mesothelioma cancer

Malignant mesothelioma (MM) is a primary tumor of the pleura and
peritoneum. Malignant mesotheliomas that are limited to other organs are
extremely rare, though several cases of pericardial MM have been reported (1). A
unique feature of MM is its strong relationship with asbestos exposure (2,3),
which has recently led to great public concern in view of the ubiquitous
presence of that mineral. Insulation, construction, shipyard industries, and
automobile brakes are among the many sources of occupational exposure (4).
Exposure can be not only occupational but also environmental, or even familial
by household contamination (5).

The mechanisms of MM pathogenesis have not been fully elucidated. Asbestos
fibers could work their way through the lung tissues to damage pleura and
produce adhesions and plaques. Changes observed in target tissues exposed to
asbestos include hyperplasia, metaplasia, DNA synthesis, and increased
production of oxygen free radicals. Acti¬vation of diacylglycerol, protein
kinase C, and ornithine decarboxylase also has been reported in a pathway
similar to classic tumor promot¬ers, such as phorbol esters (6–8). Moreover,
crocidolite fibers, which are the major tumorigenic asbestos fibers, induce
angiogenesis in the peri¬toneal lining of MM animal models (9). Thus, ingrowth
of new blood vessels around clusters of asbestos fibers may also facilitate the
later emergence of MM at these sites.

Angiogenesis plays an important role in the growth, progression, and metastasis
of solid tumors (10). Further, quantitative histologic studies have suggested
that angiogenesis, as assessed by intratumoral microvascular density (IMD) and
total microvascular area (MVA), correlates with poor prognosis in several human
neoplasms (11,12). Malignant mesothelioma demonstrated a higher IMD than colon
and breast tumors (13–15). This value was significantly and independently
related to survival adjusted for other known prognostic variables in MM, such as
histologic type, stage, and age (16). This might call for an IMD profile to be
provided as part of the pathologic evaluation of tumor specimens from patients
with MM.

Currently, many angiogenic factors have been identified to be re¬leased from
tumor-associated inflammatory cells, extracellular matrix,

or tumor cells, which support and stimulate angiogenesis (17). Vascu¬lar
endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),
platelet-derived growth factor (PDGFs), transforming growth factor-b (TGF-b),
hepatocyte growth factor/scatter factor (HGF/SF), insulin-like growth factor-I
(IGF-I), and various cytokines like inter-leukins (IL)-6 and -8 are strong
regulators of angiogenesis with a paracrine loop mechanism (18–20). All these
factors were identified in MM (21–23), which may affect tumor angiogenesis by
positively regu¬lating endothelial cell proliferation, motility, and vascular
permeabil¬ity (17). However, the primary cause of fatality in MM is local
invasion of the primary tumor, unlike other solid tumors where metastasis is
most commonly seen. Malignant mesothelioma cells show a high pro¬liferation rate
as evidenced by a high mitotic count and by methods to demonstrate proliferating
cell nuclear antigens and silver-stained nucleolar organizing regions. In
addition, MM presents with minimal central necrosis, despite its huge size
(24,25). Thus, it was postulated that several growth factors with angiogenic
capacity are also additional effects regarding the process of MM carcinogenesis,
growth depen¬dency patterns, and long latency.

Role of Vascular Endothelial Growth Factor in Malignant Mesothelioma Cell Growth
and Progression

Various autocrine stimulatory effects of angiogenic growth factors such as
IGF-I, PDGFs, and VEGF, via their receptors, have been recently pos¬tulated in
the pathogenesis of MM (26–28). Among these, VEGF seems to play a key role on MM
biology, as VEGF binds with high affinity to the tyrosine kinase receptors
VEGFR-1 (also known as flt-1) and R-2 (also known as Flk-1/KDR) expressed by
endothelial cells (29). The receptors VEGFR-1 and R-2 are also expressed by the
tumor cells of Karposi’s sarcoma, ovarian and breast cancers, and in
choriocarcinoma, melanoma, and ovarian cancer cell lines, suggesting that the
role of the VEGF signaling pathway extends beyond angiogenesis in solid tumors
(30). In addition, VEGF overexpression has been demonstrated in MM tissues and
cell lines (Fig. 9.1) and its protein production was corre¬lated with poor
survival (28). Expression of VEGFR-1 and R-2 by malig¬nant cells within MM
tumors also has been reported (28).

