Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments.

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

Cytoskeletal tubulin competes with actin to increase deformability of metastatic melanoma cells.

The formation of membrane protrusions during migration is reliant upon the cells' cytoskeletal structure and stiffness. It has been reported that actin disruption blocks protrusion and decreases cell stiffness whereas microtubule disruption blocks protrusion but increases stiffness in several cell types. In melanoma, cell migration is of concern as this cancer spreads unusually rapidly during early tumour development. The aim of this study was to characterise motility, structural properties and stiffness of human melanoma cells at radial growth phase (RGP), vertical growth phase (VGP), and metastatic stage (MET) in two-dimensional in vitro environments. Wound assays, western blotting and mitochondrial particle tracking were used to assess cell migration, cytoskeletal content and intracellular fluidity. Our results indicate that cell motility increase with increasing disease stage. Despite their different motility, RGP and VGP cells exhibit similar fluidity, actin and tubulin levels. MET cells, however, display increased fluidity which was associated with increased actin and tubulin content. Our findings demonstrate an interplay between actin and microtubule activity and their role in increasing motility of cells while minimizing cell stiffness at advanced disease stage. In earlier disease stages, cell stiffness may however not serve as an indicator of migratory capabilities.

Metformin-induced alterations in nucleotide metabolism cause 5-fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma.

5-Fluorouracil (5-FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5-year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti-cancer activity, in combination with 5-FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5-FU in WHCO1 and WHCO5 cells, by more than five- and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5-FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a "dilution" of 5-FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre-treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5-FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.

T-box transcription factors in cancer biology.

The evolutionarily conserved T-box family of transcription factors have critical and well-established roles in embryonic development. More recently, T-box factors have also gained increasing prominence in the field of cancer biology where a wide range of cancers exhibit deregulated expression of T-box factors that possess tumour suppressor and/or tumour promoter functions. Of these the best characterised is TBX2, whose expression is upregulated in cancers including breast, pancreatic, ovarian, liver, endometrial adenocarcinoma, glioblastomas, gastric, uterine cervical and melanoma. Understanding the role and regulation of TBX2, as well as other T-box factors, in contributing directly to tumour progression, and especially in suppression of senescence and control of invasiveness suggests that targeting TBX2 expression or function alone or in combination with currently available chemotherapeutic agents may represent a therapeutic strategy for cancer.

The T-box transcription factor, TBX3, is sufficient to promote melanoma formation and invasion.

The T-box transcription factor, TBX3, is overexpressed in several cancers and has been proposed as a chemotherapeutic target. Several lines of evidence suggest that TBX3 may be a key contributor to malignant melanoma, a highly aggressive and intractable disease. Using in vitro and in vivo assays we demonstrate here for the first time that overexpressing TBX3 in non-tumourigenic early stage melanoma cells is sufficient to promote tumour formation and invasion. Furthermore, we show that TBX3 may play an important role as a reciprocal switch between substrate dependent cell proliferation and tumour invasion.

Tendon-like tether formation for tongue-base advancement in an ovine model using a novel implant device intended for the surgical management of obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is a serious debilitating condition with significant morbidity and mortality affecting almost one billion adults globally. The current gold standard in the non-surgical management of airway collapse is continuous positive airway pressure (CPAP). However, non-compliance leads to a high abandon rate (27-46%). While there are multiple sites of airway obstruction during sleep, the tongue base is recognized as the key player in the pathogenesis of OSA. Poor outcomes of current tongue suspension devices are due to fracture, slippage or migration of devices. Three tongue tethering device groups, namely a polydioxanone/polyurethane combination (PDO + PU) treatment group, a PDO analytical control group, and a polypropylene (PP) descriptive control group, were implanted into 22 sheep (75-85 kg) in a two-phased study. After implant times of 8, 16, and 32 weeks, sheep were serially euthanized to allow for explantation of their tongues and chins. The PDO + PU devices remodeled during the 32-week implant period into a hybrid biological tendon-like tether through the process of gradual degradation of the PDO and collagen deposition as shown by electrophoresis, histology and mechanical testing. The control PDO device degraded completely after 32 weeks and the PP devices remained intact. The hybrid biological tendon-like tether exhibited a break-strength of 60 N, thus exceeding the maximum force to overcome upper airway collapse.

