Inflammation as a chemoprevention target in asbestos-induced malignant mesothelioma

Abstract

Malignant mesothelioma (MM) is an incurable cancer of the serosal lining that is often caused by exposure to asbestos. Therefore, novel agents for the prevention and treatment of this disease are urgently needed. Asbestos induces the release of pro-inflammatory cytokines such as IL-1β and IL-6, which play a role in MM development. IL-6 is a component of the JAK-STAT3 pathway that contributes to inflammation-associated tumorigenesis. Glycoprotein 130 (gp130), the signal transducer of this signaling axis, is an attractive drug target because of its role in promoting neoplasia via the activation of downstream STAT3 signaling. The anticancer drug, SC144, inhibits the interaction of gp130 with the IL-6 receptor (IL6R), effectively blunting signaling from this inflammatory axis. To test whether the inflammation-related release of IL-6 plays a role in the formation of MM, we evaluated the ability of SC144 to inhibit asbestos-induced carcinogenesis in a mouse model. The ability of sulindac and anakinra, an IL6R antagonist/positive control, to inhibit MM formation in this model was tested in parallel. Asbestos-exposed Nf2+/-;Cdkn2a+/- mice treated with SC144, sulindac or anakinra showed significantly prolonged survival compared to asbestos-exposed vehicle-treated mice. STAT3 activity was markedly decreased in MM specimens from SC144-treated mice. Furthermore, SC144 inhibited STAT3 activation by IL-6 in cultured normal mesothelial cells, and in vitro treatment of MM cells with SC144 markedly decreased the expression of STAT3 target genes. The emerging availability of newer, more potent SC144 analogs showing improved pharmacokinetic properties holds promise for future trials, benefitting individuals at high risk of this disease.

Graphical Abstract

Figure 1.

Kaplan–Meier survival curves of asbestos-exposed Nf2^+/−;Cdkn2a^+/− mice treated with anti-inflammatory agents. (A) Survival curves of mice treated with sulindac, anakinra or vehicle (citrate buffer). (B) Survival curves of mice treated with vehicle control (Labrafil:citrate buffer, L:C), 75 mg/kg SC144, or 150 mg/kg SC144. The outcome was time to death due to MM. Animals that were MM-free when they expired were censored at the time of death. Statistical significance between experimental groups was assessed using the log-rank test.

Figure 2.

Histopathological and immunohistochemical analyses of representative MMs from asbestos-injected Nf2^+/−;Cdkn2a^+/− mice. (A) Histopathological assessment of representative MM from asbestos-injected Nf2^+/−;Cdkn2a^+/− mouse. Left panel, H&E staining; middle, mesothelin staining and right, WT1 staining. (B) Left and middle panels, Ki67 staining of MMs from vehicle-treated and SC144-treated Nf2^+/−;Cdkn2a^+/− mice, respectively. Right panel, bar graph depicting mean Ki67 proliferation index in MMs from the two mouse treatment groups (n = 5). Error bars indicate the standard error of the means (SEM). (C) Immunohistochemical staining with phospho (P)-Stat3 antibody in representative MMs from vehicle-treated (left panel) and SC144-treated (right) mice. The original magnification of all histopathological images was ×200.

Figure 3.

Immunoblot analyses demonstrating effects of SC144 on IL-6/JAK-STAT3 signaling pathway. (A) Immunoblotting reveals robust expression of P-Stat3 in MMs from vehicle-treated control mice but diminished expression in MMs from SC144-treated mice, with expression of total Stat3 (T-Stat3) markedly reduced in tumors from mice in the 150 mg/kg SC144-treatment group. (B) SC144 inhibits activation of Stat3 by IL-6 in non-malignant mesothelial cells (NMC) from Nf2^+/−;Cdkn2a^+/− mice. Early passage (p. 4) Nf2^+/−;Cdkn2a^+/− NMC 701 and 704 were cultured overnight in medium containing 1% FBS containing 40 µM SC144. The following morning, the medium was replenished with fresh SC144 for another 4 h, and then IL-6 at 100 ng/ml was added for the final 15 min before harvesting cells to prepare lysates for immunoblot analysis. (C) Immunoblotting showing reduced Nf2/Merlin and p16Ink4a expression in NMC 701 and 704 (both haploinsufficient for Nf2 and Cdkn2a) as compared to that of wild-type NMC (NMC wt).

Figure 4.

Effect of SC144 on STAT3 target genes. (A) Immunoblot analysis documenting markedly diminished expression of T-STAT and P-Stat3 with increasing dose of SC144, with a consequent abrogated expression of two Stat3 target gene products, cyclin D1 and survivin. (B) Cartoon depiction of the effect of SC144 on the IL-6/JAK-STAT3 signaling pathway. SC144 interferes with the IL-6-gp130-IL-6 receptor (IL-6R) interaction by binding to gp130. As a result (red arrow), SC144 induces phosphorylation (P) on Ser782 (S) of gp130 and deglycosylation (release of green oval symbols) of this transmembrane protein, as well as conformational changes and decreased activity of gp130 (8). As a result of treatment with SC144, phosphorylation and nuclear translocation of Stat3 are blocked, thereby leading to diminished expression of STAT3 target gene products, such as those involved in cell proliferation and survival (e.g. cyclin D1 and survivin). Panel (B) was created in part with Biorender.com software.

Figure 5.

Effects of SC144 on human mesothelial cells and MM cell lines. (A) Immunoblot analyses demonstrating effects of SC144 on IL-6/JAK-STAT3 signaling pathway. Left panel, SC144 inhibits IL-6-induced activation of STAT3 and expression of two protein products (cyclin D1 and survivin) of STAT3 target genes in LP9 mesothelial cells. The LP9 cells were cultured overnight in medium containing 1% FBS containing 10 µM SC144. The following day, the medium was replenished with fresh SC144 for another 4 h, and then IL-6 (100 ng/ml) was added for the final 15 min before harvesting cells to prepare lysates for immunoblotting. Right two panels, immunoblot analysis depicting the effect of SC144 on P-STAT3, T-STAT3 and cyclin D1 in human MM cell lines H-MESO-1 and MM25 cultured in the presence of increasing concentrations of SC144 for 48 h. Vinculin was used as a loading control. (B) SC144 inhibits cell viability of human MM cell lines in a dose-dependent manner. Cell viability at increasing concentrations of SC144 was assessed by MTS assay 96 h after beginning treatment. (C) Colony formation in human MM cell lines treated with increasing concentrations of SC144. Cells were treated with five different concentrations of SC144 for 7 days. Each experiment was performed in triplicate, and representative stained plates for each cell line are shown.