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

Abstract

The serine proteases thrombin and trypsin are among many factors that malignant cells secrete into the extracellular space to mediate metastatic processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. Malignant cells solicit the help of other cell types, such as stromal fibroblasts, mast cells, monocytes, and vascular cells, to facilitate their invasion into the surrounding tissue 1 because unrestrained growth of the tumor, by itself, does not result in invasion and metastasis. 2 The interface between the invading malignant cells and the hosting stromal cells, referred to as the tumor microenvironment (TME), 3 possesses a vast array of well-orchestrated cell signaling molecules which function to facilitate the ability of the proliferating tumor front to invade the stroma, as well as to degrade and remodel the extracellular matrix. 1 Of the many factors secreted by tumor cells, the two proteolytic enzymes, thrombin and trypsin, have been correlated to the stage and type of carcinoma and are associated with cell invasion and extracellular matrix degradation. 4,5 Furthermore, the ratio of proteases to their inhibitors in the TME can favor capillary sprout elongation and lumen formation during angiogenesis. 2 Thrombin is known to influence the behavior of all of the cells identified within the TME. For instance, thrombin activates platelets to adhere to other cells or extracellular matrix, increases vascular permeability and expression of adhesion molecules, attracts monocytes, stimulates mitogenic activity of endothelial cells and fibroblasts, and degranulates mast cells. 6 -9 Thrombin also influences the rate of deposition of connective tissue proteins and the development of tissue fibrosis during normal wound healing; a process similar to cellular metastasis. 10,11 Many of thrombin's effects are mediated through the seven transmembrane G-protein coupled receptors, protease-activated receptor (PAR)-1, via proteolytic cleavage of the amino-terminal extension unveiling a new amino terminus that activates the receptor through a tethered peptide ligand mechanism. 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. 13-18 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. 23 Furthermore, B16F10 melanoma cells, transfected with PAR-1, enhanced thrombin-treated tumor cell adhesion to fibronectin 2.5-fold in vitro and pulmonary metastasis as high as 39-fold in vivo compared to the control thrombin-treated tumor cells. 24 Trypsin can stimulate fibroblasts to secrete procollagen, stimulate mast cells to degranulate, and is secreted

Expression of protease-activated receptors 1 and 2 in melanocytic nevi and malignant melanoma

“…Furthermore, dermal fibroblasts in normal skin and in benign common melanocytic nevi were also PAR-1-and PAR-2-positive. These findings are at variance with previous observations indicating PAR-2 negativity in normal dermal fibroblasts [36,37] as well as absence of PAR-1 and PAR-2 immunolabeling in stromal fibroblasts surrounding normal and breast benign tissues in contrast with the moderate to strong PAR-1 and PAR-2 staining observed in the stromal fibroblasts in most breast malignant tissues [20]. One possible explanation for our findings could be that resident dermal fibroblasts have acquired a myofibroblastic phenotype.…”

“…Indeed, overexpression of PAR-1 has been detected in numerous human cancers, including colon [14,15], laryngeal [16], breast [10], pancreatic [17,18], and oral cavity carcinomas [19]. Of interest, an up-regulation of PAR-1 and PAR-2 has been demonstrated in stromal fibroblasts surrounding neoplastic aggregates in human malignant tissues [20]. In cutaneous melanomas, it has been recently shown that loss of expression of the transcription factor activator protein 2a correlates with overexpression of PAR-1, which in turn contributes to the acquisition of a malignant phenotype [21].…”

Role of tissue stroma in cancer cell invasion

“…This is in contrast to PDGF, which indirectly transdifferentiates fibroblasts into myofibroblasts or stimulates the proliferation of myofibroblasts. Other direct acting cytokines that may be implicated in cancer are endothelin-1, released from endothelial cells [46,47], and thrombin, delivered by the circulation [48,49] (Figure 2 and Table 1). Directly acting cytokines described in non-cancerous pathological situations, such as in asthma, fibrosis, and wound healing, are shown in Table 1.…”

“…Only bioactive TGF-β signals through its receptors. Activation of the latent TGF-β complex is environmentally regulated • protease-activated receptors-1 and -2, the receptors for thrombin, are overexpressed in breast cancer-associated myofibroblasts [49]; h, human origin (unless mentioned otherwise); § fibroblasts were pretreated with TGF-β. Abbreviations: Act, Activin; bFGF, basic fibroblast growth factor; cCAF, chicken chemotactic and angiogenic factor; ET, endothelin; F, fibroblast; HB-EGF, heparin binding-epidermal growth factor; IFN, interferon; IL, interleukin; MF, myofibroblast; PPAR, peroxisome proliferatoractivated receptor; SMA, smooth muscle actin; TGF-β = TGF-β1, transforming growth factor-β; VN, vitronectin.…”

Lack of neurokinin-1 receptor expression affects tissue mast cell numbers but not their spatial relationship with nerves

“…The tissues were routinely processed for mast cell staining. Thereafter, the slides were subsequently processed for routine single immunohistochemistry (14). Slides were placed in PBS (pH 7.4) and treated with 3.0% H2O2 for 10 min at room temperature.…”