is abrogated (Ohuchi et al., 2000; Pepicelli et al., 1998; Sekine et al., 1999; Weaver et al., 2000).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“Rheumatoid arthritis (RA) is characterized by chronic inflammation, which primarily affects the synovial joints leading to tissue damage and physical disability and genome wide association studies have casually linked TBX3 to RA susceptibility (Freudenberg et al., 2011; Julià et al., 2008; Plenge et al., 2007).”

“Moreover, Shh null mice have hypoplastic lungs due to incorrect branching morphogenesis (Litingtung et al., 1998; Pepicelli et al., 1998).”

“Together, Fgf and Shh signalling promote digit development and control digit number and patterning (Martin, 1998; Ohuchi et al., 2000).”

“Fgf signalling is regulated by, among other pathways, Bmp4 and Shh and when Fgf signalling is disrupted, branching is abrogated (Ohuchi et al., 2000; Pepicelli et al., 1998; Sekine et al., 1999; Weaver et al., 2000).”

“Moreover, Shh null mice have hypoplastic lungs due to incorrect branching morphogenesis (Litingtung et al., 1998; Pepicelli et al., 1998).”

“They are resistant to standard chemotherapies and are thought to be one of the main contributors to cancer development, drug resistance and clinical relapse (Yu et al., 2012).”

“Furthermore, in the same study the authors show that TBX3 represses both basal and induced PTEN levels in HeLa and HEK cells (Burgucu et al., 2012).”

“5 onwards, the epithelium undergoes branching morphogenesis and eventually forms a respiratory (bronchial) tree (Cardoso and Lu, 2006).”

“It encodes a 723 amino acid protein with part of exon 1, exons 2 and 3, and part of exon 4, encoding the conserved T-box domain, exon 1 and 2 encode one of two repression domains (R2), exon 6 encodes the activation domain and part of exon 6 and 7 encode the second repression domain (R1) (Bamshad et al., 1997; Carlson…”

“During the process of looping, a chamber myocardiumspecific gene program, which includes expression of Nppa, Cx40, Cx43, and Chisel, initiates proliferation and differentiation in specific regions of the heart tube to form the chamber myocardium (Christoffels et al., 2000; Delorme et al., 1997; Van Kempen et al., 1996…”

“TBX3 is also overexpressed in advanced melanoma and can drive the transition from noninvasive RGP melanoma to invasive vertical growth phase (VGP) melanoma (Hoek et al., 2004; Peres et al., 2010; Rodriguez et al., 2008).”

“For example, Wnt signalling depends on active Bmp signalling and the loss of Wnt2/2b leads to failure of trachea and branching lung formation and inactivation of the Bmp receptors Bmpr1a and Bmpr1b leads to tracheal agenesis and ectopic primary bronchi (Domyan et al., 2011; Goss et al., 2009).”

“TBX3 may also promote proliferation by repressing PTEN, an inhibitor of PI3K/ AKT-mediated cell growth, proliferation and survival (Leslie and Downes, 2004).”

“Ectopic expression of Tbx3 in the atria of mouse models resulted in the development of functional ectopic pacemakers and induced Tbx3 expression reprogrammed terminally differentiated cardiomyocytes into pacemaker cells (Bakker et al., 2012; Hoogaars et al., 2007).”

“It is believed that Tbx3 contributes to the developing CCS by firstly modulating cell division which results in constrictions between chambers, and secondly by directly repressing chamber myocardium genes by cooperatively binding their promoters along with other transcription factors (Christoffels et al., 2004; Hoogaa…”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript (Brummelkamp et al., 2002; Jacobs et al., 2000; Lingbeek et al., 2002; Prince et al., 2004; Willmer et al., 2016a, 2016b).”

“Recently, SAN-like pacemaker cells were generated from human pluripotent stem cells without genetic manipulation and these cells were positive for Tbx3, Tbx18 and Shox2 and were shown to be able to function as a biological pacemaker in vivo (Protze et al., 2017).”

“Indeed, loss of components of the RA pathway in mutant mice leads to various forelimb abnormalities ranging from small limbs with digit anomalies to absent limbs (Lohnes et al., 1994; Sandell et al., 2007).”

“Recently, SAN-like pacemaker cells were generated from human pluripotent stem cells without genetic manipulation and these cells were positive for Tbx3, Tbx18 and Shox2 and were shown to be able to function as a biological pacemaker in vivo (Protze et al., 2017).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“Loss of Tbx3 in homozygous mutant mice results in failure of placode induction and heterozygous mutant mice have decreased ductal tree development and failed nipple formation (Davenport et al., 2003; Jerome-Majewska et al., 2005; Rowley et al., 2004).”

