79 cells in sh8-EGI-1, P = 0,02 with respect to EGI-1 and shRNA;

79 cells in sh8-EGI-1, P = 0,02 with respect to EGI-1 and shRNA; 496.50 ± 76.01 cells in sh9-EGI-1, P = 0.001) (Fig. 5F). We found no difference in proliferation rates and caspase-3 expression among the CCA cell lines and the S100A4 silenced clones. The results are shown in the Supporting Materials (text and Fig. S3). By enzyme-linked immunosorbent assay (ELISA), we found that EGI-1 cells were able to secrete MMP-9 but not MMP-2, in accordance with immunohistochemistry performed in liver metastases of SCID mice. This secretory

property was significantly reduced by lentiviral silencing of S100A4 in sh8 and sh9 clones (4141 ± 520 pg/mL in EGI-1, 1649 ± 128 in sh9, 146 ± 59 in sh8, P < 0.001 versus parental EGI-1 cells). TFK-1 ability to secrete MMP-9 is conversely negligible (74 ± 110) (Fig. S4C). Cholangiocarcinoma is ABT-737 price characterized by a poor prognosis and strong invasiveness. The availability of biomarkers of early metastatic behavior would help to allocate CCA patients to their best treatment, which may even include transplant. Unfortunately, little is known about the mechanisms that favor invasiveness in CCA. Among the molecular signatures of cancer invasiveness, S100A4, a member of the S100 family of small calcium-binding proteins, normally

expressed by mesenchymal cells, is of particular interest because it was shown to correlate with metastatic potential in breast Talazoparib clinical trial and colon cancers.7, 19 However, it is currently unknown whether S100A4 merely represents a surrogate marker of cancer invasiveness or actually plays a key role in the development of a metastatic phenotype, thereby potentially representing SPTLC1 a functional target amenable to specific therapeutic

interference. To understand the role of S100A4 as a predictor of CCA invasiveness, we studied the expression of S100A4 in a large series of resected human CCA samples and correlated it to the clinical outcomes, considered either as death or as development of metastases. We found that nuclear expression of S100A4 by neoplastic bile ducts significantly correlated with increased metastasization and reduced survival after surgery. The association of S100A4 expression with worse patient outcome has been reported earlier in colonic and breast cancers.7, 19 These studies did not distinguish between its nuclear or cytoplasmic expression, although prior data from Flatmark et al.20 indicated that nuclear localization of S100A4 was more closely correlated with advanced tumor stage at diagnosis in colorectal cancer. This is important, because expression of S100A4 in the cytoplasm of biliary epithelial cells can be seen also in nonneoplastic diseases, as a part of the epithelial reaction to damage.21 Therefore, in our study on CCA we focused on the nuclear expression of S100A4, a feature not seen in nonneoplastic biliary diseases.

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