Results
The expression of thrombin in lung cancer is closely related toclinicopathological features and the prognosis of patients.
Thrombin has also been shown to contribute to tumor progression. However, the expression of thrombin in NSCLC tissues and the relationship between thrombin expression and clinicopathological features and the prognosis of NSCLC patients have not been reported. To confirm the presence of thrombin (prothrombin) in NSCLC, 132 patients with a pathologically confirmed diagnosis of NSCLC were analyzed. We found the expression of thrombin was significantly increased in tumors of all different types of NSCLC tissues compared with their adjacent non-tumor lung tissues (Fig. 1A-C). There was no significant difference in different subtypes of NSCLC (Fig. 1C). To further evaluate the prognostic value of thrombin for NSCLC patients, univariate and multivariate analyses were performed with the clinicopathological characteristic. As shown in Table 1, thrombin expression in tumor tissue was significantly correlated with TNM stage of NSCLC. The 5-year overall survival (OS) rates of thrombin-positive patients were significantly lower than those of thrombin-negative patients (Fig. 1D). Moreover, both the mRNA and protein levels were significantly increased in comparison to normal lung cell line (BEAS-2B) detected by Q-PCR (Fig. 1E) and western blot (Fig. 1F) in three NSCLC cell lines.
Thrombin plays an important role in the progression of lung cancer.
To observe the role of thrombin in lung cancer cells, we constructed A549THR-/- and LLCThr-/- cells. Transwell assay results showed that thrombin depletion inhibited cell migration in A549 and Lewis cells (Fig. 2A-D). IL6 has been demonstrated to be involved in the development, progression and metastasis in several cancers (Huang et al., 2018). Matrix metalloproteinases (MMPs) affects the physical barrier of the tumor microenvironment (TME) and induces metastasis (Lyu, Xiao, Yin, Yang & He, 2019). We found that the expression levels of MMP9 and IL6 were decreased in thrombin deficient cells, and the expression of MMP9 and IL6 could be restored by adding exogenous thrombin (supplementary Fig. S1).
To assess the role of thrombin in vivo, we employed tumor models. In subcutaneous transplanted tumor model, tumor growth inhibition was observed in thrombin deficient group (Fig. 2E-G). In addition, the lung metastases were reduced in thrombin deficient group (Fig. 2H-I). In orthotopic lung tumor model, thrombin deficiency could markedly increase the survival time of mice (Fig. 2J). And the control LLC cells generated massive lung tumor burden in mice. Significantly, the depletion of thrombin led to smaller lung tumor burden (Figure 2K). HE staining of lung sections also revealed bigger tumor area in control mice as compared to the thrombin deficient group (Figure 2L). Thrombin deficiency also inhibited liver metastasis in mice. Together with the in vitro experiments, we concluded that thrombin plays an important role in the progression of lung cancer.
r-hirudin and DTIP inhibitthrombin-promotedcell migration, invasion and angiogenesis.
To further explore the role of thrombin in the lung cancer, we used exogenous thrombin to treat NSCLC cell lines in vitro. DTIP and r-hirudin which are direct thrombin inhibitors, were developed by our group. A549, Lewis (Fig. 3A) and 95D cells (supplementary Fig. S2A) incubated with 10 nmol/L thrombin displayed a remarkable promotion in the ability to migrate into the blank space compared with the normal control (NC) group. However, r-hirudin and DTIP blocked thrombin-enhanced wound-closure capability of NSCNC cells. Transwell assay results showed that thrombin-driven migration was inhibited by pre-treatment with r-hirudin and DTIP in A549, Lewis (Fig. 3B) and 95D cells (supplementary Fig. S2B). Rho GTPases are well known as regulators of actin cytoskeletal organization and cell motility. Therefore, we examined the effect of r-hirudin and DTIP on the activation of RhoA and the status of actin filament organization. Our findings indicated that r-hirudin and DTIP could suppress the activation of RhoA in thrombin-stimulated A549 cells (Fig. 3C). As illustrated in Fig. 3D, thrombin significantly enhanced the fluorescence intensity of polymerized actin (F-actin) compared with the NS groups. However, r-hirudin and DTIP could decrease the fluorescence intensity in thrombin-stimulated cells. The appearance of membrane ruffles and the formation of lamellipodia were also decreased in r-hirudin- and DTIP-treated cells. (Fig. 3E, 3F and supplementary Fig. S2C). These data demonstrated that r-hirudin and DTIP decreased the amount of F-actin and the formation of lamellipodia in thrombin-stimulated NSCLC cells, which consequently leads to decreased cell motility and migration ability.