“It is believed that Tbx3 contributes to the developing CCS by firstly modulating cell division which results in constrictions between chambers, and secondly by directly repressing chamber myocardium genes by cooperatively binding their promoters along with other transcription factors (Christoffels et al., 2004; Hoogaa…”

“A mouse mutation that results in a short tail identified in 1927 led to the discovery and ultimate cloning of Brachyury, the prototype of the family, in 1990 and, as shown in Table 1, there are currently 17 paralogues in human, mouse and rat that are grouped into five subfamilies, namely Brachyury (T), T-brain (Tbr1),…”

“5, expression of Tbx3 in the AER is restricted to the tips of the digits (Chapman et al., 1996; Gibson-Brown et al., 1996).”

“These results highlight the importance of the FGF/Tbx3 signalling pathway in the expansion of breast CSCs and reveal another mechanism by which TBX3 aids breast cancer progression, recurrence and drug resistance (Fillmore et al., 2010; Dong et al., 2018a, 2018b).”

“These findings are interesting because the methylation of TBX3 in glioblastoma is associated with resistance to standard therapy and metabolic pathways have been implicated as important mediators of resistance to anti-cancer agents (Etcheverry et al., 2010; Zaal and Berkers, 2018).”

“…lungs, pituitary glands and genitalia (Chapman et al., 1996; Tümpel et al., 2002; Davenport et al., 2003; Moorman et al., 2004; Cho et al., 2006; Lin et al., 2007; Bakker et al., 2008; Mesbah et al., 2008; Pontecorvi et al., 2008; Lüdtke et al., 2009, 2016; Begum and Papaioannou, 2011;…”

“The authors showed that in this gastric cancer cell line, but not in 5 other gastric cancer cell lines tested, methylation of the CpG island in the 5′ region of the TBX3 gene effectively silenced its expression (Yamashita et al., 2006).”

“In vitro studies have shown that expressing Tbx3, KLF4, SOX2, OCT4, Nanog, LIN-28A and C-MYC in somatic cells can reprogram them to form iPSCs (Okita and Yamanaka, 2011; Lee, et al., 2013).”

“Cells from the non-chamber myocardium form the cardiac conduction system (CCS), which controls the co-ordinated contraction of the heart (Christoffels et al., 2004; Greulich et al., 2011).”

“Recently, SAN-like pacemaker cells were generated from human pluripotent stem cells without genetic manipulation and these cells were positive for Tbx3, Tbx18 and Shox2 and were shown to be able to function as a biological pacemaker in vivo (Protze et al., 2017).”

“During the process of looping, a chamber myocardiumspecific gene program, which includes expression of Nppa, Cx40, Cx43, and Chisel, initiates proliferation and differentiation in specific regions of the heart tube to form the chamber myocardium (Christoffels et al., 2000; Delorme et al., 1997; Van Kempen et al., 1996…”

“Indeed, whereas TBX2 was shown to be required to maintain proliferation and suppress senescence in melanomas by repressing expression of p21WAF1/CIP1 (Vance et al., 2005), TBX3 was found to enhance melanoma invasiveness by down-regulating expression of E-cadherin (Rodriguez et al.”

“These findings are interesting because the methylation of TBX3 in glioblastoma is associated with resistance to standard therapy and metabolic pathways have been implicated as important mediators of resistance to anti-cancer agents (Etcheverry et al., 2010; Zaal and Berkers, 2018).”

“For example, Wnt signalling depends on active Bmp signalling and the loss of Wnt2/2b leads to failure of trachea and branching lung formation and inactivation of the Bmp receptors Bmpr1a and Bmpr1b leads to tracheal agenesis and ectopic primary bronchi (Domyan et al., 2011; Goss et al., 2009).”

“Their importance has been well established in the field of developmental biology where they play essential roles from as early as cell-fate determination all the way through to organogenesis (Packham and Brook, 2003; Papaioannou, 2001).”

“Their importance has been well established in the field of developmental biology where they play essential roles from as early as cell-fate determination all the way through to organogenesis (Packham and Brook, 2003; Papaioannou, 2001).”

“Indeed, Tbx3 was demonstrated to be important for self-renewal and extraembryonic endoderm specification in mESCs (Lu et al., 2011; Semrau et al., 2017).”

“It was however predicted that TBX3, like all other T-box members identified to date, will bind its biological downstream effectors as a single monomer through the half T-element of the palindrome target sequence (Coll et al., 2002; Wilson and Conlon, 2002).”

“BMPs are members of the transforming growth factor β (TGF-β) gene family and play a critical role in the formation of the non-chamber myocardium (Shi et al., 2000).”

“For example, TBX3 directly represses transcription of the tumour suppressors, p19ARF (p14ARF in humans) through CACCTCTGGTGCCA in primary breast tumours (Lingbeek et al., 2002), p21WAF1/CIP1 through a GTGTGA close to the initiator in chondrosarcoma (Willmer et al.”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript (Brummelkamp et al., 2002; Jacobs et al., 2000; Lingbeek et al., 2002; Prince et al., 2004; Willmer et al., 2016a, 2016b).”

“Sox4 to assist it binding to its target genes to regulate heart development (Bakker et al., 2008; Boogerd et al., 2008, 2011, Christoffels et al., 2000, 2004; Hoogaars et al., 2008; Stennard and Harvey, 2005).”

