Thrombin increases the metastatic potential of tumor cells

Abstract

Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that alpha-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of alpha-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-alpha-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of alpha-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the alpha11b beta 3 integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against alpha 11b beta 3 integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of alpha-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.

Role and regulation of the thrombin receptor (PAR-1) in human melanoma

“…Expression of the thrombin receptor has been detected in human carcinoma cell lines including colon adenocarcinoma (Wojtukiewicz et al, 1995), laryngeal (Kaufmann et al, 1997), breast (EvenRam et al, 1998;Henrikson et al, 1999), pancreatic (Rudroff et al, 1998), and oral squamous cell carcinoma (Liu et al, 2001). We have demonstrated that the metastatic melanoma cells A375SM and WM266-4 overexpress the thrombin receptor (Figure 1).…”

“…Furthermore, the thrombin receptor was reported to be a rate-limiting factor in thrombin-enhanced experimental pulmonary metastasis in a murine melanoma cell line, demonstrating a potential role for the thrombin receptor in the metastasis of melanoma (Nierodzik et al, 1998). In human melanoma cells, thrombin acts as a growth factor, suggesting that signaling by the thrombin receptor is involved in the biological response of these cells, which likely involves downstream regulation of gene products contributing to their metastatic phenotype (Wojtukiewicz et al, 1992;Fischer et al, 1995;Nierodzik et al, 1998).…”

“…Recent studies indicated that activation of blood coagulation factors is a common occurrence in cancer patients, and these factors have been implicated to contribute to tumor growth (Ornstein and Zacharski, 2001). The vast majority of cancer patients with metastatic disease have clinical signs of coagulation abnormalities reflecting augmented thrombin generation (Wojtukiewicz et al, 1993). For example, melanoma is one of a number of human tumor types in which the tumor is associated with an intact coagulation pathway, leading to thrombin generation and conversion of fibrinogen to fibrin in situ immediately adjacent to viable tumor cells (Ornstein and Zacharski, 2001).…”

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho–Rho kinase axis and tumor microenvironment

“…Thrombin is a potent mitogen for fibroblasts and epithelial cells, and potentiates the proliferative responses of tumor cells to classical growth factors, such as epidermal growth factor and insulin (Van Obberghen-Schilling et al, 1995). Both thrombin and proteinase-activated receptors type I (PAR-1) are expressed in invasive cancer cell lines and breast carcinoma biopsy specimens (Wojtukiewicz et al, 1995;Even-Ram et al, 1998, 2001Nguyen et al, 2002;Darmoul et al, 2003). However, the signaling pathways triggered by thrombin and PAR-1 signaling during tumor progression and invasion remain poorly documented.…”

Differential Expression of Protease-Activated Receptors-1 and -2 in Stromal Fibroblasts of Normal, Benign, and Malignant Human Tissues

“…12 In vitro studies have demonstrated increased tumor cell adhesion to endothelium, extracellular matrix and platelets, enhanced metastatic capacity of tumor cells, and activated cell growth and stimulation of angiogenesis in response to thrombin and PAR-1 agonist peptides. [13][14][15][16][17][18] PAR-1 has been localized in smooth muscle cells, 19 pancreas tumor cells, 20 -21 carcinoma and melanoma cell lines 16 and recently in human mast cells. 22 In breast carcinoma cells, the level of PAR-1 expression has been correlated to the degree of invasiveness.…”