In the current study, the functional role of Tβ10 in cell migration and tumor metastasis of CCA cell lines were investigated. Suppression of Tβ10 expression in CCA cell lines using siRNA-Tβ10 or shRNA-Tβ10 increases cell migration in vitro and enhances tumor metastasis in the nude mouse model. These results strongly suggest that suppression of Tβ10 in the primary CCA may increase its aggressiveness, possibly triggering some key signaling pathways for tumor metastasis.
There are numerous studies suggesting the critical roles for Tβ10 in tumorigenesis and progression of human cancers
[20, 23, 30–34]. Expression of Tβ10 has been shown to confer cell migratory advantage in thyroid carcinoma
[17, 18, 35, 36], and melanoma
[19, 31, 37]; but disadvantage in endothelial cells
 and ovarian cancer
. However, roles of Tβ10 in cancer development such as cell growth and apoptosis still remain controversial among cancers
[15, 16]. At present, little is known about the expression and functions of Tβ10 in CCA. Using expressed sequence tags, Tβ10 was reported to be upregulated in intrahepatic CCA compared with normal liver tissues
. In this study, however, using real-time RT-PCR, we provide evidence, for the first time, that Tβ10 is upregulated in primary CCA; while it is significantly decreased in the metastatic CCA tumors. Functionally, reducing Tβ10 expression by transiently and stably silencing technologies significantly enhanced the migration of CCA cell lines.
Recently, there have been many reports that describe the potential functional roles of Tβ10 in human cancers; however, these functions are quite different among different types of cancers. Tβ10 induces antiproliferative and proapoptotic effects in ovarian cancer; while in pancreatic cancer, Tβ10 stimulates secretion of proinflammatory cytokines interleukin (IL-7) and IL-8, which may promote pancreatic cancer pathogenesis and progression
. Tβ10 inhibits tumor growth, angiogenesis, migration, and invasion of ovarian cancer in vitro and in vivo studies by disrupting actin polymerization and by inhibiting Ras action
. In our study, we demonstrate that Tβ10 silence significantly promotes cell migration in CCA cell lines (KKU-M055 and KKU-M214 cells); while forced overexpression of Tβ10 in CCA cell lines (KKU-M055, KKU-M213) has an inhibitory effect on CCA migration. The function of Tβ10 is specific because the effect of Tβ10 silence can be reversed by overexpression of Tβ10 in CCA cell lines. Tβ10 transiently silenced by siRNA oligonucleodie in KKU-M214 cells significantly increased both migration and invasion in M214 cells in vitro. However, the invasion was increased more than the migration in M214 cells with Tβ10 silence. The reason for the difference of invasion and migration in the same cell type is not clear. It is possible that the migration and invasion have different molecular mechanisms. Invasion requires local proteolysis of the extracellular matrix (ECM), pseudopodial extension, and cell migration
From technical aspects, sh-RNA retrovirus construct for Tβ10 (sh-Tβ10) and empty control vector (sh2003) were used to infect both M214 and M055 CCA cells to establish stable silence cell lines by puromycin selection. Control vector nonspecifically reduced Tβ10 mRNA in M214 clones, but did not affect Tβ10 levels in M055. It is possible that different types of cells may contribute to this discrepancy. M214 was derived from a moderately differentiated CCA; while M055 was derived from a poorly differentiated CCA
[21, 22]. For the wound healing assay, control cells M055 Lenti-GFP had a lower wound healing rate compared with the control cells M213 Lenti-GFP although both cell types had a similar expression level of Tβ10. It is possible that different types of CCA cell lines have different mechanisms to control cell migration. Under the culture condition, M055 cells grow slower than M213 cells. In the rescue experiment, Tβ10-overexpressing plasmid was transiently transfected into the Tβ10 stable knockdown cells (M214 sh-Tβ10-GFP) and caused a 35-fold increase of Tβ10 mRNA levels compared with that in vector control cells. It is possible that the overexpression of Tβ10 from the transiently transfected plasmid was strong and overcome sh-Tβ10-mediated degradation of Tβ10 in these rescue cells.
More importantly, we also demonstrate that silence of Tβ10 in CCA cell lines enhanced tumor metastasis in the nude mouse model. These data may indicate clinical significance of the suppression of Tβ10 in metastatic CCA. Our results were consistent with previous studies in endothelial cells
 and ovarian cancer
[24, 42, 43].