“Interestingly, TBX3 is methylated in metastatic cervical cancer, DU-145 prostate cancer cells, bladder cancer, urothelial carcinoma, in the AGS gastric cancer cell line, glioblastoma and glioblastoma stem cells, and the methylation of TBX3 was associated with a poor overall survival, resistance to cancer therapy and a…”

“Ectopic expression of Tbx3 in the atria of mouse models resulted in the development of functional ectopic pacemakers and induced Tbx3 expression reprogrammed terminally differentiated cardiomyocytes into pacemaker cells (Bakker et al., 2012; Hoogaars et al., 2007).”

“Importantly, Renard et al. (2007) demonstrated that TBX3 is a direct transcriptional target of β-catenin/Tcf and that β-catenin mediated upregulation of TBX3 confers resistance to doxorubicin-induced apoptosis in U2OS osteosarcoma and HCT116 colorectal carcinoma cells.”

“Furthermore, there is evidence that TBX2 and TBX3 are differentially expressed in some cancers which appears to relate, in part, to their ability to repress one another (Mohamad et al., 2018; Rodriguez et al., 2008).”

“Furthermore, Fan et al. (2004) showed that Tbx3, and not its isoform Tbx3+2a, could immortalise MEFs and Brummelkamp et al. (2002) identified Tbx3 as a key anti-senescence factor in a genetic screen of conditionally immortalised mouse striatal cells.”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“In addition, experiments in the chick have shown that Tbx3 plays an important role in posterior digit specification, acting together with Tbx2 and the interdigital BMP signalling cascade (Suzuki et al., 2004).”

“5 with the formation of the milk line between the forelimb and hindlimb of each flank, which forms an ectodermal ridge characterized by Wnt10b expression (Veltmaat et al., 2004).”

“It was however predicted that TBX3, like all other T-box members identified to date, will bind its biological downstream effectors as a single monomer through the half T-element of the palindrome target sequence (Coll et al., 2002; Wilson and Conlon, 2002).”

“It encodes a 723 amino acid protein with part of exon 1, exons 2 and 3, and part of exon 4, encoding the conserved T-box domain, exon 1 and 2 encode one of two repression domains (R2), exon 6 encodes the activation domain and part of exon 6 and 7 encode the second repression domain (R1) (Bamshad et al., 1997; Carlson…”

“For example, anterior and posterior polarization of the limb bud mesenchyme requires the expression of Tbx3 (and Gli3) which is regulated by Hand2 and Hand2 is downregulated in the limbs of Tbx3 mutant mice (Davenport, et al., 2003; Osterwalder et al., 2014; Sheeba and Logan, 2017).”

“In early mouse adipocyte precursor cells, miRNA-93 also exhibited the ability to repress Tbx3 to prevent self-renewal (Cioffi et al., 2015).”

“In contrast to the above findings, it is interesting to note that in pancreatic ductal adenocarcinomas (PDAC), melanoma and breast carcinomas, TBX3 has no effect, or negatively regulates proliferation, in favour of promoting cell migration, a phenotypic tradeoff which is common in cancer (Gallaher et al., 2019).”

“Importantly, several lines of evidence suggest that TBX3 enhances and maintains stem cell pluripotency in vitro by preventing differentiation and enhancing self-renewal (Niwa et al., 2009; Russell et al., 2015; Saunders et al., 2013).”

“Furthermore, in vitro studies in which the RD1 was deleted resulted in decreased transcriptional activity of Tbx3 (Carlson et al., 2001).”

“Tbx3 expression is particularly important for the formation of the CCS, AVB, and the ventricular septum of the heart and Tbx3-deficient embryos develop ventricular septal defects, delay in heart looping and outflow tract malformations (Bakker et al., 2008; Mesbah et al., 2008; Ribeiro et al., 2007; Washkowitz et al.,…”

“UMS is an autosomal dominant developmental disorder, characterized by a number of clinical features including mammary and apocrine gland hypoplasia, upper limb defects, malformations of areola, dental structures, heart and genitalia (Chen and Chen, 2017).”

“Interestingly, TBX3 is methylated in metastatic cervical cancer, DU-145 prostate cancer cells, bladder cancer, urothelial carcinoma, in the AGS gastric cancer cell line, glioblastoma and glioblastoma stem cells, and the methylation of TBX3 was associated with a poor overall survival, resistance to cancer therapy and a…”

“Indeed, it interacts with HDACs 1, 2, 3 and 5 to repress the tumour suppressor p14ARF in breast cancer and with HDAC5 to repress Ecadherin to promote metastasis in hepatocellular carcinoma (Dong et al., 2018a, 2018b; Yarosh et al., 2008).”

“Rheumatoid arthritis (RA) is characterized by chronic inflammation, which primarily affects the synovial joints leading to tissue damage and physical disability and genome wide association studies have casually linked TBX3 to RA susceptibility (Freudenberg et al., 2011; Julià et al., 2008; Plenge et al., 2007).”

