Molecular Pathways: Targeting Mechanisms of Asbestos and Erionite Carcinogenesis in Mesothelioma

Pathogenesis of mesothelioma and mechanisms of asbestos carcinogenesis

The observation that only a fraction (∼5%) of workers exposed to high doses of asbestos for prolonged periods of time developed mesothelioma and the identification of clusters of mesothelioma cases within certain families suggest that genetics influences mineral fiber carcinogenesis (1). Studies of an epidemic of mesothelioma in Cappadocia, Turkey and the United States showed that the risk of developing mesothelioma is transmitted with an autosomal-dominant pattern in certain high-risk families (2). Germline mutations of the BAP1 gene have now been linked to a high incidence of malignant mesothelioma in some U.S. families (10). Individuals with heterozygous BAP1 germline mutations are affected by a novel cancer syndrome characterized by a very high risk of developing mesothelioma, uveal melanoma, and possibly additional cancers (10). Mesothelioma may become dominant in these families upon exposure to asbestos or erionite (10). The identification of BAP1 mutant carriers may be facilitated by the detection of melanocytic nevi, as described by Wiesner and colleagues (11) and confirmed by genetic testing. In addition to familial cases, BAP1 somatic mutations have been identified in 25% of sporadic mesotheliomas (10, 12) and 84% of metastasizing uveal melanomas (13), but they appear to be relatively rare in other cancers (14, 15). BAP1 is a deubiquitinating enzyme that has tumor-suppressor activity (14, 15). BAP1 seems to regulate deubiquitination during the DNA damage response and the cell cycle, thus influencing S-phase progression, cell necrosis, and apoptosis (14, 15). Mutations that abolish the deubiquitinating activity of BAP1 and/or its nuclear localization abolish BAP1 tumor-suppressor activity (15). The exact target of BAP1 remains unclear. Ventii and colleagues (15) proposed that expression of BAP1 induces early exit out of G₁, causing an accumulation of DNA damage and cell death. Along these lines, we hypothesize that by influencing DNA damage repair, BAP1 may help prevent environmental carcinogenesis caused by asbestos/erionite or UV light. This would explain the very high incidence of mesothelioma, uveal melanoma, and melanocytic tumors (rather than other not environmentally related cancer types) among BAP1 germline mutant carriers.

DNA damage may be caused directly by mechanical interference of asbestos fibers with chromosome segregation during mitosis (16) or, more likely, indirectly by asbestos-related induction of mesothelial cells and macrophages to generate mutagenic reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS), both of which are mutagenic in vitro (17, 18). The generation of oxidants by macrophages as they attempt to digest asbestos fibers may trigger the activation of several signaling pathways. Two such pathways, mitogen-activated protein kinase (MAPK) signaling and the resulting activator protein 1 (AP-1) transcriptional activity, have been linked to mesothelial cell transformation (19). Other minerals, such as crystalline silica, also elicit ROS and iNOS production from lung macrophages but do not cause mesothelioma (20). Thus, the capacity of asbestos to induce ROS and iNOS is only one of multiple factors that contribute to asbestos carcinogenesis.

In addition to triggering MAPK signaling, asbestos may directly initiate AP-1 activity through EGFR and downstream pathways (19). Similar to EGFR signaling, activation of hepatocyte growth/scatter factor (HGF) and its receptor tyrosine kinase (RTK) c-Met leads to cell proliferation and motility. Both HGF and c-Met are highly expressed in most mesotheliomas, especially those that are SV40-positive (21). HGF activation is mediated through the phosphatidylinositol-3-kinase (PI3K)/MEK5/Fos-related antigen 1 (Fra-1) feedback pathway (19). PI3K, AKT, and the downstream mTOR are involved in cell growth and survival, and they are often found to be activated in mesothelioma (22).

P16^INK4a and p14^ARF are frequently inactivated in mesothelioma (23–25), and ∼50% of mesotheliomas contain missense or nonsense mutations in the neurofibromatosis type 2 (NF2) gene (26, 27). It was shown that mice that were homozygous null for the ARF tumor suppressor rapidly developed mesothelioma upon exposure to asbestos and that mice that were heterozygous for ARF were also susceptible to mesothelioma and consistently exhibited biallelic inactivation of ARF (28, 29).