However, it is not clear why metastatic CCA has a reduced expression of Tβ10. A current study has reported that approximately 16.7% of CCA have KRAS mutations
, resulting in constitutively active Ras, which may contribute to the loss of Tβ10 expression. Other studies report that Tβ10 is differentially regulated by many factors such as retinoic acid and retinoids, growth factors and steroid hormones. For examples, vascular endothelial growth factor (VEGF), thyroid-stimulating hormones (TSH) upregulate Tβ10 expression in a dose-dependent manner
[15, 16]. Moreover, chemotherapeutic drugs such as 5-Fluorouracil (5-FU) has been shown to affect Tβ10 expression
. Thus, Tβ10 could be an important biomarker for 5-FU treatment.
Cell migration is a complex biological process involving highly orchestrated multistep process network of proteins and regulatory pathways. One of these regulatory pathways is the ERK1/2 MAPK pathway, which transduces extracellular signals into intracellular responses and is necessary for many cellular events
[46, 47]. To address regulatory pathways, which are associated with the functional role of Tβ10 silence in CCA, we determined the correlation between Tβ10 silence and activation of ERK1/2. Indeed, when Tβ10 was silenced in CCA cell lines, phosphorylation of ERK1/2 was substantially increased. It has been reported that ERK-mediated phosphorylation of FAK at Ser910 inhibits the interaction of FAK with paxillin, then regulate of the FAK-paxillin complex and it is possible that ERK-modulated disassembly of the FAK-paxillin complex is involved in focal adhesion disassembly
. This emphasizes that ERK is an important factor in the regulation of cell migration.
It is unknown how silence of Tβ10 increases cell migration and metastasis of CCA. However, it is possible that suppression of Tβ10 increases the free form of G-actin, which is available for the dynamic actin polymerization especially in the cell front, thus enhances cell migration and tumor metastasis. Furthermore, Tβ10 is a key factor that interacts with Ras and inhibits Ras-dependent ERK1/2 signaling pathway
. It is recently reported that ERK1/2 activation mediates the expression of EGR1, which subsequently increases the invasive capability of ovarian cancer cells
. EGR1 also activates expression of Snail
, a key inducer of epithelial-mesenchymal transition (EMT), which plays an important role in cancer metastasis
[51–54]. In our current study, we demonstrate that Tβ10 silence-induced cell migration and metastasis of CCA may also involve ERK12, EGR1 and Snail pathways. Silence of Tβ10 substantially activated ERK1/2, and increased mRNA and protein levels of Snail and mRNA levels of EGR1 in CCA cell lines. However, silence of Tβ10 did not increase protein levels of EGR1. It is possible that Snail binds to the EGR1 promoter and represses EGR1 transcription, as well as its own promoter, thereby establishing a negative regulatory feedback loop
[50, 55, 56].
In addition, activation of ERK1/2 can be caused by KRAS mutation in many cancer types
. Our data also confirm this possibility in CCA. The Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked the activation of ERK1/2 and the expression of Snail as well as the wound healing rate in Tβ10-silenced CCA cell lines (M055-sh-Tβ10 and M214-sh-Tβ10).
Furthermore, high expression levels and activities of MMPs contribute to the invasiveness and metastasis potential in many types of cancers
. In the current study, we determined the relationship between silence of Tβ10 and expression of MMPs in CCA cell lines. Our data showed that stable Tβ10 knockdown cells (M055-sh-Tβ10 and M214-sh-Tβ10) had a relatively higher expression of MMP3, MMP7 and MMP9 than their control cells. The loss of Tβ10 in CCA may have a causal relationship with the increased expression of MMPs, which may enhance CCA metastasis.
Currently, functional roles and regulation mechanisms of Ras, ERK1/2, EGR1, Snail and MMPs in CCA metastasis are not fully understood. Further investigation into the whole picture of signaling mechanisms and protein interactions mediated by Tβ10 is warranted. It is not clear whether the current findings obtained from the research in the fluke-associated CCA are applicable to other types of CCA with different etiology. Now, there are no reports on the relationship between Tβ10 and other types of CCA. It could be a great opportunity for future investigation.