Role of Extracellular High-Mobility Group Box-1 as a Therapeutic Target of Gastric Cancer

Abstract

Background: High-mobility group box-1 (HMGB1) is involved in the tumorigenesis and metastasis of various cancers. The present study investigated the roles of extracellular HMGB1 in the progression of gastric cancer (GC) and the therapeutic effects of recombinant human soluble thrombomodulin (rTM) targeting HMGB1. Methods: The effects of extracellular HMGB1 and rTM on GC cells were assessed using proliferation and Transwell assays. Their effects on local tumor growth and metastasis were evaluated using subcutaneous tumor and liver metastasis mouse models, respectively. Plasma HMGB1 concentrations in GC patients were measured using ELISA. The relationships between plasma HMGB1 concentrations and the prognosis and clinicopathological factors of patients were also investigated. Results: GC proliferation, migration, and invasion abilities were promoted by increases in extracellular HMGB1 concentrations and alleviated by rTM. In the subcutaneous tumor model, local tumor growth was promoted by the addition of rhHMGB1 and alleviated by rTM. Similar changes occurred in the liver metastasis model. Recurrence-free survival (p < 0.01) and overall survival (p = 0.01) were significantly worse in patients with high plasma HMGB1 concentrations. Conclusion: Plasma HMGB1 concentrations are a prognostic marker in GC patients. Extracellular HMGB1 promotes cancer progression and has potential as a novel treatment target in GC cells for rTM.

Keywords: gastric cancer; high-mobility group box-1; recombinant human soluble thrombomodulin.

Figure 1

Effects of rhHMGB1 and rTM on GC cells. (a) Cell proliferation was evaluated among non-treated NUGC4 and MKN7 cells, cells treated with rhHMGB1 (200 ng/mL), and cells pre-treated with rTM (100 ng/mL, 30 min) followed by rhHMGB1 (200 ng/mL) at the indicated times. Data represent the mean absorbance value ± SD. * p < 0.05. (b,c) The migration (b) and invasion (c) abilities of NUGC4 and MKN7 cells were evaluated among non-treated cells, cells treated with rhHMGB1 (200 ng/mL), and cells pre-treated with rTM (100 ng/mL, 30 min) followed by rhHMGB1 (200 ng/mL) using the Transwell migration assay and Matrigel invasion assay, respectively. Data represent the mean ± SD of the relative cell count. * p < 0.05 and ** p < 0.01. (d) Alterations in protein expression in the RAGE/Erk/NF-κB pathway by the rhHMGB1 and rTM treatments were evaluated using Western blotting. Whole lysates of NUGC4 and MKN7 cells were obtained under the conditions of no treatment, a treatment with rhHMGB1 (200 ng/mL, 48 h), and a pre-treatment with rTM (100 ng/mL, 30 min) followed by rhHMGB1 (200 ng/mL, 48 h).

Figure 2

Effects of rhHMGB1 and rTM on local tumor growth in a subcutaneous tumor model. (a) Comparisons of subcutaneous tumors among the following three groups: a group treated with only PBS, treated with rhHMGB1, and treated with rhHMGB1 and rTM (n = three, respectively). The treatment schedule was the same for all groups (Figure S4). Tumors were resected after sacrifice, and their volume (mm³) and weight (µg) were measured. Tumors were slightly larger in the group treated with rhHMGB1 alone. (b) Time course of increases in local tumor volumes among the 3 groups. Tumor sizes were measured from the body surface on the indicated days and tumor volumes were calculated. Data represent the mean ± SD of tumors. Alterations in the RAGE/Erk/NF-κB pathway of tumors resected from mice were examined by WB. The phosphorylation of Erk and NF-κB was slightly enhanced by the treatment with rhHMGB1 and inhibited by the addition of rTM. (c) Alterations in the RAGE/Erk/NF-κB pathway of tumors resected from mice were examined by WB. The phosphorylation of Erk and NF-κB was slightly enhanced by the treatment with rhHMGB1 and inhibited by the addition of rTM. Representative data from a few experiments are shown.

Figure 3

Effects of rhHMGB1 and rTM on tumor metastasis in the liver metastasis model. (a) Whole liver samples from all mice used in the experiments were removed and fixed in formalin after sacrifice (n = 4 or 5). The treatment schedule was the same for all groups (Figure S4). All cut planes of each liver are shown. The circled area in (a) indicates liver metastasis, and an enlarged view is also shown. (b) The number of mice with liver metastasis is summarized. All mice in the group treated with rhHMGB1 had liver metastasis, which was more frequent than in the group treated with only PBS, and the addition of rTM reduced liver metastasis. (c) Liver metastasis was confirmed by HE staining (at ×40 and ×100 magnification, respectively). High N/C ratio cells were observed around the tumor, and fibrosis and necrosis were noted in the center. Representative data are shown.

Figure 4

HMGB1 concentrations in tissue and plasma samples and prognostic effects. (a) The relative expression of HMGB1 in GC tissue (T) and paired non-cancerous mucosal tissue (NT) was evaluated by qRT-PCR (n = 33). Results were analyzed using the paired t-test. * p < 0.05. (b) Plasma HMGB1 concentrations in 56 GC patients and 26 healthy volunteers (HV) were measured by an enzyme-linked immunosorbent assay. Differences were analyzed by the Wilcoxon signed-rank test. * p < 0.05. (c) Comparison of pre- and post-operative plasma HMGB1 concentrations in 10 GC patients. Plasma HMGB1 concentrations significantly decreased after surgery. Results were analyzed by the Wilcoxon signed-rank test. * p < 0.05. (d) Plasma HMGB1 concentrations at each stage in GC patients and healthy volunteers (HV). Concentrations slightly increased with stage progression, and a significant difference was observed in concentrations between stage I and III patients (the Mann–Whitney U test). ** p < 0.01. (e) Relationships between plasma HMGB1 concentrations and HMGB1 expression levels in tumor tissue were examined (n = 18). A linear regression model was used for the analysis. (f) Kaplan–Meier curves for RFS (left) and OS (right) in GC patients according to pre-operative plasma HMGB1 concentrations. HMGB1 concentrations were divided into two groups, High and Low, by the median value. Differences were analyzed by the Log-rank test.