Thrombin Is A Therapeutic Target For Metastasic Osteopontin-positive Hepatocellular Carcinoma

Our lab previously identified osteopontin (OPN) as a promoter of hepatocellular carcinoma (HCC) metastasis and thus a potential therapeutic target to prevent HCC metastasis. The serine protease thrombin interacts with OPN and can modify the biological activity of this molecule. To explore the role of thrombin alone or in conjunction with OPN in HCC, we measured patient thrombin levels and studied the correlation of thrombin to the HCC prognosis in patients with various OPN expression levels. We found that the expression level of thrombin was strongly associated with the metastatic potential of each HCC cell line, and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent non-tumor tissue. In addition, HCC tissue from patients with recurrent disease displayed much higher thrombin levels, particularly in those with elevated OPN levels. Immunohistochemical analysis revealed that only tissues with elevated OPN levels had a significant positive correlation between high thrombin levels,the overall survival (OS; P< 0.01), and between high thrombin levels and time to recurrence of HCC (TTR; P< 0.0001). We also evaluated the effects of OPN fragments generated by thrombin cleavage on in vitro proliferation and adhesion of HCC cells. In vitro and in vivo assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+HCC cells, as well as pulmonary metastases, which were significantly suppressed by treatment with Argatroban, a thrombin inhibitor. Furthermore, thrombin activated the FAK pathway of OPN+HCC cells, which was blocked by the inhibition of integrinβ1.Conclusion:Thrombin plays an important role in OPN-mediated aggressive phenotype, and poor prognosis of, HCC through activation of integrin (31-FAK signaling. Thus, thrombin may be a potential therapeutic target to inhibit HCC progression in OPN+patients.Part A Overexpression of thrombin in hepatocellular carcinomaPurpose To investigate the relationship between the expression of thrombin and the malignant phenotype of HCCMethods The expression of thrombin was evaluated in HCC,their adjacent nontumorous and normal liver tissues by qRT-PCR, immunohistochemistry and western blot; the expression of thrombin was evaluated in six human HCC cell lines (HepG2, PLC, MHCC97L, MHCC97H, HCCLM3 and HCCLM6) with various metastatic potentials and two human nontransformed hepatic cell lines, L-02 and Chang Liver by qRT-PCR and western blot.Results Both the mRNA and protein expression levels of thrombin in six established HCC cell lines were found to be significantly increased in comparison to nontransformed hepatic cell lines (L-02 and Chang liver cell lines) detected by real-time PCR and Western blot. Among these HCC cell lines, HepG2, PLC and MHCC97-L cells had relatively lower invasive and metastatic capabilities (low-metastatic group), while MHCC97-H, HCCLM3, and HCCLM6 had higher invasive and metastatic potential (high-metastatic group). Both the mRNA and protein expression levels of thrombin in these three high-metastatic HCC cell lines were much higher than those of the three low-metastatic HCC cell lines (P< 0.0001). The mRNA expression levels of thrombin were evaluated in 72 human HCC tumors, in their adjacent non-tumor liver tissues, and 20 normal liver tissues. The amount of thrombin mRNA was significantly increased in HCC tissues compared with their adjacent non-tumor liver tissues (P= 0.005). Moreover, the higher amount of thrombin mRNA in HCC tissue was significantly positively correlated with tumor recurrence (P= 0.037). Expression of thrombin in HCC tissues (2 -ΔCt,137.4±69.1) was significantly higher than in non-tumor tissues and normal liver tissues (2 -ΔCt, 22.2±7.6 and 19.9±8.7, respectively), with the difference begins statistically significant. These findings were confirmed by elevated thrombin protein levels in 8 out of the above 72 HCC tissue samples using Western blot analyses.Conclusion The expression of thrombin is upregulated in HCC cell lines, and its increased expression is positively correlated with the malignant phenotype of HCC cells.Part B Correlation and clinical significance of overexpression of Thrombin and/or OPN in hepatocellular carcinomaPurpose To investigate the correlation and clinical significance of Thrombin and/or OPN with hepatocellular carcinoma.Methods The qRT-PCR was used to detect the expression of thrombin and OPN in HCC, the immunohistochemistry was used to detect the expression of thrombin and OPN in 230 HCC patients, then the clinical significance of overexpression of thrombin and/or OPN was analyzed by SPSS 15.0.Results A scatter plot of thrombin and osteopontin expression revealed no correlation between thrombin and osteopontin mRNA levels in cancerous tissue (r=-0.1 7,p= 0.15).To determine whether thrombin influences OPN-related prognosis, we divided the HCC patients into low-OPN and high-OPN groups using the median OPN expression level as the cutoff. Among the tissues in the high-OPN group, the expression