1 2 Asbestos-Induced Mesothelioma Maria E. Ramos-Nino, Marcella Martinelli, Luca Scapoli, and Brooke T. Mossman Asbestos, a group of chemically and physically distinct fibers, is one of the most notorious carcinogens in the lung and pleura. The National Institutes of Health in 1978 estimated that approximately 11 million individuals had been exposed to asbestos in the United States since 1940 (1). Although widely employed in World Wars I and II, the use of asbestos has undergone major changes in recent decades, with severe restrictions in most countries on amphiboles. In developed countries, with the exception of Japan, asbestos production is controlled or banned, while in developing countries, consumption has leveled off or increased (2). Between the 1940s and 1970s, asbestos was utilized extensively in insulation applications (primarily in the building construction industry), and in asbestos-cement pipes. Current usage is generally confined to chrysotile in four products: asbestos cement, friction materials, roof coating and cements, and gaskets. In 1992 approximately 28 million tons of asbestos-cement products were produced in approximately 100 countries (3). Properties of Asbestos Fibers Asbestos is a naturally occurring group of fibers, each with its own unique structure and chemical composition (Table 2.1). There are two subgroups: (1) the serpentine group, consisting of chrysotile; and (2) the amphiboles, a group of rod-like fibers including crocidolite, amosite, tremolite, anthophyllite, and actinolite (4). Asbestos fibers are ubiquitous in certain geographic areas and become problematic to human health when they are inhaled. It is unclear how they get to the pleura to cause mesothelioma. Epidemiology of Asbestos-Induced Mesotheliomas The most important causal factor for the development of human mesothelioma is exposure to asbestos, primarily the amphiboles crocidolite and amosite. Malignant mesothelioma is presently a worldwide 21
2 22 Chapter 2 Asbestos-Induced Mesothelioma Table 2.1. Types, composition and characteristics of asbestos fibers Type Composition Source Morphology Chrysotile* Mg 6 Si 4 O 10 (OH) 8 Northern hemisphere (U.S. and Curly, pliable Canada) Crocidolite Na 2 (Fe 3+ ) 2 (Fe 2+ ) 3 Si 8 O 22 (OH) 2 South Africa, Western Australia Rodlike, durable Amosite (Fe, Mg) 7 Si 8 O 22 (OH) 2 South Africa Rodlike, durable Anthophyllite (Mg, Fe) 7 Si 8 O 22 (OH) 2 Finland Rodlike, durable Tremolite Ca 2 Mg 5 Si 8 O 22 (OH) 2 Exists in some deposits of Rodlike, durable Canadian chrysotile Actinolite Ca 2 (Mg, Fe) 5 Si 8 O 22 (OH) 2 Not mined * Only member of the serpentine family. Other types of asbestos are classified as amphiboles. problem (5). Although mesothelioma is a rare disease, with an annual incidence in the United States of 2000 to 3000 cases, a steady rise in cases has been reported (6). In Europe, the incidence of malignant pleural mesothelioma has risen for decades and is expected to peak between the years 2010 and 2020 (7). In Germany, a study conducted on 1605 patients in the mesothelioma register ( ), found that 70% had a history of exposure to asbestos (8). In the United Kingdom, asbestos reportedly accounts for some 600 cases of mesothelioma and 100 cases of bronchial carcinoma per year (9). The incidence of mesothelioma has been rapidly increasing and is expected to increase even more from the present total of 1300 to more than 3000 cases per year. Exposure to fibers is associated with most of these cases (10). The link between amphibole asbestos exposure and pleural mesothelioma is the result of the pioneering work of Wagner and colleagues (11), who found a relationship between the high incidence of the disease and people working at or living near crocidolite (blue) asbestos mines, with intermediate levels of disease near amosite mines, and no tumors in chrysotile miners. Lung burden studies (see Chapter 1) have also confirmed that the amphibole subgroup of asbestos (crocidolite, amosite) is the one more strongly associated with the development of both malignant mesothelioma and lung cancers (12). In a recent study on 1445 cases of mesothelioma in the United States, it was determined that commercial amphiboles were responsible for most of the mesothelioma cases observed (13). Chrysotile asbestos may produce mesothelioma in humans, but the number of cases is small and the required exposures large (12). Heavy exposures to chrysotile asbestos alone, or with negligible amphibole contamination, can cause malignant mesothelioma and other lung cancers in humans (14), but studies evaluating worker populations that are transient and may be exposed to different types of fibers over a lifetime are difficult to interpret. Some studies have implicated tremolite fibers as the likely etiologic factor in mesotheliomas associated with chrysotile exposure (15 17). However, others suggest that chrysotile does cause mesothelioma, although it may be far less potent than amphibole asbestos (18). Although the association between amphibole asbestos exposure and the development of malignant mesothelioma is well documented (19), available information suggests that other factors contribute to its etiol-