“BRAFV600E and AKT3 are constitutively activated in approximately 50% and 70% of melanomas respectively and they play critical roles in melanoma formation and invasion (Dhawan et al., 2002; Palmieri et al., 2015; Siroy et al., 2016).”

“Tbx3 expression is particularly important for the formation of the CCS, AVB, and the ventricular septum of the heart and Tbx3-deficient embryos develop ventricular septal defects, delay in heart looping and outflow tract malformations (Bakker et al., 2008; Mesbah et al., 2008; Ribeiro et al., 2007; Washkowitz et al.,…”

“Furthermore, the coexpression of Tbx3, with Myc or H-RasVal17 can transform MEFs and bypass Myc-induced apoptosis through the repression of p19ARF/p53/ p21WAF1/CIP1 (Carlson et al., 2002).”

“Loss of Tbx3 in homozygous mutant mice results in failure of placode induction and heterozygous mutant mice have decreased ductal tree development and failed nipple formation (Davenport et al., 2003; Jerome-Majewska et al., 2005; Rowley et al., 2004).”

“TBX3 is also overexpressed in advanced melanoma and can drive the transition from noninvasive RGP melanoma to invasive vertical growth phase (VGP) melanoma (Hoek et al., 2004; Peres et al., 2010; Rodriguez et al., 2008).”

“4, they both have a T-box DNA binding domain, 2 repression domains and an activation domain (Paxton et al., 2002; Sinha et al., 2000).”

“Loss of Tbx3 in homozygous mutant mice results in failure of placode induction and heterozygous mutant mice have decreased ductal tree development and failed nipple formation (Davenport et al., 2003; Jerome-Majewska et al., 2005; Rowley et al., 2004).”

“4, they both have a T-box DNA binding domain, 2 repression domains and an activation domain (Paxton et al., 2002; Sinha et al., 2000).”

“Furthermore, Nrg3 transmits signals downstream of Tbx3 and Fgf signalling from somite to the overlying ectoderm to promote their local aggregation in the mammary placode and subsequent placode formation (Howard et al., 2005; Howard and Ashworth, 2006).”

“Indeed, Tbx3 was demonstrated to be important for self-renewal and extraembryonic endoderm specification in mESCs (Lu et al., 2011; Semrau et al., 2017).”

“Importantly, patients with UMS have shown symptoms consistent with ARC neuron dysfunction, including deficiency in growth hormone production leading to impaired puberty and obesity (Linden et al., 2009).”

“In addition, EGR1 has been identified as a TBX2 co-factor in breast cancer and rhabdomyosarcoma and their interaction has been shown to drive cell proliferation by inhibiting EGR1 dependent gene expression of p21WAF1/CIP1, PTEN, NDRG1 and CST6 (D’Costa et al., 2014; Mohamad et al., 2018; Redmond et al., 2010).”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“Furthermore, phosphorylation of TBX3 by the p38 MAP kinase at SP692 in embryonic kidney cells enhances its ability to transcriptionally repress its well-known target, E-cadherin, to promote migration (Yano et al., 2011).”

“Furthermore, Nrg3 transmits signals downstream of Tbx3 and Fgf signalling from somite to the overlying ectoderm to promote their local aggregation in the mammary placode and subsequent placode formation (Howard et al., 2005; Howard and Ashworth, 2006).”

“While these mutations can occur throughout TBX3, those which occur within or upstream of the T-domain (DNA binding) are associated with the most severe phenotype (Meneghini et al., 2006).”

“Finally, Tbx2 and Tbx3 regulate mesenchymal proliferation by maintaining pro-proliferative Wnt signalling through direct repression of the Wnt antagonists Frzb and Shisa3 (Lüdtke et al., 2016).”

“Interestingly, TBX3 is methylated in metastatic cervical cancer, DU-145 prostate cancer cells, bladder cancer, urothelial carcinoma, in the AGS gastric cancer cell line, glioblastoma and glioblastoma stem cells, and the methylation of TBX3 was associated with a poor overall survival, resistance to cancer therapy and a…”

“Interestingly, two studies showed that Tbx3/TBX3 is not entirely critical for the tenacity or generation of iPSCs (Klingenstein et al., 2016; Russell et al., 2015).”

“TBX2 and TBX3 are also overexpressed in numerous cancers and they both can contribute to similar oncogenic processes including bypassing senescence and apoptosis, promoting proliferation and EMT and conferring drug resistance (Wansleben et al., 2014).”

“It was however predicted that TBX3, like all other T-box members identified to date, will bind its biological downstream effectors as a single monomer through the half T-element of the palindrome target sequence (Coll et al., 2002; Wilson and Conlon, 2002).”

“Tbx3 expression is particularly important for the formation of the CCS, AVB, and the ventricular septum of the heart and Tbx3-deficient embryos develop ventricular septal defects, delay in heart looping and outflow tract malformations (Bakker et al., 2008; Mesbah et al., 2008; Ribeiro et al., 2007; Washkowitz et al.,…”

“Similarly, the upregulation of TBX3 in breast and melanoma cells stimulated with RA or TGFβ1 led to diminished proliferative rates but increased migration (Ballim et al., 2012; Li et al., 2013).”

