Nuclear expression of S100A4 calcium-binding protein... : Hepatology (original) (raw)

Hepatobiliary Malignancies

Nuclear expression of S100A4 calcium-binding protein increases cholangiocarcinoma invasiveness and metastasization

Fabris, Luca1,2,3; Cadamuro, Massimiliano1,3; Moserle, Lidia4; Dziura, James5; Cong, Xiangyu5; Sambado, Luisa3; Nardo, Giorgia4; Sonzogni, Aurelio3; Colledan, Michele3; Furlanetto, Alberto6; Bassi, Nicolò1,7; Massani, Marco7; Cillo, Umberto1; Mescoli, Claudia8; Indraccolo, Stefano9; Rugge, Massimo8; Okolicsanyi, Lajos1,2; Strazzabosco, Mario3,10,11,*

1_Department of Surgical and Gastroenterological Sciences, University of Padova, Italy_

2_Gastroenterology Division, Regional Hospital, Treviso, Italy_

3_Center for Liver Research (CeLiveR), Ospedali Riuniti, Bergamo, Italy_

4_Department of Oncology and Surgical Sciences, University of Padova, Padova, Italy_

5_Yale Center for Analytical Sciences, Yale University, New Haven, CT, USA_

6_Department of Pathology, Regional Hospital, Treviso, Italy_

7_Surgery Division 4th, Regional Hospital, Treviso, Italy_

8_Department of Pathology, University of Padova, Italy_

9_Istituto Oncologico Veneto, IRCCS, Padova, Italy_

10_Section of Digestive Diseases, Yale University, New Haven CT, USA_

11_Department of Clinical Medicine, University of Milano-Bicocca, Milan, Italy_

*Address reprint requests to: Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, 333 Cedar Street LMP 1080, New Haven, CT 06520

Email: [email protected]

Received 25 November 2010; accepted 16 May 2011

Grant sponsor: Telethon; Grant Number: GGP09189; Grant sponsor: Federazione Amici Di Epatologia (FADE); Grant sponsor: Progetto di Ricerca Ateneo 2008; Grant Number: CPDA083217; Grant sponsor: Associazione Scientifica Gastroenterologica di Treviso (ASGET, “associazione di promozione sociale senza scopo di lucro”; Grant sponsor: Yale University Liver Center; Grant sponsor: NIH; Grant Number: DK34989; Grant sponsor: Fondazione S. Martino, Bergamo.

Potential conflict of interest: Nothing to report.

Supported by Telethon (grant GGP09189) and Federazione Amici Di Epatologia (FADE) to L.F. Supported by Progetto di Ricerca Ateneo 2008 (grant CPDA083217 to L.F., M.C., and L.O.). Associazione Scientifica Gastroenterologica di Treviso (ASGET, “associazione di promozione sociale senza scopo di lucro” to L.F. and L.O.). Yale University Liver Center (NIH DK34989 to M.S.). Fondazione S. Martino, Bergamo.

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Additional Supporting Information may be found in the online version of this article.

Abstract

Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca2+-binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI-1 and TFK-1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI-1 CCA cells showed stronger metastatic properties than TFK-1 when xenotransplanted in SCID mice. S100A4-silenced EGI-1 cells showed significantly reduced motility, invasiveness, and MMP-9 secretion in vitro, without changes in cell proliferation. Conclusion: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target. (HEPATOLOGY 2011; 54:890–899)

Abbreviations: CCA, cholangiocarcinoma; DAPI, 4′,6-diamidino-2-phenylindole; EMT, epithelial-mesenchymal transition; H&E, hematoxylin and eosin; HR, hazard ratios; K19, keratin 19; SCID, severe combined immunodeficiency; Sh, short hairpin; WB, western blot.