In previous studies, we found thrombin deficiency could reduce the expression of MMP9 and IL6. Furthermore, the expression of MMP9 and IL6 also could be increased by exogenous thrombin, which could be inhibited by r-hirudin and DTIP (Fig. 3G and supplementary Fig. S3E-G). RhoA could be activated by thrombin (Fig. 3C). CCG, an inhibitor of RhoA, could inhibit thrombin-induced expression of MMP9 and IL6. LPA, an activator of RhoA, could prevent the inhibition induced by r-hirudin and DTIP (Fig. 3G and supplementary Fig. S3E-G).
Thrombin has been reported to activate NF-κB signaling in human pleural mesothelial. The effect of r-hirudin and DTIP on the NF-κB pathway in thrombin-stimulated NSCLC cells was analyzed. The results indicated that thrombin could activate NF-κB signaling in NSCLC cells. Compared with the thrombin-treated group, r-hirudin and DTIP exhibited diminished IκBα and p65 phosphorylation, suggesting that r-hirudin and DTIP can inhibit thrombin-induced NF-κB activation. CCG could inhibit the thrombin-induced NF-κB activation, while RhoA or NF-κB activatiors (LPA and LPS) could prevent the inhibition induced by r-hirudin and DTIP (supplementary Fig. S3A-D). The results indicated that thrombin could activate NF-κB signaling via RhoA in NSCLC cells.
Studies have shown that NF-κB pathway activation upregulates the expression of cell adhesion molecules and inflammatory cytokines. We also found RhoA and NF-κB inhibitor could inhibit thrombin-induced expression of MMP9 and IL6, RhoA and NF-κB activator could prevent the inhibition induced by r-hirudin and DTIP (Fig. 3G and supplementary Fig. S3E-G), suggesting that thrombin can regulate the expression of MMP9 and IL6 via RhoA and NF-κB pathway.
Thrombin is known to promote the release VEGF and induce angiogenesis. Hence, we examined the effects of r-hirudin and DTIP on angiogenesis using a tube formation assay. After thrombin treatment, the tubule formation was promoted, r-hirudin and DTIP significantly inhibit thrombin-induced tube formation (Fig. 3H). These results demonstrated that r-hirudin and DTIP possessed an anti-angiogenic potential.
r-hirudin and DTIP exert anti-invasive andanti-metastatic abilities in a mouse lung cancer model.
Our aforementioned results suggest anti-metastatic and anti-angiogenic activity of r-hirudin and DTIP in vitro. We further confirmed the effects in vivo. In orthotopic lung tumor model, DTIP could improve the survival time of mice compared with the control group, (Fig. 4A), and mice in r-hirudin or DTIP-treated groups had smaller tumor burden (Fig. 4B), had fewer mice with liver metastases (Fig. 4C).
Murine models of experimental metastasis have been used frequently to investigate the effects of anti-haemostatic agents on cancer metastasis. Although such artificial models do not encompass the entire metastatic process, they remain useful for ‘proof-of-concept’-experiments, focusing on the haematogenous phase of tumor dissemination (Mammadova-Bach et al., 2020; Sjoberg et al., 2019; Vuong et al., 2019). Gross examination of the lungs harvested from r-hirudin- or DTIP-treated mice revealed a median of 21 (n=12) or 20 (n=12) surface pulmonary metastases per animal. In contrast, the lungs harvested from normal saline treated mice (n=16) had confluent metastases that were too numerous to count and were clearly enlarged (Fig. 4D, 4E). Micro-PET scan and histologic analyses revealed scattered small foci of tumor tissue within the lungs harvested from r-hirudin- or DTIP-treated mice, while the lungs harvested from normal saline-treated mice were nearly completely effaced by tumor tissue (Fig. 4F, 4G). We found the number of mice with tumor cells colonized in the liver was largely reduced in the r-hiruin and DTIP group compared with the normal saline group (Fig. 4I, 4J). It is important to note 78% of control mice (n=9) were dead at 24 days, with all dead at 32 days, whereas 25% of r-hirudin-treated mice (n=12) and 30% of DTIP-treated mice (n=10) were dead at 24 days (Fig. 4H).