level of thrombin was significantly increased in HCCs from the patients with tumor recurrence compared with those without recurrence (P= 0.027). Importantly, this difference was not statistically significant in the low-OPN group (P = 0.457). We used immunohistochemical staining to assess the correlation between the expression levels of thrombin and OPN HCC tumor tissues from 230 HCC patients. We also analyzed the association with HCC prognosis in the same 230 HCC patients. Positive staining for thrombin and OPN was found in 33%(77/230) and 39% (90/230) of patients, respectively. HCC tissue from 36 (15.7%) patients was positive for both thrombin and OPN. Thrombin-positive tumor tissue was significantly correlated with tumor size (P= 0.0438), vascular invasion (P= 0.0317), and TNM stage (P= 0.0187). However, no statistically significant association was found between the thrombin expression and other clinical characteristics. In the patients with positive OPN (OPN+), positive thrombin staining in the tumor tissue was significantly correlated with preoperative serum AFP (P= 0.0304), tumor size (P= 0.0024), vascular invasion (P= 0.0018), TNM stage (P= 0.0008), tumor differentiation (P= 0.0373), and tumor encapsulation (P= 0.0477). However, no statistically significant correlation was found between thrombin expression and these characteristics in the patients with undetectable OPN expression (OPN-).Thrombin protein levels were significantly associated with TTR (P=0.021), and this association was much stronger in OPN+patients (P= 0.0001), but not in OPN-patients (P= 0.728). The 1-,3-, and 5-year tumor recurrence rates of those thrombin-positive (thrombin+) patients were 41.6,67.5, and 68.8%, respectively; these tumor recurrence rates were much higher than those of thrombin-negative (thrombin-) patients (24.8, 43.1, and 47.1%, respectively; P= 0.0001). The 1-,3-, and 5-year OS rates of thrombin+patients (75.3,42.9, and 40.2%, respectively) were significantly lower than those of thrombin-patients (85.6,59.5, and 57.5%, respectively; P= 0.005).We then stratified the 230 HCC patients by OPN expression and found that, in OPN+patients, positive thrombin expression (thrombin+/OPN+) was associated with a much shorter TTR compared with those of thrombin-HCC (P= 0.001). The 1-,3-, and 5-year recurrence rates of thrombin+/OPN+patients were 47.2,86.1, and 88.9%, respectively, which were significantly higher than those of patients with thrombin-/OPN+(20.4,42.6, and 46.4%, respectively; P< 0.0001). The 1-,3-, and 5-year OS rates of thrombin+/OPN+patients (72.2,27.8, and 22.2%, respectively) were significantly lower compared with those of thrombin-/OPN+patients (85.2,61.1, and 55.2%, respectively; P= 0.001). However, no significant difference in the survival and recurrence rates was observed between the thrombin-and thrombin+ patients within the OPN-group (TTR, P= 0.728; OS, P= 0.596).Conclusion The overexpression of thrombin and high expression of thrombin/OPN can be a new market in predicting the prognosis of HCC.Part C Relationship of overexpression thrombin with the invasion and metastasis of hepatocellular carcinomaPurpose To invesgate the relationship of thrombin with the invasion and metastasis of HCC cells.Methods The qRT-PCR,western blot was used to detect the expression of thrombin and OPN in different HCC cell lines with different metastatic potential. We then transfected the pOPN-GFP plasmid and Pegfp-N1 control plasmid into the non-metastatic cell line PLC, the expression of osteopontin was determined by western blot in the stable transfection HCC cell lines.The ability of proliferation and adhesion was detected by CCK-8 and in the stable transfection HCC cell lines respectively.At the last,metastasis assays in vivo was performed.Results PLC cells were stably transfected with either wild-type OPN (PLC-OPN) or an empty vector control (PLC-CON); level of OPN expression of transfected cells was detected by fluorescent inverted microscope and western blot. PLC-OPN cells were confirmed to have an elevated level of OPN protein compared with PLC-CON cells. In vitro cell proliferation assays were performed to evaluate the difference between PLC-CON and PLC-OPN cells in response to thrombin treatment (2 U/ml). PLC-CON and PLC-OPN cells had similar growth kinetics in normal culture. When grown in the presence of thrombin, PLC-OPN cells demonstrated a significant increase in cell proliferation during the exponential growth phase (P< 0.05); however, PLC-CON cells demonstrated no significant proliferation changes when treated with thrombin. PLC-CON and PLC-OPN cells were evaluated for altered cell adhesion to various ECM molecules in response to thrombin (2 U/ml). Thrombin treatment significantly increased the adhesion of PLC-OPN cells, but not PLC-CON cells. To investigate the effects of thrombin on in vivo HCC metastasis, PLC-OPN and PLC-CON cells treated with 2 U/ml thrombin were injected i.v. into nude mice. The rate of pulmonary metastasis and presence of metastatic lesions were evaluated at the