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

Cholangiocarcinoma (CCA) is the second most common primary malignancy of the liver; the incidence of intrahepatic CCA in Western countries has been steadily growing in the last two decades.1 In spite of the rising incidence, treatment options for CCA remain unsatisfactory,1,2 particularly because of the strong and early invasiveness of the tumor. In many patients, lymphnodal or distant metastasis or micrometastasis are present already at the time of the diagnosis, limiting and worsening the prognosis in patients otherwise eligible for surgical resection. However, a subset of patients with less aggressive CCA may even undergo liver transplantation after neoadjuvant radiochemotherapy and have excellent survival. Biomarkers able to predict tumor invasiveness and prognosis would be an important decision-making tool. Unfortunately, mechanisms that determine CCA invasiveness are largely unknown. Cancer invasiveness and metastasization requires tightly adherent epithelial cells to convert to a more motile phenotype expressing several mesenchymal features. 3 During this process, some molecular programs typical of the mesenchymal phenotype are activated, as shown by the expression of specific cell surface proteins, cytoskeletal proteins, extracellular matrix-degrading enzymes, and transcription factors.4 One such proteins is S100A4, a member of the S100 family of small calcium-binding proteins, expressed by mesenchymal cells, macrophages,5 and by epithelial cells in mesenchymal transition (EMT). Expression of S100A4 was shown to be a predictor of metastasization in colon cancer.6,7 The mechanisms of action of A100A4 depends on the cellular localization of the protein. In the cytoplasm S100A4 interacts with a number of partner proteins in cytoskeleton and in the plasma membrane (such as myosin IIa or liprin-β1). When localized in the nucleus, S100A4 may exert transcriptional functions that affect several genes, including matrix metalloproteinase (MMP)-98 and E-cadherin.9 However, the mechanism of action of S100A4 remains largely unknown, as it remains unclear whether S100A4 is just a biomarker of cancer cell aggressiveness or actually represents a functional target amenable of therapeutic intervention. While examining the expression of EMT markers in CCA specimens, we noticed that a subgroup of CCAs expressed S100A4 in the nucleus. In this study we addressed: (1) the prognostic significance of S100A4 nuclear expression in a large series of patients undergoing surgical resection, and (2) the functional relevance of S100A4 expression on the metastatic potential, motility, and invasiveness of CCA cell lines in vivo and in vitro. Our results show that nuclear expression of S100A4 by neoplastic bile ducts significantly correlated with increased metastasization and reduced survival after surgery, that human CCA cells with nuclear expression of S100A4 have a much stronger metastatic ability when xenotransplanted into severe combined immunodeficiency (SCID) mice, and that silencing S100A4 in CCA cells that originally overexpressed S100A4 significantly reduced motility and invasive capabilities in vitro.