Cancer cell secretion of the DAMP protein HMGB1 supports progression in malignant mesothelioma

Abstract

Human malignant mesothelioma is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor because of late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival. Malignant mesothelioma cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in malignant mesothelioma patient sera were higher than that found in healthy individuals. The motility, survival, and anchorage-independent growth of HMGB1-secreting malignant mesothelioma cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products, a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of malignant mesothelioma xenografts in severe-combined immunodeficient mice and extended host survival. Taken together, our findings indicate that malignant mesothelioma cells rely on HMGB1, and they offer a preclinical proof-of-principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for malignant mesothelioma treatment.

Figure 1. HMGB1 is highly expressed in MM tissues and sera of MM patients

(A) Strong expression of HMGB1 was detected in the nuclei of 31/31 MM biopsies representing all 3 main histological subtypes of MM: epithelial (a, b), biphasic (c, d) and sarcomatoid (e, f). In 26/31 MM biopsies HMGB1 is detected in both nucleus and cytoplasm. In the single-cell mesothelial layer of normal pleura (g, h), HMGB1 is only detected in nucleus. Rectangles in x100 magnification pictures (top) indicate the area shown in x400 magnification (bottom). Scale bar, 100 μm. (B) HMGB1 levels in sera of 20 mesothelioma patients are significantly higher (P < 0.0001) than in 20 healthy individuals. Bars show mean of HMGB1 levels.

Figure 2. HMGB1 and RAGE expression are both upregulated in MM cells

(A) HMGB1 mRNA levels are higher in MM than in HM. Different primary HM cell cultures have similar low HMGB1 levels (three representatives HM cells are shown). *P < 0.05; MM versus HM. (B) RAGE mRNA levels are higher in MM than in HM. Experiments were performed as in (A). *P < 0.05; MM versus HM. (C) Cell compartmentalization. Total HMGB1 protein levels are higher in MM than in HM, and HMGB1 is localized in both nucleus and cytoplasm of MM but mainly in the nucleus of HM. α-Tubulin and Lamin B, loading controls for the cytoplasmic and nuclear fractions, respectively. HMGB1 relative densitometry units were calculated. *P < 0.05; MM versus HM. (D) Western blotting shows higher RAGE expression in MM than in HM. α-Tubulin, loading control. RAGE relative densitometry units were calculated. *P < 0.05; MM versus HM. All the experiments were performed three times; error bars represent SEM. (E) Immunocytochemistry. HMGB1 is detected in both nucleus and cytoplasm of MM but mainly in the nucleus of HM (three representatives MM and three representatives HM cells are shown). Original magnification, x400. Scale bar, 100 μm. (F) HMGB1 and RAGE transcript levels show significant positive correlation in five different MM cell lines tested (r = 0.93, P = 0.022).

Figure 3. HMGB1 secreted by MM cells is biologically active

(A) MM cells release larger amounts of HMGB1 in the culture media than HM, as measured by ELISA. Culture media for the different cells were collected and concentrated under identical condition. Experiments were done in duplicate and performed twice. *P < 0.05; MM versus HM. (B) Concentrated conditioned media (CCM) from REN cells induce the migration of REN cells themselves. Migration is blocked by BoxA. (C) CCM collected from PPM-Mill cells does not induce a significant chemotactic response in REN cells. All CCM were collected under identical conditions. Migrated cells were counted using ImageJ software and represent mean values per field from at least three fields. Experiments were done in duplicate. *P < 0.05; HMGB1 and CCM from REN/PPM-Mill versus 0% FBS. **P < 0.05; CCM alone versus CCM plus BoxA. In all panels, error bars represent SEM.

Figure 4. MM cells require HMGB1 for survival and migration

(A) Viability, determined by MTS assay. Inhibition of HMGB1 by BoxA or anti-HMGB1 or anti-RAGE antibodies substantially decreases the viability of REN and PPM-Phi cells. *P < 0.05; treated versus untreated. (B) Cytotoxicity, determined by LDH assay. Both anti-HMGB1 and anti-RAGE antibodies induce substantial cytotoxicity in REN and PPM-Phi cells. Experiments were done in quadruplicate and performed twice. *P < 0.05; treated versus untreated. (C and D) Wound healing assay. One hour prior to scratching the monolayer, the cells were treated with either BoxA (100 ng/ml), anti-HMGB1 (1.0 μg/ml), anti-RAGE (1.7 μg/ml), or IgG control (1.7 μg/ml). (D) For quantification of wound closure, the scratched area covered by the cells after 48 hours was measured using ImageJ software and normalized to control. All HMGB1 antagonists reduced wound healing by REN (C and D) and PPM-Phi cells (D). Original magnification, x40. Figure shows representative images of wound healing (REN) from three experiments done in duplicate. *P < 0.05; treated versus untreated. In all panels, error bars represent SEM.

Figure 5. HMGB1 supports the malignant phenotype of MM cells

(A) Matrigel invasion assay. Exogenous recombinant HMGB1 induces REN cells invasion. Anti-RAGE monoclonal antibodies significantly inhibited HMGB1-induced cell invasion. Invading cells were counted using ImageJ software and represent mean values per field from at least three fields. *P < 0.05; HMGB1 and 10% FBS versus 0% FBS. **P < 0.05; HMGB1 alone versus HMGB1 plus anti-RAGE. (B and C) Anchorage-independent growth. REN cells treated with HMGB1 antagonists BoxA (a), anti-HMGB1 (b), and anti-RAGE (c) formed fewer (B) and smaller (C) colonies in soft agar than IgG (d) and untreated (e) controls. Original magnification, x40. Figure shows representative images from two experiments done in duplicate. Colonies larger than 0.1 mm in diameter were counted using ImageJ software. *P < 0.05; treated versus IgG control. In all panels, error bars represent SEM. Scale bar, 0.5 mm.

Figure 6. Anti-HMGB1 monoclonal antibody reduces tumor growth and extends survival in a mouse model of MM

(A) Bioluminescence imaging of mice xenografted i.p. with 5 × 10⁵ REN/luc shows tumor development. One representative mouse from each group is shown. (B) Tumor growth rate is reduced in mice injected with anti-HMGB1 mAb in comparison with controls. Mean ± SEM of each group is shown (n = 7 per group). P < 0.05; two-way ANOVA analysis. (C) Survival curve. Anti-HMGB1 mAb enhances animal survival in comparison with PBS and IgG control groups. Differences across groups (P < 0.05) were assessed by fitting a parametric model to the survival time data. Black triangles on (B) and (C) graphs represent first (day 4) and last (day 38) injections.