6862

T

The epithelial-mesenchymal transition (EMT) is a process associated with the metastasis of solid tumors as well as with the acquisition of resistance to standard anticancer modalities. A major initiator of EMT in carcinoma cells is TGF-beta, which has been shown to induce the expression of several transcription factors ultimately responsible for initiating and maintaining the EMT program. We have previously identified Brachyury, a T-box transcription factor, as an inducer of mesenchymal features in human carcinoma cells. In this study, a potential link between Brachyury and TGF-beta signaling has been investigated. The results show for the first time that Brachyury expression is enhanced during TGF-beta1-induced EMT in various human cancer cell lines, and that a positive feedback loop is established between Brachyury and TGF-beta1 in mesenchymal-like tumor cells. In this context, Brachyury overexpression is shown to promote upregulation of TGF-beta1 at the mRNA and protein levels, an effect mediated by activation of the TGF-beta1 promoter in the presence of high levels of Brachyury. Furthermore, inhibition of TGF-beta1 signaling by a small-molecule inhibitor of TGF-beta receptor type I decreases Brachyury expression, induces a mesenchymal-to-epithelial transition, and renders cancer cells more susceptible to chemotherapy. This study thus has implications for the future development of clinical trials using TGF-beta inhibitors in combination with other anticancer agents.

BACKGROUND: epithelial-to-mesenchymal transition (EMT) induced by TGF-beta1 is one of well-recognized factors contributing to renal fibrosis. However, the underlying molecular mechanisms of EMT are not fully understood. Brachyury, an evolutionarily conserved transcription factor, was recently identified as an important factor promoting EMT in human carcinoma cell lines. There is no evidence that Brachyury is involved in renal tubular EMT. RESULTS: Our results demonstrated that Brachyury was prominently induced in TGF-beta1-treated human proximal tubular epithelial (HK-2) cells and that this induction was accompanied by changes characteristic of EMT. Blockage of Brachyury expression by short interfering RNA (siRNA) in HK-2 cells effectively reversed the TGF-beta1-induced EMT phenotype. Brachyury induction repressed E-cadherin transcription; the E-cadherin promoter contains a Brachyury binding site, and decreased expression of E-cadherin occurred in Brachyury-overexpressing cells when they were transfected with reporter constructs using the promoter. This effect was partially mediated by Slug and Snail, as knockdown of Snail and Slug by siRNA effectively reversed Brachyury-mediated EMT and partially restored E-cadherin expression. The expression of Brachyury also presented in a rat model of obstructive nephropathy and in tubulointerstitial fibrosis tissues of IgA nephropathy, suggesting that it may have a role in EMT and renal fibrosis in vivo. CONCLUSION: Our results demonstrate for the first time that Brachyury plays an important role in regulating TGF-beta1-mediated renal EMT and could be an attractive target for progression of renal disease therapies.

AIMS: Brachyury overexpression has been reported in various human malignant neoplasms, but its expression and function in hepatocellular carcinoma progression and metastasis remains unknown. The present study aimed to evaluate the critical role of Brachyury in HCC metastasis. METHODS: The expression of Brachyury in human HCC (SMMC7721, HepG2, FHCC98, and Hep3B) and control cell lines was analyzed using quantitative reverse-transcriptase polymerase chain reaction and immunoflourence methods. cancerous tissues collected from patients with HCC (n = 112) were analyzed using immunohistochemical method; a microarray analysis of HCC tissues was performed to explore the clinicopathological variables of HCC. The migratory and invasive capacities of Brachyury-SMMC7721 and Brachyury-HepG2 transfected cells were evaluated using in vitro scratch wound healing and Matrigel invasion assays, respectively. Further, six-week-old male BALB/c nude mice (n = 10) model was used in vivo assay. RESULTS: Elevated expression of Brachyury was detected in HCCs (62.5%) compared with that in adjacent nontumorous tissues. Clinicopathological analysis revealed a close correlation of Brachyury expression with distant metastasis and poor prognosis of HCC. Overexpression of Brachyury promoted epithelial-mesenchymal transition (EMT) and metastasis of HCC cells in vitro and in vivo. Brachyury overexpression enhanced Akt activation by inhibiting phosphatase and tensin homolog (PTEN), which led to subsequent stabilization of Snail, a critical EMT mediator. CONCLUSION: The study findings suggest that elevated Brachyury facilitates HCC metastasis by promoting EMT via PTEN/Akt/Snail-dependent pathway. Brachyury plays a pivotal role in HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target.

Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, which is characterized by the negative form of estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (HER2). TNBC accounts for ~15% of all breast cancer forms, and often leads to high mortality and poor prognosis. Structural maintenance of chromosome 1 (SMC1) is a subunit of the cohesion protein complex. Brachyury is a protein that is encoded by the T gene in humans, which is a transcription factor within the T-box complex of genes. epithelial-mesenchymal transition (EMT) is a ubiquitous process in the body, and in particular, induces metastasis and the proliferation of cancer cells. In the present study, we found that SMC1 expression in TNBC tissues exceeded its expression in adjacent non-tumor tissues. Similarly, the expression of SMC1 in TNBC cell lines (hs578T and HCC1937) was found to be higher than in MCF10a and MCF7 cells. Subsequently, SMC1 was overexpressed and silenced in hs578T and HCC1937 cells through plasmid and siRNA transfection, respectively. The results showed that the high expression of SMC1 often promoted EMT, accompanied by the enhanced expression of Brachyury. Besides, upregulated expression of Brachyury through plasmid transfection also significantly improved the level of EMT, which further indicated that SMC1 increased EMT in TNBC through the induction of Brachyury expression. Taken together, these results contributed to a better understanding of the pathogenesis of TNBC, which also provided an experimental basis for the prevention, diagnosis and treatment of TNBC.

BACKGROUND: The formation of the primitive streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic stem cells (ESCs) provide a good system for understanding the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both nodal/TGFbetaR and Wnt/beta-catenin signalling pathways specify cells to adopt a primitive-streak-like fate and direct them to undertake an epithelial-to-mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear to what extent events in the embryo are able to be reproduced in culture. RESULTS: Here, we combine flow cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs towards a primitive streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of brachyury, whose expression is dependent on the EMT and beta-catenin activity. CONCLUSIONS: As a consequence of our analysis, we propose that a major output of brachyury expression is in controlling the velocity of the cells that are transiting out of the primitive streak.

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1alpha (HIF-1alpha) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury on tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling.CI - Copyright (c) 2015 Elsevier Inc. All rights reserved.