“Tbx3 is upregulated in rat bladder carcinoma cells and depleting Tbx3 in these cells dramatically reduced cell growth and cell adhesion while promoting apoptosis (Ito et al., 2005).”

“TBX3 has been implicated in human diseases including ulnar mammary syndrome, rheumatoid arthritis, obesity and cancer (Frank et al., 2013; Quarta et al., 2019; Sardar et al., 2019; Willmer et al., 2017).”

“Hypo-phosphorylated Rb interacts with and sequesters the E2F family of transcription factors which results in a G1 cell cycle arrest and the maintenance of the senescence phenotype (DeGregori, 2004).”

“Interestingly, TBX3 is methylated in metastatic cervical cancer, DU-145 prostate cancer cells, bladder cancer, urothelial carcinoma, in the AGS gastric cancer cell line, glioblastoma and glioblastoma stem cells, and the methylation of TBX3 was associated with a poor overall survival, resistance to cancer therapy and a…”

“For example, anterior and posterior polarization of the limb bud mesenchyme requires the expression of Tbx3 (and Gli3) which is regulated by Hand2 and Hand2 is downregulated in the limbs of Tbx3 mutant mice (Davenport, et al., 2003; Osterwalder et al., 2014; Sheeba and Logan, 2017).”

“[PubMed: 21779450] Peres J, Mowla S, Prince S, 2015 The T-box transcription factor, TBX3, is a key substrate of AKT3 in melanomagenesis.”

“During differentiation of mESCs into neural cells, miR-137 is upregulated and it was found to bind the 3′ UTR of Tbx3 which resulted in the repression of Tbx3 levels, the inhibition of self-renewal and increased differentiation of mESCs in vitro (Jiang et al., 2013).”

“This raises the possibility that TBX3 may also repress some of its target genes through interfering with transcriptional activators (Burgucu et al., 2012).”

“Ectopic expression of TBX3 alone in RGP cells was sufficient to drive them to assume a VGP phenotype and knockdown of TBX3 in advanced melanoma cells inhibited their tumour forming ability and their aggressive phenotype (Peres et al., 2010; Peres and Prince, 2013).”

“Lineage tracing showed that the SAN originates from Tbx3 positive cells in the early heart tube (Mohan et al., 2018).”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“differentiation potential of mESCs and its subunit Dpf2 was recently found to directly activate Tbx3 expression (Zhang et al., 2019).”

“…2004; Cho et al., 2006; Lin et al., 2007; Bakker et al., 2008; Mesbah et al., 2008; Pontecorvi et al., 2008; Lüdtke et al., 2009, 2016; Begum and Papaioannou, 2011; Colasanto et al., 2016; Emechebe et al., 2016; Ichijo et al., 2017; López et al., 2018; Quarta et al., 2019; Karolak et al., 2019).”

“The mechanism by which TBX3 mediated the antiproliferative effect downstream of TGFβ1 was shown to be through it directly repressing a T-element in the TBX2 promoter (Li et al., 2014).”

“Indeed, uncontrolled cell proliferation and the bypass of senescence and apoptosis are early events in oncogenesis, and TBX3 has been shown to impact these processes as well as to promote tumour formation, angiogenesis and metastasis (Dong et al., 2018b, 2018a; Feng et al., 2018; Krstic et al., 2019; Wang, 2018; Willm…”

“This is consistent with the limb defects seen in UMS which suggests the involvement of TBX3 in bone development pathways which are closely associated with immune pathways (D’Amelio and Sassi, 2016; Frank et al., 2013).”

“In human bladder cancer cells, the regulation of E-cadherin by TBX3 occurs in a PLCε/PKC-dependent manner (Du et al., 2014).”

“Importantly, knockdown of either CAPERα or TBX3 increased senescence-associated βgalactosidase activity in human foreskin fibroblasts which was accompanied by an increase in p21WAF1/CIP1, p16INK4a and pRb (Kumar et al., 2014b).”

“Phosphorylation of TBX3 While there are several predicted post-translational modification sites for TBX3 including 10 ubiquitination, 1 acetylation, 2 methylation and 29 phosphorylation sites only the SP190, SP692 and S720 phosphorylation sites have been fully characterised.”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“This is consistent with the limb defects seen in UMS which suggests the involvement of TBX3 in bone development pathways which are closely associated with immune pathways (D’Amelio and Sassi, 2016; Frank et al., 2013).”

“Levels of miR-137 and TBX3 mRNA correlate inversely in a panel of melanoma cell lines as well as a cohort of primary melanoma patients and miR-137 was shown to be an important component of the TBX3/E-cadherin axis in melanomagenesis (Peres et al., 2017).”

“Ectopic overexpression of TBX3 enhanced the migratory and invasive ability of human PDAC cell lines and promoted angiogenesis in vitro and in vivo which correlated with increased expression of angiogenesis-associated genes such as FGF2 and VEGF-A (Perkhofer et al., 2016).”