Chronic inflammation and mesothelioma

Inflammation is the hallmark of asbestos deposition in tissue and contributes to asbestos carcinogenesis (1, 30, 31). The inflammatory infiltrate into tissue areas containing asbestos deposits consists largely of phagocytic macrophages that internalize asbestos and release numerous cytokines and mutagenic ROS (32). Two such cytokines, TNF-α and interleukin (IL)-1β, have been convincingly linked to asbestos-related carcinogenesis (31, 33). Both IL-1β and TNF-α were shown to enhance erionite fiber-induced transformation of the immortalized nontumorigenic human mesothelial cell line MeT-5A (33). Caspase-1 activation promotes the secretion of IL-1β and requires the assembly of high-molecular-weight complexes known as inflammasomes (34). The Nalp3 inflammasome, the best characterized of these complexes, is activated by a wide variety of stimuli, including exogenous pathogen-associated molecular patterns (PAMP) and endogenous damage-associated molecular patterns [DAMP (34, 35)]. Asbestos induces cell necrosis, causing the release of high-mobility group protein B1 [HMGB1 (36)], a typical DAMP and a key mediator of inflammation (37). This, in turn, can trigger activation of Nalp3 inflammasome and subsequent IL-1β secretion, a process that is enhanced by asbestos-induced production of ROS (38, 39). In one study, Nalp3-deficient mice exhibited decreased pulmonary pathology in response to silica and asbestos exposure compared with wild-type controls (38).

In addition, upon asbestos exposure, HMGB1 is released by reactive macrophages and other inflammatory cells, as well as by human mesothelial cells (HMC; 36). HMGB1 is a critical regulator in the initiation of asbestos-mediated inflammation leading to the release of TNF-α and subsequent NF-κB signaling (36). TNF-α is a central mediator in asbestos-, silica- and bleomycin-induced models of pulmonary fibrogenesis (40). In one study, asbestos-exposed TNF-α receptor knockout mice did not develop the fibroproliferative lesions found in asbestos-exposed wild-type mice (41). TNF-α–induced NF-κB signaling was shown to be the critical link between inflammation and carcinogenesis in multiple cancer models, including mesothelioma (31). Asbestos-mediated TNF-α signaling induces the activation of NF-κB–dependent mechanisms, thus promoting the survival of HMCs after asbestos exposure (31). This allows HMCs with accumulated asbestos-induced genetic damage to survive, divide, and propagate genetic aberrations in premalignant cells that can give rise to a malignant clone.

In healthy cells, HMGB1 is found primarily in the nucleus, where it stabilizes chromatin and plays multiple roles in DNA transcription, replication, and recombination. During programmed necrosis, HMGB1 translocates from the nucleus to the cytosol and the extracellular space, where it binds several proinflammatory molecules and triggers the inflammatory responses that distinguish this type of cell death from apoptosis. These findings provide mechanistic links between asbestos-induced cell death, chronic inflammation, and mesothelioma. Secreted HMGB1 stimulates RAGE, TLR2, and TLR4 (the 3 main HMGB1 receptors) expressed on neighboring inflammatory cells such as macrophages and induces the release of several inflammatory cytokines, including TNF-α and IL-1β. In addition, HMGB1 enhances the activity of NF-κB, which promotes tumor formation, progression, and metastasis (42). An investigation into the targeting of extracellular HMGB1 as a novel strategy for mesothelioma prevention and/or therapy is currently underway (43). As illustrated in Fig. 1, we hypothesize that HMGB1 functions as a master switch that initiates a series of inflammatory responses leading to malignant transformation of asbestos- or erionite-damaged HMCs.

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Figure 1.

Working hypothesis for mesothelioma carcinogenesis. Asbestos causes necrotic HMC death, leading to the release of HMGB1 into the extracellular space. As a typical DAMP and a key mediator of inflammation, HMGB can induce activation of Nalp3 inflammasome and subsequent IL-1β secretion, as well as eliciting macrophage accumulation and triggering the inflammatory response and TNF-α secretion, which increases the survival of asbestos-damaged HMCs. This allows key genetic alterations to accumulate within HMCs that sustain asbestos-induced DNA damage, leading to the initiation of mesothelioma.

Molecular therapies

Although increased expression of EGFR has been noted in human mesothelioma, a phase II clinical trial of the EGFR signaling inhibitor gefitinib yielded disappointing results (44). Because RTKs are frequently activated in mesothelioma, investigators tested the possible benefits of small-molecule RTK inhibitors, including erlotinib and imatinib (Fig. 2). However, the results of such studies to date have not been promising (45, 46). The accumulation of cytoplasmic β-catenin, a downstream component of the Wnt signaling pathway, in the majority of human mesotheliomas indicates that Wnt signaling is abnormally activated (47). Moreover, the dishevelled proteins (also downstream of Wnt) are often overexpressed in mesothelioma, and siRNA knockdown of dishevelled proteins was shown to suppress mesothelioma growth (48). These data suggest that agents that target components of the Wnt signaling pathway could benefit mesothelioma patients. The inverse correlation between VEGF serum levels and mesothelioma patient survival (49) suggests that VEGF signaling contributes to mesothelioma. However, a phase II clinical trial of the humanized anti-VEGF monoclonal antibody bevacizumab plus erlotinib (Fig. 2) in mesothelioma patients yielded no clinical benefits (50). PI3K, AKT, and the downstream mTOR are often found to be activated in mesothelioma, and inhibition of mTOR using rapamycin enhances the apoptosis of mesothelioma cells in vitro (22), which suggests that mTOR may serve as a target for mesothelioma therapies (Fig. 2). The NF-κB signaling pathway is critical for the pathogenesis of mesothelioma. Therapies aimed at inhibiting NF-κB activity, such as ranpirnase and bortezomib, may benefit a small subset (10%) of patients (51–53).