TBX3 Promotes Melanoma Migration by Transcriptional Activation of ID1, which Prevents Activation of E-Cadherin by MITF.

In melanoma, a phenotype switch from proliferation to invasion underpins metastasis, the major cause of melanoma-associated death. The transition from radial to vertical growth phase (invasive) melanoma is characterized by downregulation of both E-cadherin (CDH1) and MITF and upregulation of the key cancer-associated gene TBX3 and the phosphatidylinositol 3 kinase signaling pathway. Yet, whether and how these diverse events are linked remains poorly understood. Here, we show that TBX3 directly promotes expression of ID1, a dominant-negative regulator of basic helix-loop-helix transcription factors, and that ID1 decreases MITF binding and upregulation of CDH1. Significantly, we show that TBX3 activation of ID1 is necessary for TBX3 to enhance melanoma cell migration, and the mechanistic links between TBX3, ID1, MITF, and invasion revealed here are reflected in their expression in human melanomas. Our results reveal that melanoma migration is promoted through a TBX3-ID1-MITF-E-cadherin axis and that ID1-mediated repression of MITF activity may reinforce maintenance of an MITFLow phenotype associated with disease progression and therapy resistance.

The c-Myc/AKT1/TBX3 Axis Is Important to Target in the Treatment of Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma is a highly aggressive malignant cancer that arises from skeletal muscle progenitor cells and is the third most common solid tumour in children. Despite significant advances, rhabdomyosarcoma still presents a therapeutic challenge, and while targeted therapy has shown promise, there are limited options because the molecular drivers of rhabdomyosarcoma are poorly understood. We previously reported that the T-box transcription factor 3 (TBX3), which has been identified as a druggable target in many cancers, is overexpressed in rhabdomyosarcoma patient samples and cell lines. To identify new molecular therapeutic targets to treat rhabdomyosarcoma, this study investigates the potential oncogenic role(s) for TBX3 and the factors responsible for upregulating it in this cancer. To this end, rhabdomyosarcoma cell culture models in which TBX3 was either stably knocked down or overexpressed were established and the impact on key hallmarks of cancer were examined using growth curves, soft agar and scratch motility assays, as well as tumour-forming ability in nude mice. Our data show that TBX3 promotes substrate-dependent and -independent proliferation, migration and tumour formation. We further reveal that TBX3 is upregulated by c-Myc transcriptionally and AKT1 post-translationally. This study identifies c-Myc/AKT1/TBX3 as an important axis that could be targeted for the treatment of rhabdomyosarcoma.

The roles and regulation of TBX3 in development and disease.

TBX3, a member of the ancient and evolutionary conserved T-box transcription factor family, is a critical developmental regulator of several structures including the heart, mammary glands, limbs and lungs. Indeed, mutations in the human TBX3 lead to ulnar mammary syndrome which is characterized by several clinical malformations including hypoplasia of the mammary and apocrine glands, defects of the upper limb, areola, dental structures, heart and genitalia. In contrast, TBX3 has no known function in adult tissues but is frequently overexpressed in a wide range of epithelial and mesenchymal derived cancers. This overexpression greatly impacts several hallmarks of cancer including bypass of senescence, apoptosis and anoikis, promotion of proliferation, tumour formation, angiogenesis, invasion and metastatic capabilities as well as cancer stem cell expansion. The debilitating consequences of having too little or too much TBX3 suggest that its expression levels need to be tightly regulated. While we have a reasonable understanding of the mutations that result in low levels of functional TBX3 during development, very little is known about the factors responsible for the overexpression of TBX3 in cancer. Furthermore, given the plethora of oncogenic processes that TBX3 impacts, it must be regulating several target genes but to date only a few have been identified and characterised. Interestingly, while there is compelling evidence to support oncogenic roles for TBX3, a few studies have indicated that it may also have tumour suppressor functions in certain contexts. Together, the diverse functional elasticity of TBX3 in development and cancer is thought to involve, in part, the protein partners that it interacts with and this area of research has recently received some attention. This review provides an insight into the significance of TBX3 in development and cancer and identifies research gaps that need to be explored to shed more light on this transcription factor.

