Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma

Background: Thymosin beta 10 (T beta 10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of T beta 10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of T beta 10 in CCA tumor tissues and cell lines as well as molecular mechanisms of T beta 10 in tumor metastasis of CCA cell lines. Methods: T beta 10 expression was determined by real time RT-PCR or immunocytochemistry. T beta 10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing T beta 10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied. Results: Ten pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, T beta 10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of T beta 10. Silence of T beta 10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of T beta 10 reduced cell migration compared with control cells (P<0.05). In addition, silence of T beta 10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of T beta 10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked T beta 10 silence-associated ERK1/2 activation, Snail expression and cell migration. Conclusions: Low expression of T beta 10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.