“In addition, TBX3 was shown to mediate breast cancer cell migration downstream of the PKC and TGF-β signalling pathways (Li et al., 2014, 2013; Mowla et al., 2011).”

“…expression of TBX3 in chondrosarcoma cells also enhanced their ability to form tumours in mice and knockdown of TBX3 in liposarcoma, rhabdomyosarcoma and chondrosarcoma, resulted in diminished substratedependent and -independent cell proliferation and migration (Willmer et al., 2016a, 2016b).”

“Indeed, Tbx3 expression promoted the proliferative ability of normal and tumorigenic mammary epithelial cells (MECs) by transcriptionally repressing p19ARF which was accompanied by the downregulation of p21WAF1/CIP1 (Platonova et al., 2007).”

“Indeed, TBX3 overexpression in HNSCC cells increased their resistance to anoikis thus enabling them to survive without appropriate ECM interaction (Humtsoe et al., 2012).”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“Furthermore, there is evidence that TBX2 and TBX3 are differentially expressed in some cancers which appears to relate, in part, to their ability to repress one another (Mohamad et al., 2018; Rodriguez et al., 2008).”

“Indeed, uncontrolled cell proliferation and the bypass of senescence and apoptosis are early events in oncogenesis, and TBX3 has been shown to impact these processes as well as to promote tumour formation, angiogenesis and metastasis (Dong et al., 2018b, 2018a; Feng et al., 2018; Krstic et al., 2019; Wang, 2018; Willm…”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript senescence and apoptosis (Berkovich et al., 2003; Brugarolas et al., 1995; Inoue et al., 1999; Pomerantz et al., 1998). p16INK4a, like other members of the Ink4 family, functions by blocking the kinase active sites of…”

“Furthermore, Tbx3 and Hand2 are both regulated by the microRNAprocessing enzyme Dicer to ensure proper limb bud positioning (Zhang et al., 2011a, 2011b).”

“In chondrosarcoma cells, TBX3 is upregulated transcriptionally by c-Myc and post-translationally by cyclin A/CDK2 and it is required for transition through S-phase (Willmer et al., 2015).”

“Interestingly, Tbx3 is expressed in these hypothalamic neurons and has been implicated in the differentiation of human embryonic stem cells into hypothalamic Pomc neurons (Eriksson and Mignot, 2009; Linden et al., 2009; Quarta et al., 2019).”

“Recently Tbx3 was also shown to be highly expressed in the interfollicular epidermal stem cells and it was shown to promote proliferation of these cells and to be required for abdominal skin expansion in mice during pregnancy and regeneration during wound repair (Ichijo et al., 2017).”

“Furthermore, Weidgang et al. (2013) showed that in mouse embryonic stem cells (mESCs), Tbx3 directly bound highly conserved T-elements to activate the promoters of Eomes, T and Sox17 which are essential for mesoderm differentiation.”

“In addition, co-expression of Tbx3 and Nr5α2 with Oct4, Sox2, Klf4 and c-Myc enhanced the generation of porcine iPSCs which resembled mESCs (Wang et al., 2013).”

“More recently, TBX3 was shown to promote proliferation of papillary thyroid carcinoma cells through repressing p57KIP2 (Li et al., 2018a, 2018b).”

“Consistent with this data, naïve cynomolgus monkey (Cm) iPSCs was shown to express Oct3/4, DPPA5, SOX2, TBX3, KLF4, and KLF5 and expression of these genes in Cm ESCs was LIF-dependent (Honda et al., 2017).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“It was however predicted that TBX3, like all other T-box members identified to date, will bind its biological downstream effectors as a single monomer through the half T-element of the palindrome target sequence (Coll et al., 2002; Wilson and Conlon, 2002).”

“UMS is an autosomal dominant developmental disorder, characterized by a number of clinical features including mammary and apocrine gland hypoplasia, upper limb defects, malformations of areola, dental structures, heart and genitalia (Chen and Chen, 2017).”

“Similarly, the upregulation of TBX3 in breast and melanoma cells stimulated with RA or TGFβ1 led to diminished proliferative rates but increased migration (Ballim et al., 2012; Li et al., 2013).”

“Interestingly, in two different studies TBX2 and TBX3 were separately implicated in gastric cancer and their levels were shown to correlate inversely with E-cadherin levels (Liu et al., 2019; Miao et al., 2016).”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“On the other hand, the ectopic overexpression of TBX3 or TBX3+2a in human mesangial cells inhibited apoptosis (Wensing and Campos, 2014).”

“Furthermore, while Tbx3 levels are high in undifferentiated mESCs, its levels are downregulated in mESCs undergoing retinoic acid induced differentiation (Ivanova et al., 2006).”

“TBX3 in human disease TBX3 has been implicated in human diseases including ulnar mammary syndrome, rheumatoid arthritis, obesity and cancer (Frank et al., 2013; Quarta et al., 2019; Sardar et al., 2019; Willmer et al., 2017).”

