Aspirin delays mesothelioma growth by inhibiting HMGB1-mediated tumor progression

Abstract

High-mobility group box 1 (HMGB1) is an inflammatory molecule that has a critical role in the initiation and progression of malignant mesothelioma (MM). Aspirin (acetylsalicylic acid, ASA) is the most widely used nonsteroidal anti-inflammatory drug that reduces the incidence, metastatic potential and mortality of many inflammation-induced cancers. We hypothesized that ASA may exert anticancer properties in MM by abrogating the carcinogenic effects of HMGB1. Using HMGB1-secreting and -non-secreting human MM cell lines, we determined whether aspirin inhibited the hallmarks of HMGB1-induced MM cell growth in vitro and in vivo. Our data demonstrated that ASA and its metabolite, salicylic acid (SA), inhibit motility, migration, invasion and anchorage-independent colony formation of MM cells via a novel HMGB1-mediated mechanism. ASA/SA, at serum concentrations comparable to those achieved in humans taking therapeutic doses of aspirin, and BoxA, a specific inhibitor of HMGB1, markedly reduced MM growth in xenograft mice and significantly improved survival of treated animals. The effects of ASA and BoxA were cyclooxygenase-2 independent and were not additive, consistent with both acting via inhibition of HMGB1 activity. Our findings provide a rationale for the well documented, yet poorly understood antitumorigenic activity of aspirin, which we show proceeds via HMGB1 inhibition. Moreover, the use of BoxA appears to allow a more efficient HMGB1 targeting while eluding the known gastrointestinal side effects of ASA. Our findings are directly relevant to MM. Given the emerging importance of HMGB1 and its tumor-promoting functions in many cancer types, and of aspirin in cancer prevention and therapy, our investigation is poised to provide broadly applicable information.

Figure 1

Treatment with ASA significantly reduces MM tumor growth in vivo. Fourteen SCID mice were injected intraperitoneally with 5 × 10⁵ REN/luc cells. After formation of detectable tumor nodules by IVIS imaging (4 days after cells injection), mice were weighed and randomly assigned to control (gavage of vehicle solution) and treatment (gavage of 25 mg/kg per day ASA); groups of seven animals each. (a) Representative IVIS images. (b) Tumor growth curves of the two groups. Based on a slope comparison test, ASA treatment resulted in significant suppression of MM growth compared with controls (t(67)=4.8, P<0.0001). (c) Female SCID mice were randomly assigned to receive ASA- (25 mg/kg) or vehicle-supplemented gavage feeding. Blood was collected by cheek bleeding and serum levels of HMGB1 were measured by ELISA. *P<0.05. (d) SA plasma levels in mice receiving ASA. Fifteen female SCID mice were randomly assigned to receive ASA (25 or 50 mg/kg) or vehicle by gavage feeding in groups of five mice. Blood was collected by cheek bleeding at different time points ranging from 30 min to 24 h after feeding, and plasma levels of SA were measured with a specific ELISA. (e) Representative IVIS images at different days postinjection of REN/luc cells shows reduced tumor growth in ASA-treated mice. Sixty SCID mice were injected intraperitoneally with 5 × 10⁵ REN/luc cells. After formation of tumor nodules detectable by IVIS imaging (4 days after cells injection), mice were weighed and randomly assigned to control (gavage of vehicle) and treatment (gavage of ASA at 25 or 50 mg/kg per day for the first 2 weeks and three times a week thereafter), groups of 20 animals each. (f) Tumor growth curves of the three animal groups up to 76 days. Measurement was halted thereafter as most mice in the control group had died. Based on a slope comparison test, treated animals showed a marked reduction in tumor growth (t(443)=9.9, P<0.0001). (g) Kaplan–Meier survival plot of the three animal groups. Median survival was 76 days in the control group, 87.5 days in the ASA 25 mg/kg group and 91 days in the ASA 50 mg/kg group. The increase in survival of treated animals is statistically significant for both ASA -treated groups (χ²(2)=38.0, P<0.0001, log-rank test)

