Cancer metastasis is the most crucial event directly influencing patient prognosis. Recent studies suggest that the EMT is strongly correlated with cancer invasion and metastasis
]. In contrast, CSCs have gained attention as targets for cancer treatment because they show chemo- and radioresistance
]. More recently, EMT was reported to promote the CSC signature
]; however, the regulatory mechanism of CSC and EMT is still unclear.
We demonstrated a direct correlation between EMT and CSCs in AdCC cells. Importantly, the EMT we analyzed in this study was developed from an in vivo
model and was not artificially isolated
], or genetically promoted
], as described previously. Therefore, the findings that we report here strongly support the hypothesis that CSCs are involved in the EMT. This study is the first to identify Brachyury as a regulator for both EMT and CSC characteristics. This conclusion is based on the observation that Brachyury knockdown resulted in simultaneous loss of all stem cell markers and loss of EMT and CSC phenotypes in morphological and biochemical assays.
The classification of EMT into 3 subtypes based on the biological and biomarker context in which they occur has been proposed
]. EMT associated with organ development is referred to as type 1 EMT, and EMT associated with wound healing and tissue regeneration are type 2 EMT. EMT in cancer progression and metastasis is categorized as type 3 EMT. Multiple extracellular signals including TGF-β, receptor tyrosine kinases, Notch, nuclear factor kappa B (NFκB), and Wnt can initiate the type 3 EMT program. The downstream intracellular signaling pathways and transcription factors that constitute this complex program demonstrate significant crosstalk, including multiple positive feedback loops
This principle of EMT suggests that the phenomenon may be reversible if such extracellular signals are removed. However, our established cell line, ACCS-M GFP, is stable and does not change to a nonmetastatic phenotype after several passages. Recent data from mammary epithelial cells also demonstrate that continuous activation of the EMT leads to epigenetic alterations in cells that induce heritable effects to maintain the EMT state even after EMT-inducing signals or factors are no longer present
]. Hence, under certain conditions such as in vivo
selection, EMT can yield stable changes in phenotype and thus the lineage identity of cells. In these cells, all possible pathways initiating EMT are constitutively active without any stimulation, as shown in Figure
. This characteristic may make the cells self-renewing, the most important phenotype of CSCs. This type of phenotypic alteration or cell selection is proposed to occur upon repeated chemotherapy or radiotherapy for cancer treatment in vivo
Although much is known about the mechanisms or signals involved in type 1 and type 2 EMT
], type 3 EMT-specific signaling still remains to be resolved in epithelial carcinoma cells. Our study indicates that one such possibility is the constitutive upregulation of TGF-β2 in ACCS-M GFP cells. TGF-β appears to be responsible for the induction or functional activation of a series of EMT-inducing transcription factors in cancer cells, notably Snail, Slug, ZEB1, Twist, Goosecoid, and FOXC2
]. Constitutive upregulation of TGF-β2 would therefore maintain the EMT or CSC status in an autocrine manner.
Brachyury is a T-box transcription factor with an evolutionarily conserved function in vertebrate development, whereby it is required for mesoderm formation
]. Brachyury is also highly expressed in various human epithelial tumors and human tumor cell lines (lung, colon, and prostate carcinomas), but not in human normal adult tissues
]. However, no studies have analyzed the role of Brachyury in tumor cells. Recently, Fernando et al.
] reported that Brachyury promotes EMT in human carcinoma cell lines. Their study demonstrated that overexpression of Brachyury in human carcinoma cells induced EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and increase in cell migration and invasion.
Downregulation of E-cadherin transcription is induced by Brachyury overexpression and partially mediated by Slug. In our model, Brachyury was overexpressed in the ACCS-M GFP (EMT cell line), and the expression level was 2-fold greater than that of the parental cell line. In contrast, overexpression of ZEB1 and ZEB2 in the EMT cell line was 5- and 9-fold higher, respectively, compared to parental cells. Surprisingly, Brachyury silencing by shRNA in ACCS-M GFP cells resulted in an almost complete inhibition of EMT-related genes and stem cell markers, including ZEB1 and ZEB2. This significant change induced by Brachyury silencing promoted the mesenchymal to epithelial transition (gain of E-cadherin and loss of vimentin) and loss of the CSC phenotype (sphere formation and tumorigenicity).
