Introduction
Lung cancer is the leading cause of cancer-related deaths among humans worldwide (Kyu et al., 2018), and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases (Yuan, Huang, Chen, Wu & Xu, 2019). Even when NSCLC is diagnosed at an operable stage and treatment with chemotherapy is applied, the incidence of recurrence and metastasis remains high with a median survival of less than 10-12 months. Most patients initially respond to platinum or gemcitabine-based chemotherapy (Mlak et al., 2016; Pelayo Alvarez, Westeel, Cortes-Jofre & Bonfill Cosp, 2013; Wang et al., 2018), but usually relapse and acquire chemo-resistant disease, such that the 5-year survival is < 0.5% (Morabito et al., 2014). Thus, targeting tumor metastasis and drug-resistant may represent a promising strategy to treat NSCLC (Vyse & Huang, 2019).
It has long been presumed that tumors may take advantage of the hemostatic system. Many significant hemostatic abnormalities have been described in cancer patients, including disseminated intravascular coagulation, hemorrhagic events, and migratory thrombophlebitis (Soff, 2019; Tan et al., 2019). Indeed, hemostatic complications are a common cause of death in patients with cancer(Joseph S. Palumbo, 2000). Previous studies in mice have unequivocally shown that tumor cell-associated tissue factor (Yokota et al., 2014), circulating prothrombin (Horowitz et al., 2011), and several downstream thrombin procoagulant targets (i.e., platelets, fibrinogen, factor XII) strongly promote tumor cell metastatic potential (Joseph S. Palumbo, 2000; Plantureux, Crescence, Dignat-George, Panicot-Dubois & Dubois, 2018; Yokoyama, Mori & Matsuura, 2008). However, at the level of primary tumor growth, the contribution of hemostatic factors has been less clear. Thrombin is an allosteric enzyme with an elaborate structure that exerts diverse biological effects by interacting with various receptors on the surface of vascular and nonvascular cells (Lane, Philippou & Huntington, 2005). Thrombin has also been shown to contribute to tumor progression in manners both coagulation-dependent and coagulation-independent (Xue et al., 2010). A substantial amount of data supports the idea that thrombin plays important roles in tumorigenesis, contributing to inflammation, angiogenesis, and metastatic dissemination of tumor cells through its PAR-1 receptor (Battinelli, Markens, Kulenthirarajan, Machlus, Flaumenhaft & Italiano, 2014; Reddel et al., 2017; Yokota et al., 2014). Nevertheless, thrombin expression in NSCLC primary tumor tissues and the association with prognosis of NSCLC patients remain largely unknown.
PAR-1, the prototypic member of the PAR family, has been shown to respond to a highly select group of serine proteases. Cleavage of PAR-1 by thrombin initiates potent inflammatory responses, including the up-regulation of cell surface adhesion molecules, induction of hyperpermeability (Feistritzer & Riewald, 2005), and the activation of the nuclear factor κB (NF-κB) pathways (Jeffers et al., 2015).
At present, chemotherapy is an established multimodal therapy for NSCLC, however, its benefits are limited due to a low response rate or acquired tumor resistance. In the present study, Arnold et. al have shown that PAR-1 in the tumors induces the chemo-resistance of cancer. As thrombin is the prototypical PAR-1 agonist, which indicates that targeting thrombin may be particular effective in combination with routine chemotherapy.
Direct Thrombin Inhibitor Peptide (DTIP) and recombinant hirudin (r-hirudin) which are derivatives of wild-type hirudin variant 2, were developed by our group (Mo, Zhang, Chen, Wang & Song, 2009; Zhao et al., 2017). DTIP and r-hirudin bind to exosite I and to the apolar region of thrombin, while the N-terminal moiety of r-hirudin and DTIP blocks access to the thrombin active site to inhibit the activity of thrombin. r-hirudin has entered phase I clinical trials, and DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk for subcutaneous injection.
Here, we investigated the protein levels of thrombin in clinical NSCLC samples, explored the relationship between thrombin expression level and clinicopathological features, prognosis of NSCLC patients. We evaluated the effects of r-hirudin and DTIP on tumor progression, dissemination and spontaneous metastasis in vitro and in vivo. We also demonstrated the presence of thrombin and PAR-1 accounts for the majority of the invasive signal. The novel findings presented here indicate the roles of r-hirudin and DTIP, anticoagulant drugs, could be expanded for anti-tumor therapy. We speculate that the use of r-hirudin and DTIP in combination would be a new breakthrough in cancer treatment.