Recent evidence indicates that, similar to other S100 proteins, i.e., S100A1 and S100B,23, 24 S100A4 may also have transcriptional PD98059 cost activity, either by direct DNA binding or by interacting with other DNA-binding proteins. For example, S100A4 regulates the transcriptional activation of MMP-9 in human prostate cancer.8 S100A4 also negatively regulates expression of E-cadherin, an important prerequisite for cancer cell

motility.9 To better understand the functional effects of S100A4 in CCA, we then studied if silencing S100A4 expression in the EGI-1 cell line interfered with cell motility, invasion, cell proliferation, and apoptosis. Our results demonstrate a significant reduction in both cell migration and invasiveness as measured by transwell migration through Matrigel in Boyden chambers in the absence of chemotactic check details stimuli. Contrary to motility and invasion, we observed no effects on cell proliferation and apoptosis in S100A4-silenced EGI-1 cells. Thus, S100A4 appears

to be a key determinant of CCA invasiveness, given its involvement in the regulation of cellular motility and invasion, without affecting the local growth of the tumor that depends on the balance between cell proliferation and apoptosis. Noteworthy, expression of MMP-9 was significantly reduced in EGI-1 clones with silenced S100A4, indicating that S100A4 modulates MMP-9, an important mediator of cancer invasiveness.8

In summary, our study demonstrates that nuclear expression of S100A4 identifies a subtype of CCA with poor response to surgical resection. Furthermore, MCE the functional data generated in this work strongly suggest that S100A4 is a mechanistic determinant of CCA invasiveness. In fact, nuclear expression of S100A4 was associated with enhanced metastatic potential of CCA cell lines xenotransplanted into SCID mice, and silencing of S100A4 with reduced motility, invasiveness, and expression of MMP9 in vitro. If validated prospectively, nuclear expression of S100A4 may eventually become a tool in clinical decision making to allocate patients with CCA that are candidates for potentially curative therapies, including liver transplantation. On the other hand, down-regulation of S100A4 may become an attractive strategy to reduce CCA progression. This could eventually be achieved also using small molecules.25 The authors thank Dr. Luigi Dall’Olmo (Department of Surgical and Gastroenterological Sciences, University of Padova) for surgical assistance in experiments with SCID mice, and Dr.