Targeting the Oncogenic TBX2 Transcription Factor With Chromomycins.

The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.

Disulfiram with or without metformin inhibits oesophageal squamous cell carcinoma in vivo.

Oesophageal squamous cell carcinoma (OSCC) is highly prevalent in developing countries but there has been little recent progress into efficacious yet affordable treatment strategies. Drug repurposing is one attractive approach for cancer therapy. Disulfiram (DSF), used to treat alcoholism, inhibits cancer growth and we previously found that DSF perturbs protein degradation/turnover pathways in vitro. This was enhanced by combining DSF with the anti-diabetic drug metformin (Met). Here, we investigated DSF with/without Met, against OSCC in vivo. Nude mice injected subcutaneously with the human OSCC cell line WHCO1, were treated with 30 mg/kg or 50 mg/kg DSF three times per week and with/without Met, for 21 days. DSF and DSF/Met-treated animals had significantly smaller tumours compared to untreated, vehicle and positive control cisplatin-treated groups. This effect for DSF was independent of copper, with no significant accumulation of copper in tumours, together with maintained proteasome activity. However, increases in total ubiquitinated proteins, LC3B-II, LAMP1 and p62 in DSF and DSF/Met groups, indicate that autophagy is inhibited. These findings show that DSF and DSF/Met significantly impede OSCC tumour growth in vivo and offer prospective alternative chemotherapy approaches for OSCC.

Doxorubicin resistance in breast cancer: A novel role for the human protein AHNAK.

Understanding the response of cancer cells to anti-cancer therapies is crucial to unraveling and preventing the development of therapeutic resistance. The human AHNAK protein is a giant scaffold protein implicated in several diverse cellular functions. The role of AHNAK in cancer is however unclear as the protein has previously been described as a tumor suppressor, as well as being essential for tumor metastasis and invasion, while also being implicated in selected chemotherapeutic responses. To clarify the role of AHNAK in cancer, we investigated the effect of doxorubicin treatment on AHNAK in doxorubicin-sensitive MCF-7 and doxorubicin-resistant MDA-MB-231 breast cancer cell lines, as well as in a tumor-bearing mouse model. The role of AHNAK in the cellular response of breast cancer cells to doxorubicin was also investigated. We report here, for the first time, an association between AHNAK and resistance to doxorubicin. While treatment with doxorubicin modulated AHNAK protein expression both in vitro and in vivo in a dose-dependent manner, no changes in its cellular localization were observed. AHNAK knockdown prevented doxorubicin-induced modulation of cleaved caspase 7 protein expression and cell cycle arrest, while its overexpression decreased cleaved caspase 7 and cleaved PARP levels and induced S-phase arrest, changes that were comparable to the effects of doxorubicin. This novel association was restricted to doxorubicin-resistant cells, implicating the protein in therapeutic resistance. These findings confirm that AHNAK does indeed function in the chemotherapeutic response of breast cancer cells while also emphasizing the need for further investigation into potential implications for AHNAK in terms of predicting and modulating treatment response.

The tumour suppressor, miR-137, inhibits malignant melanoma migration by targetting the TBX3 transcription factor.