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Figure 2.

Potential targets and strategies for mesothelioma therapy that have been proposed based on recent studies. PDGFR, platelet-derived growth factor receptor.

Molecular therapies that target aspects of tumor immunity may also have a significant impact on the course of mesothelioma because the altered microenvironment will affect the ability of the immune system to mount antitumor responses. Sterman and colleagues (54) led several clinical trials examining the effects of intrapleural delivery of type I IFN-encoded adenoviruses. These trials showed that high local concentrations of IFN-α or IFN-β were well tolerated and induced strong cellular and humoral antitumor immune responses, leading to tumor cell death. Some patients with mesothelioma experienced prolonged survival.

In summary, molecular therapies have not affected the average survival of mesothelioma patients, although in several trials 5% to 10% of the patients responded and experienced prolonged survival. Investigators in clinical trials normally look at averages, and therefore no significant benefit can be detected for any therapy when the benefit occurs in only a small fraction of patients. Thus, the challenge ahead of us is to identify the subset of patients who will respond to a given type of therapy.

Targeting asbestos-induced inflammation to prevent or treat mesothelioma

Chronic inflammation has been associated with an increased risk of developing numerous types of cancer. In this regard, daily treatment with aspirin for ≥5 years was shown to reduce tumor burden in several common malignancies (55), and results from animal experiments support a beneficial role for anti-inflammatory therapies in mesothelioma (56). Thus, we hypothesize that prolonged aspirin treatment may help reduce the incidence of mesothelioma and other asbestos-related malignancies among high-risk cohorts with either a lengthy history of exposure and/or genetic predisposition.

On the basis of recent findings, it is tempting to speculate that HMGB1 and the Nalp3 inflammasome act as critical initiators of chronic inflammation in asbestos- and erionite-exposed individuals, with the secretion of IL-1β and TNF-α acting as the key downstream driving force. Therefore, HMGB1, Nalp3, TNF-α, and IL-1β can all serve as potential targets for inhibitors of asbestos-induced inflammation leading to mesothelioma. Indeed, Hamada and colleagues (57) showed that bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis exhibited elevated levels of HMGB1 and that treatment with an anti-HMGB1 antibody prevented bleomycin-induced lung fibrosis in mice. In addition to mesothelioma, several solid tumors, including melanoma, prostate, pancreatic, breast, and gastrointestinal cancers (42), display elevated levels of HMGB1; therefore, therapies that seek to block HMGB1 signaling would likely prove effective in other cancer types as well as in mesothelioma.

Treatment with an IL-1 receptor antagonist can protect mice from developing fibrosis upon exposure to bleomycin or silica (58). In a murine model of bleomycin- or silica-induced pulmonary fibrosis, infusion with the human recombinant soluble TNF receptor rsTNFR-β was effective not only in preventing the development of pulmonary fibrosis but also in the treatment of established fibrosis (40). Similar results were observed with the use of anti-TNF-α antibodies (59). Specific U.S. Food and Drug Administration–approved reagents that inhibit these molecules are available. Anakinra, an IL-1 receptor antagonist, is used in therapies for patients with autoimmune diseases and gout. Infliximab, a chimeric human–mouse anti-TNF-α, and etanercept, a soluble TNF receptor fusion protein, have both been used to treat patients with rheumatoid arthritis and other diseases, including plaque psoriasis and ankylosing spondylitis. Glyburide, the most widely used sulfonylurea drug for type 2 diabetes in the United States, inhibits the Nalp3 inflammasome (60). Specific molecules that target the activity of HMGB1 are anti-HMGB1 and anti-RAGE antibodies, recombinant HMG Box A, and ethyl pyruvate, an inhibitor of HMGB1 secretion (ref. 42; Fig. 2).

Early detection of mesothelioma is associated with improved clinical outcomes (1). The finding of significantly higher serum levels of HMGB1 in asbestos-exposed individuals compared with cohorts of smokers with histologically proved bronchial inflammation and dysplasia (36) suggests that HMGB1 may be a potential marker of exposure to carcinogenic mineral fibers. Moreover, soluble mesothelin-related peptide (SMRP) and osteopontin were proposed to be candidate markers for the early detection of mesothelioma (i.e., before the appearance of clinical symptoms).