Figure 2

Salicylates inhibit anchorage-independent colony formation of high HMGB1-secreting REN and HMESO cells but not of low HMGB1-secreting PPM-MILL cells. Five thousand REN (a and b), HMESO (c and d) and PPM-MILL (e and f) cells were seeded on soft agar-coated plates. Fresh medium (DMEM plus 1% FBS) supplemented with different concentrations of salicylates or vehicle control was added every 2 days for 4 weeks. Representative photographs of colonies (a, c and e) and quantification of number of colonies (b, d and f). Cells were treated with vehicle or salicylates. For each well, all colonies larger than 0.1 mm in diameter were counted using the ImageJ software (NIH). In all panels, experiments were carried out in triplicate and repeated three times. *P<0.05

Figure 3

Salicylates inhibit MM cell migration, cell motility and invasion induced by HMGB1. (a) Quantification of wound healing assay of REN cells in the presence of HMGB1 (100 ng/ml) with or without different concentrations of ASA or SA after 48 h. The percentage of wound reduction was analyzed using the ImageJ software (NIH). Experiments were carried out in triplicate. (b) Both ASA and SA inhibit HMGB1-induced MM cell migration. Quantification of REN cells migrating towards HMGB1 in the presence of different concentrations of ASA or SA. Bars represent mean values per field from three fields. (c) Both ASA and SA inhibit HMGB1-induced MM cell invasion. Quantification of Matrigel invasion of REN cells in the presence of HMGB1 and different concentrations of ASA or SA. Bars represent mean values per field from three fields. (d and e) ASA and SA do not inhibit HMGB1-induced MM cell proliferation at 48 h (d), but significantly inhibit HMGB1-induced MM cell proliferation at 72 h (e). One thousand REN cells were treated with HMGB1 (100 ng/ml) in the presence or absence of different concentrations of ASA or SA. Cell metabolic activity was assessed by Alamar Blue assay after 48 and 72 h. Effects of treatments were assessed by analysis of variance (ANOVA) with Bonferroni-corrected posttests. (Here we are comparing MM cells treated with HMGB1 in the presence or absence of ASA/SA.) **P<0.01, ***P<0.001 and ****P<0.0001

Figure 4

Salicylates inhibit N-cadherin and β-catenin expression induced by HMGB1. (a) Representative western blots show N-cadherin and β-catenin expression in REN MM cells treated with HMGB1 in the presence or absence of different concentrations of ASA or SA. (b and c) The expression of N-cadherin (b) and β-catenin (c) relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels was evaluated as band density using the ImageJ software (NIH). ^#Significantly different compared with MM cells not treated with HMGB1. *Significantly different compared with MM cells treated with HMGB1 in the absence of salicylates (P<0.05)

Figure 5

Effect of salicylates on HMGB1-induced cell migration is COX-2 independent and is not additive with BoxA. (a and b) SA suppresses HMGB1's chemoattractant activity on mouse 3T3 fibroblasts (a) and MEFs knock out for COX-2 (Ptgs2−/−) (b). Cell migration was performed in Boyden chambers 3 h after stimulation with 1 nM reduced HMGB1, 0.1 μM fMLP or buffer (phosphate-buffered saline (PBS)). Bars represent the mean of triplicate samples (P<0.001 in analysis of variance (ANOVA)). (c) Quantification of wound healing assay of PHI cells in the presence of HMGB1 and ASA or BoxA alone or in combination after 48 h. The percentage of wound reduction was analyzed using the ImageJ software (NIH). Experiments were carried out in triplicate. **P<0.002, HMGB1-treated cells compared with vehicle. ^#P=0.01 and ^##P<0.005, MM cells treated with HMGB1 in the presence of BoxA, ASA or BoxA+ASA compared with cells treated with HMGB1 alone

Figure 6

BoxA significantly reduces tumor growth in an MM xenograft model. (a) Tumor growth curves of BoxA-treated mice and controls. Based on a slope comparison test, tumors in BoxA-treated mice grew significantly slower than controls (t(146)=4.8, P<0.0001). (b) Survival curve of the two groups. BoxA-treated mice had a median survival of 142 days, compared with 76.5 days median survival in the control group in which the longest survival was 96 days. Based on the log-rank test, this difference was statistically significant (χ²(1)=18.4, P<0.0001)