The transcription factor, TBX3, is a key driver of malignant melanoma and any drug that impacts its expression is likely to have an impact on the treatment of this highly aggressive and treatment resistant cancer. Replacement of miRNAs that target oncogenes has gained much attention as a therapy because it is anticipated to be effective with little side-effects since miRNAs are naturally occurring and often target large set of genes in the same oncogenic pathway. Here we show that miR-137 levels correlate inversely with TBX3 mRNA levels in a panel of melanoma cell lines and in a cohort of patients with primary melanoma. Low levels of miR-137 and high levels of TBX3 are shown to be associated with poor patient survival. We show that miR-137 binds a conserved site in the TBX3 3' untranslated region and that a miR-137 mimic significantly reduces endogenous levels of TBX3 and inhibits anchorage independent growth and migration of malignant melanoma cells. Novel data are provided that the miR-137/TBX3/E-cadherin axis plays an important role in melanomagenesis and that miR-137 replacement is a potential therapeutic approach for treating melanomas.

The T-Box transcription factor 3 in development and cancer.

T-box factors comprise an archaic family of evolutionary conserved transcription factors that regulate patterns of gene expression essential for embryonic development. The T-box transcription factor 3 (TBX3), a member of this family, is expressed in several tissues and plays critical roles in, among other structures, the heart, mammary gland and limbs and haploinsufficiency of the human TBX3 gene is the genetic basis for the autosomal dominant disorder, ulnar-mammary syndrome. Overexpression of TBX3 on the other hand has been linked to several cancers including melanoma, breast, pancreatic, liver, lung, head and neck, ovarian, bladder carcinomas and a number of sarcoma subtypes. Furthermore, there is strong evidence that TBX3 promotes oncogenesis by impacting proliferation, tumour formation, metastasis as well as cell survival and drug resistance. More recently, TBX3 was however shown to also have tumour suppressor activity in fibrosarcomas and thus its functions in oncogenesis appear to be context dependent. Identification of the upstream regulators of TBX3 and the molecular mechanism(s) underpinning its oncogenic roles will make valuable contributions to cancer biology.

The T-box transcription factor TBX3 drives proliferation by direct repression of the p21(WAF1) cyclin-dependent kinase inhibitor.

BACKGROUND: TBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. TBX3 is overexpressed in several cancers and has been shown to contribute directly to tumour formation, migration and invasion. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. The cyclin-dependent kinase inhibitor p21(WAF1) plays a pivotal role in a myriad of processes including cell cycle arrest, senescence and apoptosis and here we provide a detailed mechanism to show that it is a direct and biologically relevant target of TBX3. RESULTS: Using a combination of luciferase reporter gene assays and in vitro and in vivo binding assays we show that TBX3 directly represses the p21(WAF1) promoter by binding a T-element close to its initiator. Furthermore, we show that the TBX3 DNA binding domain is required for the transcriptional repression of p21(WAF1) and that pseudo-phosphorylation of a serine proline motif (S190) located within this domain may play an important role in regulating this ability. Importantly, we demonstrate using knockdown and overexpression experiments that p21(WAF1) repression by TBX3 is biologically significant and required for TBX3-induced cell proliferation of chondrosarcoma cells. CONCLUSIONS: Results from this study provide a detailed mechanism of how TBX3 transcriptionally represses p21(WAF1) which adds to our understanding of how it may contribute to oncogenesis.

The T-box transcription factor, TBX3, is a key substrate of AKT3 in melanomagenesis.

The AKT3 signalling pathway plays a critical role in melanoma formation and invasion and components of this signalling cascade are therefore attractive targets for the treatment of malignant melanoma. Recent evidence show that the embryonically important TBX3 transcription factor is upregulated in a subset of melanomas and plays a key role in promoting melanoma formation and invasion, in part by repressing the cell adhesion molecule E-cadherin. We have identified TBX3 as a key substrate of AKT3 in melanomagenesis. Briefly, using site-directed mutagenesis and in vitro kinase assays, we have identified the AKT3 target site at serine residue 720 in the TBX3 protein and show that this site is phosphorylated in vivo. Importantly, we show by western blotting, immunofluorescence, reporter, migration and invasion assays that the phosphorylation at S720 promotes TBX3 protein stability, nuclear localization, transcriptional repression of E-cadherin, and its role in cell migration and invasion. Our results identify a novel signalling and transcriptional network linking AKT3, TBX3 and E-cadherin during melanoma migration and invasion and reveals TBX3 as a potential target for anti-metastatic therapeutics.