The mechanisms of Brachyury regulation of the EMT and stem cell-related genes are not certain. Brachyury and other members of the T-box transcription family preferentially bind to the palindromic consensus element AATTTCACACCTAGGTGTGAAATT, and a half-site (TCACACCT) of this consensus sequence is located at position −645 of the human E-cadherin promoter. Brachyury is able to bind to the E-cadherin promoter in vitro
, although with low efficiency
]. Other reports have suggested low-affinity binding of T-box proteins to a half consensus site, such as the one present in the E-cadherin promoter
]. However, the in vivo
binding of Brachyury to the half-site on the E-cadherin promoter could be greatly improved by interactions with accessory proteins or cofactors. Brachyury overexpression in tumor cells induces a concurrent enhancement of Slug expression, followed by the effective silencing of E-cadherin
transcription as a result of Brachyury and Slug association within the E-cadherin promoter region
The transcription factor Slug, but not Snail, has been shown to control desmosomal disruption during the initial and necessary steps of EMT in addition to repressing E-cadherin
]. Induction of EMT by FGF-1 treatment or Slug overexpression in the rat bladder carcinoma cell line NBT-II is also characterized by dissociation of desmosomes, with no change in E-cadherin expression
]. Therefore, Slug may mainly control desmosomal proteins such as plakoglobin during the initial step of EMT and associate with Brachyury to regulate E-cadherin and accomplish EMT.
During the developmental process in vertebrates, Brachyury regulates downstream genes that are components of signaling pathways such as noncanonical Wnt/planar cell polarity (Wnt/PCP), NFκB, and TGF-β signaling
]. Sox2 (SRY Sex Determining Region Y-Box2) is a member of the Sox (SRY-related HMG box) family of transcription factors. Sox2 regulates expression of multiple genes, especially stable expression of Oct-3/4, which is also a transcription factor that maintains stemness and pluripotency in normal stem cells. Recently, an association between SOX2 and EMT was also reported. Activation of SOX2 induces TGF-β downstream signaling including activation of Wnt, Notch, and Hedgehog signals, followed by induction of Snail
mRNA expression to ultimately result in inhibition of E-cadherin
transcription through induction of ZEB1/2 expression. This phenomenon is consistent with our mRNA expression results after SOX2 knockdown. Importantly, unlike Brachyury knockdown, SOX2 knockdown only inhibited genes downstream of TGF-β and failed to inhibit Brachyury expression. In contrast, Brachyury knockdown inhibited almost all the genes tested including Sox2
and its downstream genes. Also of note, silencing of SOX2 inhibited EMT but not tumorigenicity and metastasis. Therefore, it is possible that Brachyury controls multiple functional signals related to EMT and CSC simultaneously. The impact of the simultaneous silencing effect of Brachyury on EMT and CSC phenotypes observed in this study support this hypothesis. Additionally, these data suggest the existence of a partial but direct link between the EMT and CSC and that Brachyury is one of the central regulators of EMT and CSC maintenance in AdCC cells.
The use of a single cell line is a limitation of this study. It is quite difficult to establish CSC-like cell lines in vitro
and this is an obstacle to research in this field. However, parallel data from clinical samples support our hypothesis in part. Brachyury expression in clinical AdCC samples was extremely high (positive expression rate
100%), and the data suggested a close relationship with EMT (loss of E-cadherin and gain of vimentin). Therefore, at least the regulation mechanism of EMT by Brachyury demonstrated in this study may also occur in clinical AdCC.
From a clinical perspective, CSC-targeted therapy should have strict selectivity for CSCs, which is a serious obstacle for most molecular targeted therapies presently used. Selective expression of Brachyury has been reported in various human tumors of epithelial origin, but not in most human normal adult tissues
], a fact that strongly encourages the use of this molecule as a clinical therapeutic target.