We also examined spontaneous metastasis through subcutaneous inoculation of tumor cells in mice, which involves a more comprehensive process. Treatment of r-hirudin and DTIP for one week inhibited tumor growth slightly (Fig. 5A, 5B). Six of the 9 tumors analyzed from normal saline-treated mice showed signs of panniculus invasion, whereas only 2 of 9 tumors from r-hirudin-treated mice and 2 of 10 tumors from DTIP-treated mice had any noticeable signs of panniculus invasion (Fig. 5C, 5D).
And we also administered 1.0 mg/kg DTIP or 0.5 mg/kg r-hirudin for 21 consecutive days after one week of the injection of LLC cells. r-hirudin and DTIP significantly inhibited tumor growth (Fig. 5E-G). The number of mice with lung and liver metastases was largely reduced in r-hirudin or DTIP treated groups (Fig. 5H-K). Tumor angiogenesis was assessed using IHC analysis for CD31. The r-hirudin or DTIP treated groups showed a significant reduction of CD31-positive microvessels versus controls (Fig. 5L).
We performed immunohistochemical analysis on tumor samples to determine the expression levels of MMP9 and IL6. There is decreased expression of MMP9 and IL6 after treatment with r-hirudin and DTIP compared with normal saline-treated mice (Fig. 5M). We also examined phosphorylation levels of p65 and the key downstream signaling molecules intratumorally. We observed a marked inhibition of phospho-p65, phospho-Erk, phospho-STAT3, and phospho-Akt levels in the r-hirudin- and DTIP-treated groups (Fig. 5N).
Furthermore, we did not find increased bleeding after administration of DTIP, slight subcutaneous hemorrhage was observed after r-hirudin administration for three weeks continuously (data are not shown). These results show that DTIP, a direct thrombin inhibitor, could be extended to anti-cancer therapy.
PAR-1 is a major determinant in thrombin-promotedmetastatic of lung cancer.
Thrombin is the main activator of PAR-1 (Vu, Hung, Wheaton & Coughlin, 1991). Overexpression of PAR-1 has been detected in various types of cancers, including ovarian (Grisaru-Granovsky, Salah, Maoz, Pruss, Beller & Bar-Shavit, 2005), breast cancer (Boire, Covic, Agarwal, Jacques, Sherifl & Kuliopulos, 2005), lung cancer, prostate cancer (Black et al., 2007), and melanoma. Our previous experimental results also showed PAR-1 was highly expressed in human and mouse tumors compared with normal lung tissues (supplementary Fig. S4). However, we did not find an obvious relationship between the PAR-1 expression levels of tumors and clinical variables, such as the stage of NSCLC differentiation status and disease progression (supplementary Table 1).
To observe the role of PAR-1 in thrombin-mediated invasion and metastasis more clearly, we constructed A549PAR-1-/- and LLCPar-1-/- cells. PAR-1 depletion almost completely abrogated thrombin-promoted cell migration, and the effect of r-hirudin and DTIP on thrombin-induced migration and invasion was abolished (Fig. 6A-D). PAR-1 is a G protein coupled receptor and has been shown to induce cellular invasion through RhoA-dependent signaling. After depleting PAR-1, the activation of RhoA was inhibited and the ability of thrombin to activate RhoA was also inhibited (Fig. 6E). Similar responses were also observed via immunofluorescence staining of F-actin, as PAR-1 depletion decreased the formation of membrane ruffles (Fig. 6F). These data suggest thrombin-enhanced cell motility and migration can be completely abrogated by PAR-1 depletion in vitro. PAR-1 deficiency exhibited diminished IκBα phosphorylation and p65 phosphorylation (supplementary Fig. S5A). Importantly, thrombin-driven NF-κB activation were inhibited by pre-treatment with the specific PAR-1 inhibitor ML161 (supplementary Fig. S5A). LPA or LPS could rescue the activation of NF-κB, but thrombin could not. r-hirudin and DTIP could not inhibit NF-κB activation induced by LPA or LPS (supplementary Fig. S5B,5C), suggesting r-hirudin and DTIP inhibit thrombin-induced RhoA and NF-κB activation via PAR-1 signaling. We also found PAR-1 deficiency exhibited deceased expression of MMP9 and IL6. Besides thrombin, LPA or LPS could increase MMP9 and IL6 expression in PAR-1 deficient cells. r-hirudin and DTIP could not inhibit MMP9 and IL6 expression induced by LPA or LPS (supplementary Fig. S6), suggesting r-hirudin and DTIP inhibit thrombin-induced MMP9 and IL6 expression through RhoA and NF-κB activation via PAR-1 signaling.