fourth week post-injection. In spite of the fact that pulmonary metastatic lesions were detected in all mice (100%), the metastatic lesions per mouse was significantly greater in the thrombin-treated PLC-OPN group than in the untreated PLC-OPN group (P< 0.05); however, there was no significant difference in the number of metastatic lesions post-thrombin treatment in the PLC-CON groups.To further confirm the role of thrombin in inducing HCC metastasis, the thrombin inhibitor Argatroban was administered to the nude mice in combination with subcutaneous implantation of HCCLM3 cells. All nude mice successfully formed tumors after subcutaneous implantation of HCCLM3 cells. There was no significant difference in primary tumor size between the Argatroban-treatment group and the controls. In the 6th week, pulmonary metastatic lesions were detected in all control mice, with 16.7±8 metastatic lesions in each lung, the majority of which were gradeⅠ-Ⅱ, and some being gradeⅢ-Ⅳ. In contrast, only about half of the mice in the Argatroban-treatment group developed lung metastases, with 5.2±2.5 lesions in each lung; most of the lung metastases were gradeⅠ. These data indicate that Argatroban significantly reduces both the incidence and degree of lung metastasis in this mouse model of hepatocellular carcinoma (P<0.01).Conclusion Thrombin increases Hepatocellular Carcinoma malignancy via mediating osteopontin-dependent tumor growth and adhesion activityPart D Role of thrombin on the integrinβ1/FAK signaling transduction pathway in Hepatocellular carcinoma cellsPurpose To expatiate the relation between thrombin and OPN in HCC cells, and the role of thrombin on integrinβ1/FAK signaling pathway in hepatocellular carcinoma (HCC) cells.Methods We evaluated the effects of OPN fragments generated by thrombin cleavage on in vitro proliferation and adhesion of HCC cells. The phosphorylation and non-phosphorylation of FAK, which are key molecules in integrinβ1/FAK signaling pathway, were tested by western blot after thrombin acting on the OPN-positive HCC cells.Results Both recombinant N-terminal fragment and full-length human OPN were able to significantly increase the PLC-CON cell proliferation in the exponential growth phase (P< 0.05). The N-terminal fragment had a much stronger positive influence on proliferation than full-length OPN (P< 0.05). However, the recombinant C-terminal OPN fragment had no effect on PLC-CON cell proliferation (P> 0.05). To determine the effect of OPN on cell adhesion, purified OPN or its fragments were immobilized on microtiter plates and adhesion of PLC-CON cells to each recombinant protein was compared. More HCC cells adhered to N-terminal fragment of OPN compared with intact OPN (P< 0.05), while there was no significant adhesion to the C-terminal fragment or the negative control BSA. We found upregulated integrin-β1 mRNA expression in OPN-overexpression transfectants after thrombin treatment. As shown in Fig.6D, thrombin treatment induced FAK phosphorylation in OPN-overexpression transfectants (PLC-OPN) in a dose-dependent manner. FAK was maximumally phosphorylated at 2U/ml thrombin. However, no significant FAK phosphorylation was observed after thrombin treatment of control PLC-CON cells. Our results also show that thrombin treatment did not significantly change the total protein level of FAK. Moreover, integrin-β1 neutralizing antibody AIIB2 (10μg/ml) (Developmental Studies Hybridoma Bank, Iowa City, IA) significantly inhibited the thrombin-induced FAK phosphorylation.ConclusionThrombin promotes the growth and invasion of OPN+HCC cells through the activation of integrin-β1/FAK pathway.Conclusions1. The expression of thrombin is upregulated in HCC2. Thrombin plays an important role in OPN-mediated aggressive phenotype, and poor prognosis of HCC.3. Thrombin promotes the growth and invasion of OPN+HCC cells through the activation of integrin-β1/FAK pathway.4. Thrombin may be a potential therapeutic target to inhibit HCC progression in OPN+patients.The potential application of this work1.The thrombin expression level could predict HCC patients'prognosis, especially those osteopontin-positive patients. 2.Thrombin is a therapeutic target for metastasic osteopontin-positive HCC.3. Thrombin is a therapeutic target for metastasic osteopontin-positive tumors apart from HCC.Originalities of this work1. The expression level of thrombin was strongly associated with the metastatic potential and postoperative tumor recurrence of HCCs, particularly in those with elevated OPN levels; and a significant correlation was found between high thrombin levels and the survival and time to recurrence of HCCs with elevated OPN levels.2. In vitro and in vivo assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+HCC cells, as well as pulmonary metastases, which were significantly suppressed by a thrombin inhibitor.3. Thrombin activated the FAK pathway of OPN+HCC cells, which was blocked by the inhibition of integrinβ1. These indicate that thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN+patients.