3 ogy. Some studies suggest that genetic factors may play an important role in the etiology of the disease (20,21). Also, compelling multiinstitutional studies suggest that SV40 tumor (T)-antigen (Tag) is present in a large percentage of human mesotheliomas. Approximately 60% of mesotheliomas in the United States are positive for SV40 Tag (22,23), and possible mechanisms are discussed in other chapters of this volume (see Chapter 3). Properties of Asbestos Associated with Carcinogenic Potential The carcinogenic potential of asbestos fibers has been linked to their geometry, size, and chemical composition. Because of the increased potential of long (>5 mm) fibers to cause mesothelioma and fibrosis after intrapleural or intraperitoneal administration to rodents (24), health concerns for long respirable fibers [World Health Organization (WHO) criteria: length >5 mm, diameter <3 mm] are considerable (25). In addition to size, the chemical composition of fibers plays an important role in determining the durability, biopersistence, and biodegradability of asbestos types. The greater durability of amphiboles compared to chrysotile appears to be one of the principal reasons for their greater carcinogenic potential. Amphibole fibers persist at sites of tumor development and may serve as stimuli for neoplastic growth of cells (26,27). Studies on the retention of asbestos fibers in lung tissues of asbestos workers show that concentrations of amphibole fibers increase with durations of exposure, whereas chrysotile concentration does not (28). Studies also indicate that the lung fiber content of amphiboles is less than that required for chrysotile in the induction of mesothelioma (29). The persistence of the amphibole fibers at the site of tumor formation is important to both tumor induction and promotion because the mean latency period between initial exposure to asbestos and the development of mesothelioma is around 30 to 40 years (19,30). Role of Reactive Oxygen and Nitrogen Species (ROS/RNS) in Asbestos Bioreactivity An important unresolved issue is whether asbestos fiber carcinogenicity is through direct effects of asbestos on mesothelial cells or through indirect mechanisms involving oxidative stress (31,32). A ramification of interaction of long (>5 mm) fibers with cells is frustrated phagocytosis and a prolonged oxidative burst (Fig. 2.1) (33). The increased durability and high iron content of the amphiboles crocidolite and amosite also may contribute to their higher carcinogenic potential through oxidants catalyzed by iron or surface reactions occurring on the fiber. Iron-rich durable fibers such as crocidolite, which contain as much as 36% iron by weight, also may have increased reactivity because of the oxidation state of iron, i.e., increases in ferrous iron, aiding in its chelation (34). The cytotoxicity of crocidolite fibers in M.E. Ramos-Nino et al 23
4 24 Chapter 2 Asbestos-Induced Mesothelioma Figure 2.1. Scanning electron microscopy showing phagocytosis of long asbestos fibers by alveolar macrophages. human lung carcinoma cells is directly linked to iron mobilization and is followed by increased ferritin synthesis, a perpetual feedback system for uptake of iron by cells (35,36). Studies on animal models and cell cultures have confirmed that asbestos fibers generate ROS and RNS (19,32,37), and these effects may be potentiated by the inflammation associated with fiber exposures (38). Asbestos also activates redox-sensitive transcription factors such as nuclear factor kappa B (NF-kB) (39) and activator protein-1 (AP-1) (40), which lead to increased cell survival, inflammation, and, paradoxically, the upregulation of antioxidant enzymes such as manganese superoxide dismutase (38). This enzyme is also overexpressed in asbestos-related mesotheliomas (41,42), rendering them highly resistant to oxidative stress in comparison to normal mesothelial cells. Moreover, its overexpression prevents cell injury by asbestos (43). In human pleural mesothelial cells in vitro, crocidolite asbestos causes oxidative stress and DNA single-strand breaks (44), but these are not exacerbated by pretreatment with inhibitors of antioxidant enzymes. Other studies have demonstrated overexpression of enzymes related to oxidative stress, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (NOS-2) (45,46), and endothelial nitric oxide synthase
