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
exten¬sively 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 mate¬rials, roof coating
and cements, and gaskets. In 1992 approximately 28 million tons of
asbestos-cement products were produced in approxi¬mately 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 croci-dolite and amosite.
Malignant mesothelioma is presently a worldwide

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
(1987–1999), 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 mesothe-lioma is the
result of the pioneering work of Wagner and colleagues (11), who found a
relationship between the high incidence of the dis¬ease 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 meso-thelioma 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 neg¬ligible
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 etiology. Some studies suggest
that genetic factors may play an important role in the etiology of the disease
(20,21). Also, compelling multiinstitu-tional 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
(>5mm) 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 >5mm,
diameter <3mm] are considerable (25).

In addition to size, the chemical composition of fibers plays an im¬portant role
in determining the durability, biopersistence, and biode-gradability 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 amphi-boles 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 promo¬tion 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 carcinogenic-ity 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 phagocyto¬sis and a prolonged
oxidative burst (Fig. 2.1) (33).

The increased durability and high iron content of the amphiboles cro-cidolite
and amosite also may contribute to their higher carcinogenic potential through
oxidants catalyzed by iron or surface reactions occur¬ring on the fiber.
Iron-rich durable fibers such as crocidolite, which contain as much as 36% iron
by weight, also may have increased reac¬tivity because of the oxidation state of
iron, i.e., increases in ferrous iron, aiding in its chelation (34). The
cytotoxicity of crocidolite fibers in

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,
para¬doxically, 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 resis¬tant 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

(eNOS) in malignant mesotheliomas (47). Thioredoxin, a small
redox-active 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 differen¬tiation 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). Over-expression of gamma-glutamylcysteine
synthetase, a rate-limiting enzyme in glutathione-associated pathways, could
also play an im¬portant role in the primary drug resistance of mesotheliomas
(49). Catalytically active 5-lipooxygenase could also be involved in the
reg¬ulation of proliferation and survival in mesotheliomas via a vascular
endothelial growth factor (VEGF)-related circuit (50).

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 fibro-blasts (52), it is unclear whether
individual sensitivity to asbestos fibers is due to specific genetic traits. For
example, the glutathione-S-trans-ferase 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 cyto-genetic 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-p14ARF 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 muta¬tion coupled with allelic loss) is also
frequently altered in mesothe-liomas (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 fre¬quent
mutations have been found (61–63).

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 mesothe-liomas.
Carcinogenesis was classically thought to be a proliferation-driven 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 mesothe-lial 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 meso-thelioma cell lines (68). Inhibitor of
apoptosis protein-1 (IAP-1) pro¬motes mesothelioma cell survival, whereas
reduced IAP-1 results in increases in apoptotic pathways and reduced resistance
to chemother-apeutic 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 expres¬sion (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 pro¬duced in an autocrine
fashion in mesotheliomas (72). Autophosphory-lation 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,
overexpres-sion 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 malig¬nant
mesothelioma including transforming growth factor-a (TGF-a),

which binds to the EGFR (79). Although normal mesothelial cells,
asbestos-transformed mesothelioma cells, and spontaneously trans¬formed
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 addi¬tion, TGF-a acts as an autocrine growth
factor for asbestos-induced mesotheliomas, and their growth is inhibited with
use of a neutraliz¬ing 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
pro¬liferation 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)-b1, 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-onco-gene product whose activation leads to cell
growth and altered mor¬phogenesis, is activated in SV40-positive human
mesothelioma cells. Also, high expression levels of c-met have been detected in
rat mesothe-lioma cells and are fra-1 dependent (89).

Effects of Asbestos on Extracellular Matrix

Malignant mesotheliomas exhibit elevated amounts of hyaluronan, and hyaluronan
synthesis enhances cell proliferation, anchorage indepen¬dent growth and cell
migration in a number of tumor types (90). The hyaluronan receptor gene cd44 is
detected in high amounts by oligonu-cleotide 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 metallopro-teinases
(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).

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 dura¬bility 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, progres¬sion, 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 pro¬liferation, a focus on common
downstream signaling molecules is merited in prevention and therapy of
mesotheliomas.

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