To further analyze the effects of PAR-1 on lung cancer growth and metastasis, we established lung cancer model in mice using LLC cells infected by gRNA-PAR-1 lentivirus (Par-1-/- group) or LV-negative control (NC, vehicle group). In orthotopic lung tumor model, PAR-1 deficiency could markedly increase the survival rate and inhibit tumor growth in lung (Fig. 6G-I).
In the metastatic colonization model, our results confirmed PAR-1 deficient lung cancer cells lead to less lung metastatic nodes than control cells (supplementary Fig. S7A, 7B), as well as significantly decreased signal intensity in the lungs, as seen on micro-PET scans (supplementary Fig. S7C). In addition, PAR-1 deficiency could markedly increase the survival rate (supplementary Fig. S7D).
In subcutaneous tumors, PAR-1 deficient group were significantly smaller than control group (Fig. 6J, 6K). The lung metastases and liver metastases were both largely reduced in PAR-1 deficient group (supplementary Fig. S7E). In addition, based on the results of CD31 staining, we confirmed that CD31-positive microvessels decreased in the PAR-1 deficient tumors (Fig. 6L). We also found the levels of phospho-p65, phospho-Erk, phospho-STAT3, and phospho-Akt were reduced in PAR-1 deficient tumors (supplementary Fig. S7F). Together with the in vitro experiments, we concluded that PAR-1 plays an important role in the thrombin-induced progression of lung cancer.
DTIP potentiateschemotherapy-induced growth and metastasis inhibition and inhibits chemotherapeutic drug tolerance of NSCLC in mice.
Chemotherapy has been commonly prescribed in the treatment of patients with NSCLC, however, its benefits are limited due to a low response rate or acquired tumor resistance. Arnold et. al have shown that PAR-1 in the tumor induces the chemo-resistance of cancer (Queiroz et al., 2014). Meanwhile, chemotherapy such as gemcitabine, cisplatin, and paclitaxel are associated with a significant increase in the risk of arterial thromboembolic events (Zaborowska-Szmit, Krzakowski, Kowalski & Szmit, 2020). we hypothesized that DTIP could potentiate chemotherapy-induced inhibition of tumor progression.
When combination DTIP and gemcitabine, the tumor volume and tumor weight in the combination treatment group was significantly smaller than that in the groups administered DTIP alone or gemcitabine alone (Fig. 7A-C). The number of mice with lung metastases was largely reduced in the combination treatment group (Fig. 7D, 7E). We also counted the survival rates of mice in different groups. It is important to note 85% of control mice (n=7) were dead at 60 days, 57% of r-hirudin-treated mice (n=7) and 50% of gemcitabine -treated mice (n=8) were dead at 60 days, whereas, 16% combination -treated mice (n=6) were dead at 60 days (Fig. 7F). Paclitaxel could not significantly inhibit the growth of LLC in vivo. However, when combined with DTIP, the growth (Fig. 7G-I) and metastasis (Fig. 7J, 7K) of LLC were significantly inhibited. Combination of DTIP and cisplatin had a smaller tumor volume (supplementary Fig. S8A-C), but there was no significant difference in metastasis and survival time of mice compared with the group administered cisplatin alone (supplementary Fig. S8D, 8E). In addition, we evaluated the chemotherapy effects in thrombin deficient NSCLC mouse models. We found gemcitabine or paclitaxel treated mice in thrombin deficient group had smaller tumors (supplementary Fig. S9A, 9C) and longer survival time (supplementary Fig. S9B, 9D) compared with control group treated with gemcitabine or paclitaxel. These results indicated that combination therapy of DTIP and chemotherapy might achieve a better therapeutic effect.