5 (enos) in malignant mesotheliomas (47). Thioredoxin, a small redoxactive protein reduced by the selenoprotein thioredoxin reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH), is associated in other models of cancer with cell growth and differentiation and is also overexpressed in mesothelioma cells. This protein might be a factor governing the poor prognosis of mesotheliomas and their reduced responsiveness to conventional therapies (48). Overexpression of gamma-glutamylcysteine synthetase, a rate-limiting enzyme in glutathione-associated pathways, could also play an important role in the primary drug resistance of mesotheliomas (49). Catalytically active 5-lipooxygenase could also be involved in the regulation of proliferation and survival in mesotheliomas via a vascular endothelial growth factor (VEGF)-related circuit (50). M.E. Ramos-Nino et al 25 Cytogenetic Changes by Asbestos Fibers in Mesothelial Cells and Mesotheliomas Chromosomal changes and cytogenetic responses to asbestos have been observed in rodent and human mesothelial cells in culture (51 53). Although human mesothelial cells may be more sensitive to the cytotoxic effects of asbestos than bronchial epithelial cells or fibroblasts (52), it is unclear whether individual sensitivity to asbestos fibers is due to specific genetic traits. For example, the glutathione-s-transferase M1 (GSTM1) genotypes of patients with mesothelioma suggest that the lack of the GSTM1 gene does not render human mesothelial cells more sensitive to chromosomal damage by amosite asbestos fibers. However, GSTM1 null cells are more susceptible than GSTM1- positive cells to growth inhibitory effects of fibers (54). A complex profile of somatic genetic changes has been revealed in human malignant mesotheliomas. These changes implicate a multistep process of tumorigenesis. The occurrence of multiple, recurrent cytogenetic deletions suggests that loss or inactivation of tumor suppressor genes are critical to the development and progression of mesothelioma. Deletions of specific regions in the short (p) arms of chromosomes 1, 3, and 9 and long (q) arms of 6, 13, 15, and 22q are repeatedly observed, and loss of a copy of chromosome 22 is the single most consistent numerical change (55). Relatively little is known about the early changes in the genesis of mesothelioma. Of the known cytogenetic changes, the most frequent is loss of p16/cdkn2a-p14 ARF at 9p21(by homozygous deletion) (56), adversely affecting both Rb and p53 pathways, respectively. NF2 (merlin), a tumor suppressor located at 22q12 (by an inactivating mutation coupled with allelic loss) is also frequently altered in mesotheliomas (57 60). Other conventional proto-oncogenes and tumor suppressor genes have been investigated including N-ras (61), Ha- and Ki-ras (62), and the tumor suppressor gene p53, but no consistently frequent mutations have been found (61 63).
6 26 Chapter 2 Asbestos-Induced Mesothelioma Cell Signaling Pathways, Growth Factors, and Early Response Proto-Oncogenes The studies cited above suggest that cell proliferation by asbestos may play a more critical role in the promotion and progression of mesotheliomas. Carcinogenesis was classically thought to be a proliferationdriven process. However, it is now recognized that neoplastic growth is an imbalance between apoptosis and proliferation. In support of this concept, a dynamic balance between apoptosis and cell proliferation is observed in mesothelial cells exposed to crocidolite asbestos (64). Studies in vitro indicate that asbestos can induce apoptosis in mesothelial cells through formation of ROS (65,66) and mitochondrial pathways (31,67). Malignant mesothelioma (MM) routinely expresses the antiapoptotic protein Bcl-xl and the proapoptotic proteins Bax and Bak. Moreover, antisense oligonucleotides against Bcl-xl engender apoptosis in mesothelioma cell lines (68). Inhibitor of apoptosis protein-1 (IAP-1) promotes mesothelioma cell survival, whereas reduced IAP-1 results in increases in apoptotic pathways and reduced resistance to chemotherapeutic drugs (69). Cell signaling pathways induced by asbestos through receptors on the cell surface trigger early-response proto-oncogenes, activation of transcription factors such as AP-1, and AP-1 dependent gene expression (40,70). Studies in our group have found that the epidermal growth factor receptor (EGFR) is an important target of asbestos. This growth factor is required for proliferation of human mesothelial cells (71), and is produced in an autocrine fashion in mesotheliomas (72). Autophosphorylation of the EGFR occur in mesothelial cells after in vitro exposures to asbestos. Moreover, aggregation and phosphorylation of the EGFR by long fibers initiates cell signaling cascades linked to asbestos-induced injury and mitogenesis (73,74). Increased expression of EGFR in rat pleural mesothelial cells correlates with the carcinogenicity of mineral fibers (75). We have also shown that the EGFR is causally linked to activation of the mitogen-associated protein kinase (MAPK) cascade and increased expression of the proto-oncogenes c-fos and c-jun (73,76). Expression of both Fos and Jun family members (components of the transcription factor AP-1 complex) is required for transition through the G1 phase and entry into the S phase of the cell cycle (70). Moreover, overexpression of c-jun induces cell proliferation and transformation (77). Most recently, extracellular signal-regulated kinase (ERK-1/2) induced activation by asbestos has been linked to the induction of Fra-1, an important component of the AP-1 complex that is causally related to anchorage-independent growth in mesothelioma (41). Complementary DNA (cdna) microarray analyses have shown increased expression of c-myc, egfr, and fra-1 in rat mesotheliomas (78). Other growth factors and their receptors also are important in malignant mesothelioma including transforming growth factor-a (TGF-a),