The T-Box factor TBX3 is important in S-phase and is regulated by c-Myc and cyclin A-CDK2.

The transcription factor, TBX3, is critical for the formation of, among other structures, the heart, limbs and mammary glands and haploinsufficiency of the human TBX3 gene result in ulnar-mammary syndrome which is characterized by hypoplasia of these structures. On the other hand, the overexpression of TBX3 is a feature of a wide range of cancers and it has been implicated in several aspects of the oncogenic process. This includes its ability to function as an immortalizing gene and to promote proliferation through actively repressing negative cell cycle regulators. Together this suggests that TBX3 levels may need to be tightly regulated during the cell cycle. Here we demonstrate that this is indeed the case and that TBX3 mRNA and protein levels peak at S-phase and that the TBX3 protein is predominantly localized to the nucleus of S-phase cells. The increased levels of TBX3 in S-phase are shown to occur transcriptionally through activation by c-Myc at E-box motifs located at -1210 and -701 bps and post-translationally by cyclin A-CDK2 phosphorylation. Importantly, when TBX3 is depleted by shRNA the cells accumulate in S-phase. These results suggest that TBX3 is required for cells to transit through S-phase and that this function may be linked to its role as a pro-proliferative factor.

The palladacycle, AJ-5, exhibits anti-tumour and anti-cancer stem cell activity in breast cancer cells.

Breast cancer is the most common malignancy amongst women worldwide but despite enormous efforts to address this problem, there is still limited success with most of the current therapeutic strategies. The current study describes the anti-cancer activity of a binuclear palladacycle complex (AJ-5) in oestrogen receptor positive (MCF7) and oestrogen receptor negative (MDA-MB-231) breast cancer cells as well as human breast cancer stem cells. AJ-5 is shown to induce DNA double strand breaks leading to intrinsic and extrinsic apoptosis and autophagy cell death pathways which are mediated by the p38 MAP kinase. This study provides evidence that AJ-5 is potentially an effective compound in the treatment of breast cancer.

The Highly Homologous T-Box Transcription Factors, TBX2 and TBX3, Have Distinct Roles in the Oncogenic Process.

The T-box transcription factors TBX2 and TBX3 are overexpressed in several cancers and are able to bypass senescence by repressing ARF and p21(WAF1/CIP1/SDII). Although these studies suggest that they may both contribute to the oncogenic process by repressing common targets, whether they have redundant or distinct roles in cancers where they are both overexpressed remains to be elucidated. Importantly, when Tbx2 function is inhibited in melanoma cells lacking Tbx3, the cells senesce, but whether this is possible in melanoma cells overexpressing both proteins is not known. An understanding of this issue may have important implications for the design of an effective pro-senescence therapy. In this study, the authors used a sh-RNA approach to knock down TBX2 and TBX3 individually in 2 human melanoma cell lines that overexpress both these factors and then examined their specific involvement in the oncogenic process. They demonstrate, using in vitro and in vivo cell proliferation, as well as colony- and tumor-forming ability and cell motility assays, that TBX2 and TBX3 have distinct roles in melanoma progression. In the tested lines, although TBX2 could promote proliferation and transformation and was required by primary melanoma cells for immortality, TBX3 was required for tumor formation and cell migration. These findings were reproducible in a human breast cancer cell line, which confirms that TBX2 and TBX3, although highly homologous, do not have redundant roles in the transformation process of cancers where they are both overexpressed. These results have important implications for the development of new cancer treatments and in particular for melanoma, which is a highly aggressive and intractable cancer.