Low-Dose Paclitaxel Reduces S100A4 Nuclear Import to Inhibit Invasion and Hematogenous Metastasis of Cholangiocarcinoma

Abstract

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma (CCA), a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low dose paclitaxel (PTX), a microtubule stabilizing agent, inhibits CCA invasiveness and metastatic spread. Administration of low dose PTX to established (EGI-1) and primary (CCA-TV3) CCA cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low dose PTX did not affect cellular proliferation, apoptosis or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of lose dose PTX was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low dose PTX was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression and MMP-9 secretion. In a SCID mouse xenograft model, low dose metronomic PTX treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by CCA cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in CCA and establish a mechanistic rationale for the use of low dose PTX in blocking metastatic progression of cholangiocarcinoma.

Molecular Mechanisms Driving Cholangiocarcinoma Invasiveness: An Overview

“…Conversely, the growth of the primary tumor was not affected. Importantly, the selective reduction in S100A4 nuclear expression by low-dose paclitaxel was shown to rely on the inhibition of SUMOylation, a post-translational modification that we identified as the main contributor to S100A4 nuclear import in CCA cells 101 . Indeed, the covalent attachment of SUMO modifiers essentially affects the intramolecular and intermolecular interactions of substrate proteins, thereby altering their activity, subcellular localization, or stability.…”

“…In fact, high-dose paclitaxel exerted cytotoxic effects also on CCA cells, by reducing cell proliferation and viability, and increasing apoptosis. Conversely, paclitaxel at small doses did not affect cell viability or cytoskeletal integrity, but strongly reduced the nuclear import of S100A4 (through inhibition of its SUMOylation) and, more importantly, the invasive properties of CCA cells 101 . If confirmed in clinical studies, these observations may suggest the use of low-dose paclitaxel as an adjuvant therapy to prevent the metastatic spread of CCA after surgery, an exemplary case of drug repositioning 187 .…”

“…Nuclear expression of S100A4 first represents a useful tool in clinical decision-making, allowing for a proper stratification of CCA patients into groups at high-risk or low-risk for disease recurrence and progression. Furthermore, the inhibition of S100A4 nuclear import by low-dose paclitaxel was able to inhibit metastatization in CCA 100,101 . Paclitaxel given at conventional doses is well-known to promote microtubule polymerization and stabilization, thereby causing mitotic arrest and consequently, cell death.…”

“…Importantly, we further demonstrated that nuclearization of S100A4 is not merely a prognostic indicator, but rather a mechanistic determinant of the invasive phenotype of CCA cells. Indeed, either the down-regulation of S100A4 by lentiviral silencing or the interference with its nuclear import by paclitaxel at nanomolar doses significantly reduced the migratory and invasive capabilities of CCA cells, without affecting cell proliferation, viability and apoptosis, in vitro 100,101 . Furthermore, pharmacological inhibition of S100A4 nuclearization markedly blunted the activation of RhoA and Cdc42, the secretion of active MMP-9, and the expression of membrane-type (MT)1-MMP 101 .…”

“…Indeed, either the down-regulation of S100A4 by lentiviral silencing or the interference with its nuclear import by paclitaxel at nanomolar doses significantly reduced the migratory and invasive capabilities of CCA cells, without affecting cell proliferation, viability and apoptosis, in vitro 100,101 . Furthermore, pharmacological inhibition of S100A4 nuclearization markedly blunted the activation of RhoA and Cdc42, the secretion of active MMP-9, and the expression of membrane-type (MT)1-MMP 101 . Unlike secreted MMPs, MT1-MMP (also known as MMP-14) is tethered to the plasma membrane through a transmembrane domain, and is capable of both degrading pericellular ECM components (e.g., fibrillar collagens) and proteolytically processing adhesion molecules, growth factors and inactive precursors of other MMPs, including pro-MMP-2 and pro-MMP-13 102 .…”