7 which binds to the EGFR (79). Although normal mesothelial cells, asbestos-transformed mesothelioma cells, and spontaneously transformed mesothelial cells express functional EGFR (55), only cell lines derived from asbestos-induced mesotheliomas express and secrete TGF-a, which binds to the EGF receptor with high affinity. In addition, TGF-a acts as an autocrine growth factor for asbestos-induced mesotheliomas, and their growth is inhibited with use of a neutralizing TGF-a antibody (79). Insulin-like growth factor-ii, which functions as an autocrine growth factor in normal mesothelial and mesothelioma cells (71,80), and its corresponding receptor also are important in proliferation of mesothelioma cells (81). Platelet-derived growth factor (PDGF) (82) may also be an autocrine growth factor for human mesothelioma cells as both PDGF A- and B- chain messenger RNAs (mrnas) are expressed at higher levels in mesothelioma as opposed to normal mesothelial cell lines (83), and PDGF-like mitogenic activity is observed using mesothelioma cell line conditioned medium (84). Transforming growth factor (TGF)-b 1, responsible for regulatory functions in many pathologic processes including pleural fibrosis, increases pleural fluid formation in part by stimulating production of VEGF, a regulator of pleural inflammation and cell proliferation (85); VEGF is important in vascular permeability and pleural effusion formation as well as growth of mesothelioma cells (86,87). Increased levels of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), known growth factors for mesothelial cells, have been detected in pleural lavage fluids of patients (88). Although HGF is produced in general by mesenchymal cells, recent work by Cacciotti and colleagues (87) shows that the HGF receptor Met, a proto-oncogene product whose activation leads to cell growth and altered morphogenesis, is activated in SV40-positive human mesothelioma cells. Also, high expression levels of c-met have been detected in rat mesothelioma cells and are fra-1 dependent (89). M.E. Ramos-Nino et al 27 Effects of Asbestos on Extracellular Matrix Malignant mesotheliomas exhibit elevated amounts of hyaluronan, and hyaluronan synthesis enhances cell proliferation, anchorage independent growth and cell migration in a number of tumor types (90). The hyaluronan receptor gene cd44 is detected in high amounts by oligonucleotide microarray analysis of human and rat mesothelioma cell lines and may play a role in mesothelial cell motility and migration (89). Other extracellular components such as fibrin deposition via increased expression of tissue factor (TF) may play a role in pleural injury or neoplasia (91). In a study on 16 patients in whom matrix metalloproteinases (MMP)-1, -2, -3, -7, and -9 and tissue inhibitors -1 and -2 were evaluated, MMP-1 and -2 were related directly to invasion and spread of pleural malignant mesothelioma (92).
8 28 Chapter 2 Asbestos-Induced Mesothelioma Figure 2.2. Hypothetical schema of cell signaling in mesothelial cells by asbestos. Conclusion The reports cited in this chapter provide much insight into mechanisms of asbestos-induced mesotheliomas and the properties of amphibole asbestos fibers that initiate injury and compensatory mesothelial cell hyperplasia. The chemical composition of these fibers and their durability at sites of tumor development may induce chronic activation of cell signaling pathways and transcription factors linked to expression of a number of genes critical to tumor initiation, promotion, progression, and angiogenesis (Fig. 2.2). Many of these pathways have been reported after infection of human mesothelial cells with SV40 (72). Regardless of their etiology, since human mesotheliomas appear to have a number of autocrine growth factor pathways governing proliferation, a focus on common downstream signaling molecules is merited in prevention and therapy of mesotheliomas. References 1. Manning C, Vallyathan V, Mossman B. Diseases caused by asbestos: mechanisms of injury and disease development. Int Immunopharmacol Cancer Res 2002;2 3: Algranti E: Asbestos: current issues related to cancer and to uses in developing countries. Cad Saude Publica 1998;14(suppl 3): Pigg B. The uses of chrysotile. Ann Occup Hyg 1994;38: Guthrie GT, Mossman BT. Health effects of mineral dusts. In: Ribbe PH, ed. Reviews in Mineralogy. Washington, DC: Mineralogical Society of America, 1993;28:1 584.
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