“Recently, Oh et al. (2019) showed that TBX3 is expressed at very low levels in alveolar and embryonal rhabdomyosarcoma cells and the ectopic overexpression of TBX3 resulted in the inhibition of proliferation and migration of these cells.”

“During mouse embryonic development, Tbx3 is expressed in the inner cell mass of the blastocyst, in the extraembryonic mesoderm during gastrulation, and in the developing heart, limbs, musculoskeletal, mammary glands, nervous system, skin, eye, liver, pancreas, lungs, pituitary glands and genitalia (Chapman et al., 199…”

“Ke et al. (2018) also showed that LIF enhanced the levels of p-AKT as well as Tbx3 in marmoset iPSCs and an inhibitor of PI3K drastically reduced this regulation.”

“Interestingly, two studies showed that Tbx3/TBX3 is not entirely critical for the tenacity or generation of iPSCs (Klingenstein et al., 2016; Russell et al., 2015).”

“BRAFV600E and AKT3 are constitutively activated in approximately 50% and 70% of melanomas respectively and they play critical roles in melanoma formation and invasion (Dhawan et al., 2002; Palmieri et al., 2015; Siroy et al., 2016).”

“…of TBX3 was associated with a poor overall survival, resistance to cancer therapy and a more invasive phenotype (Beukers et al., 2015; Eriksson et al., 2015; Etcheverry et al., 2010; Kandimalla et al., 2012; Lee et al., 2014; Lyng et al., 2006; White-Al Habeeb et al., 2014; Yamashita et al., 2006).”

“Indeed, uncontrolled cell proliferation and the bypass of senescence and apoptosis are early events in oncogenesis, and TBX3 has been shown to impact these processes as well as to promote tumour formation, angiogenesis and metastasis (Dong et al., 2018b, 2018a; Feng et al., 2018; Krstic et al., 2019; Wang, 2018; Willm…”

“TCF7L1 and TBX3 are indeed well-known repressors in development 51,52 and, while it has been previously reported that Tcf7l1 and Tbx3 expression is zonated in the adult mouse liver 43,53 , here we show their direct implication in liver zonation regulation by enhancer-GRN mapping.”

“005), a transcriptional repressor involved in development and overexpressed in multiple cancer types, including squamous tumors [37, 38]; MET (25% T-LUAD versus 4.”

“Previous studies have shown that abnormal expression of TBX3 in humans can cause papillary carcinoma of the skin, ulnar-mammary syndrome, and arrhythmia (Frank et al., 2012; Khan et al., 2020).”

“Indeed, while large evidence suggests oncogenic roles for TBX3 41,42,44,45 , a few studies have indicated that it may also have tumor suppressor functions 46,47 , suggesting that TBX3 function may depend on cellular context-specific factors and on its protein partners/ regulators.”

“In adult tissues, TBX3 is mainly expressed in cancers of epithelial or mesenchymal origin [49], such as head and neck SCC, GC, ovarian cancer, cervical cancer (CC), pancreatic cancer, bladder cancer, and HCC [30, 50, 51].”

“Tbx3 protein contains a T-box domain required for DNA binding and transcriptional initiation, two repression domains, and an activation domain ( 18 ).”

“Co-embeddings of mutant and wild-type cells of these sub-trajectories, together with regression analysis, identify multiple keratin genes as positively correlated with lochNESS, consistent with a role for Tbx3 in epidermal development 38 (Extended Data Fig.”

“TBX3 plays a role in regulation of several structures in embryonic development, including the limbs, mammary glands, lungs, and heart [40].”

“Parathyroid hormone resistance Alstrom Syndrome [62] 2p13, ALMS1 gene Visual, hearing loss, cardiac pulmonary, renal dysfunction Ulnar mammary syndrome [68] 12q23-24.”

“As a developmental factor, Tbx3 is speci fi cally and dynamically expressed during embryonic organogenesis, while its expression goes relatively low in adult organs 9 .”

“TBX3 , a transcriptional repressor of the T-box gene family, plays an oncogenic role by inducing angiogenesis, activating metastasis, and inhibiting cell death [46].”

“26,27 This ancient early expression pattern likely reflects a conserved role in patterning structures within the post-axial zone of the forelimb bud.”

“Moreover, TBX3 regulates organ growth and development, and is also involved in the regulation of a variety of diseases [42].”

“Further, TBX3 has been shown to lead to uncontrolled cell proliferation and evasion of senescence and apoptosis, thereby promoting cancer initiation, angiogenesis, and metastasis [29].”

“TBX3 is implicated in senescence bypass partly through its ability to repress cell cycle checkpoint genes such as CDKN2B (Khan et al., 2020).”

“The ends of the ducts form terminal ductal units (TDUs), which drive further growth and branching until they reach the boundaries of the fat pad [53,73,74].”

“Lastly, organismal growth is strongly affected by CTNNB1 , HGF , and TBX3 , pointing to their influence on developmental biology [75–77].”

“TBX3, along with TBX2, TBX4, and TBX5, belongs to the TBX2-family [1, 2].”

“TBX2 was shown to prevent senescence by a mechanism involving their ability to transcriptionally repress these cell cycle inhibitors [15, 16].”

“Tbx3 -null embryos die in utero (12–14), highlighting its importance during embryonic development.”

“1) and TBX3 (supplementary Table 3) were downregulated in CPAM1 compared to healthy tissue [25, 26].”

“In the following section, we present a hypothetical research avenue to explore how maternal environmental cues, particularly maternal nutrition, influence fetal mammary biological processes, potentially shaping breast cancer susceptibility in adulthood.”

“, early-stage cessation leads to initial failure and deformity of the limb, whereas late-stage termination leads to loss of fingers (13).”

“Eighteen TBX3 variants associated with UMS phenotype are described in the literature; four nonsense, eight frameshift, three missense, and three splice site variants [1,18,20] (Figure 2).”

“This creates a mutual activation loop among Tbx2, Tbx3 , and Tbx5 , with the notable exception that Tbx3 represses Tbx2 , as was reported for cancer cells (42, 43).”

“…including Tcf21 , Spop , Foxf1 , and Tbx3a , showed similar expression profiles in both the lungs and swim bladder, supporting their homology (Acharya et al. 2011; Bian et al. 2023; Dong et al. 2019; Inoue et al. 2019; Khan et al. 2020; Lu et al. 1998; Park et al. 2019; Quaggin et al. 1998).”

“These include the bypass of senescence and apoptosis, promotion of proliferation, tumour formation, invasion, and migration, and conferring tumour drug resistance [9, 10].”

“A study also showed that pacemakers originate from TBX3-positive cells in early heart organ development [8].”

“For the endoderm, this enabled us to identify definitive endoderm progenitors (DEPs) ( Sox4 and Foxa2 ) ( 40 , 41 ), as well as extraembryonic endoderm (XEnd) ( Hnf4a and Tbx3 ) ( 42 , 43 ) at LB stage (Fig.”

“Mutations in the TBX3 gene can result in ulnar mammary syndrome.(50) Furthermore, studies have verified that TBX3 play roles in congenital heart disease,(51) hepatocellular carcinoma,(52,53) breast cancer,(54) embryonic skin development,(55) and rhabdomyosarcoma.”

“Speci fi cally, we hydrodynamically injected Akt/NICD/sgEGFP or Akt/NICD/sgMad2l1 plasmids in FVB/N mice (Supplementary Fig.”

“The transcriptional repressor Tbx3, a gene required for embryonic liver development (Khan et al. 2020; Ludtke et al. 2009; Suzuki et al. 2008), is also expressed in a zonated pattern during postnatal liver growth (fig.”

“Interestingly, overexpression of TBX3 occurs somatically in various types of cancer including carcinomas and sarcomas (Khan et al. 2020; Kumar et al. 2014).”

“TBX3 is required for embryonic mammary development, and its loss is associated with mammary aplasia or hypoplasia together with limb and nipple anomalies [125].”

“TBX3 acts as an oncogene; its overexpression or mutation enhances activity to promote proliferation, angiogenesis, and evasion of apoptosis and senescence (66), and is associated with progression or short-lived response to fi rst-line immunotherapy in metastatic non-squamous NSCLC (67).”

“The role of TBX3 has been investigated in liver and breast tumors (Khan et al., 2020), but to our knowledge, only one study has examined the function of this factor in prostate cancer cells (Hwang et al., 2022).”

“It plays an important role in the control of developmental signal systems ( 25 ) involved in critical structure formation of organs, such as the mammary glands, heart, lungs and limbs ( 26 ).”

“TBX3 function is dose-sensitive; heterozygous loss-of-function mutations in this gene cause Ulnar Mammary syndrome, and it is frequently found to be ectopically expressed in a wide range of epithelial-and mesenchymal-derived cancers [103].”

“Based on the established role of TBX3 in promoting tumour formation and metastasis [8], we propose that TBX3 overexpression in VGP cell clusters was imperative to mediate their collective migratory modes and the merging with adjacent clusters (see Figure 3).”

“No-tably, TBX3 is a critical developmental regulator directly implicated in limb and tissue development [35].”

“By contrast, Luminal A BC responders displayed higher expression of FN1 , SEC61G , and TBX3 , indicating a distinct CAF-associated context potentially related to development transcriptional programs [99], and epithelial–stromal crosstalk [100] in hormone receptor–positive disease.”

“Due to the strong enhancer activity differences between the bat and mouse Tbx3 sequences and the known role of this gene in early limb development 28 , we swapped the mouse sequence with the bat sequence using a modified protocol for the Precise Integration into Target Chromosome (PITCh) system 29…”

“TBX3 , another predicted upstream regulator, is overexpressed in various epithelial and mesenchymal tumors and promotes proliferation by repressing cell cycle inhibitors such as p14 ARF /p19 ARF , p21 WAF1/CIP1 , p57 KIP2 or phosphatase and tensin homolog ( PTEN ) (25).”

“The transcription factor TBX3 has been shown to be overexpressed in multiple subtypes of tumors and exerts direct oncogenic effects [31].”