Human recombinant (rh)-VEGF was used to treat several MM cells to demonstrate
that VEGF can stimulate DNA synthesis and cell pro¬liferation. Additionally,
monoclonal antibodies that neutralize VEGF retard the induction of DNA synthesis
by rh-VEGF in MM cells, sup¬porting the contention that VEGF directly stimulates
MM cell growth (28). In this and other studies, VEGF induction of cell
proliferation appears to be mediated by VEGFR-2 (28,31). Also, VEGF induces
higher levels of VEGFR-2 autophosphorylation in MM cells and initi¬ates a range
of cellular responses, including proliferation (32). Al¬though not yet studied,
VEGF binding to VEGFR-1 may mediate other responses in MM cells. In monocytes,
which express only VEGFR-1, VEGF induces cell migration and the production of
tissue factor,

whereas production of matrix metalloproteinases 1, 3, and 9 in smooth muscle
cells also may be mediated via the VEGFR-1 receptor (32). Inter¬estingly,
VEGFR-1 demonstrates higher affinity ligand binding than R-2 and may act
competitively to regulate VEGF-induced mitogenesis (32). Consequently, binding
of VEGF to both VEGFR-1 and R-2 expressed by MM cells may have implications for
a variety of processes involved in tumor progression, including stimulation of
tumor cell proliferation, degradation of extracellular matrix, and tumor cell
migration.

Regulation of Vascular Endothelial Growth Factor Expression by Potential
Transforming Factors in Malignant Mesothelioma

The mechanism for VEGF upregulation in MM tumors is unknown. Previous studies
have shown that p53 represses VEGF transcription by preventing the binding of
Sp-1 to the VEGF promoter (33). More recently the involvement of Src kinase
activity in p53 inhibition of VEGF transcription has been assessed (34). In
addition, p16 and Rb

family members can inhibit VEGF expression (35). Therefore, it is pos¬sible that
cell cycle regulatory proteins generally acting in the G1 phase of the cell
cycle could have similar effects, or that these proteins con¬trol distinct
pathways with a common end point. We have recently observed that VEGF
upregulation is involved in the cell cycle pertur¬bation caused by p53
inactivation in response to simian virus 40 (SV40) infection (36).

Simian virus 40 encodes two transforming proteins, the large-tumor antigen (Tag)
and the small-tumor antigen (tag); SV40-Tag, a 90-kd nuclear phosphopolypeptide,
is essential for virus growth and suffi¬cient to induce mesothelial cell
transformation in the absence of cell lysis (37). Although SV40-Tag displays
pleotropic actions on multiple potential mechanisms of cell transformation (38),
it has been proposed that it may facilitate cell transformation by binding and
inactivating p53 and retinoblastoma (Rb) tumor suppressor proteins (39). The
pres¬ence of nucleic acids and proteins of SV40 has been observed in most MMs
(36). Moreover, SV40 infection represents a negative prognostic cofactor for
patients affected by MM (40). This provocative finding is intriguing and its
significance is as yet unknown, although it was noted that the early polio
vaccines from 1954 until 1960 were contaminated with SV40. More than 50 studies
have confirmed that at least 60% of MMs contain and express SV40. These results
suggest that SV40 may intervene in the pathogenesis of MM. We found a
physiologic rela¬tionship between SV40-related proteins and VEGF expression in
MM cells. Moreover, since VEGF also acts as a potent autocrine growth factor to
MM cells, we antagonized VEGF activity in Tag-expressing MM cells using an
adenoviral vector encoding a soluble form of Flt-1 (Ad.sFlt-1). sFlt-1
expression abrogated both Flk-1/KDR phosphoryla-tion and DNA synthesis induced
by SV40-Tag in MM cells (Fig. 9.2). These data strongly indicate that VEGF
signaling induced by SV40-Tag contributes to cell cycle modulation promoted by
SV40.

In addition, the involvement of 5-lipoxygenase (5-LO) activity in induction of
VEGF transcription has been also observed; 5-LO is suggested to be involved in
the mechanisms of mesothelial cell car-cinogenesis (41). Lipoxygenase isoforms
are expressed in human mesothelial cells, and a metabolically active 5-LO is
selectively upreg-ulated in neoplastic phenotypes of these cells. It also
observed that 5-LO inhibition resulted in MM growth arrest and apoptosis.
Finally, VEGF release and messenger RNA (mRNA) levels are regulated by 5-LO
activity in MM cells, and this regulation is a crucial mechanism of 5-LO actions
on proliferation and apoptosis. Since VEGF simul¬taneously can be induced by
both SV40-Tag and 5-LO function to ac¬complish cellular transformation, its
upregulation could represent common molecular strategies for potential
transforming factors to reg¬ulate proliferation and tumor progression. However,
the roles of these molecules in tumorigenesis need to be studied more closely.

Antiangiogenic Agents as Therapeutic Tools for Malignant Mesothelioma

Antiangiogenic Agents as Therapeutic Tools for Malignant Mesothelioma

Given the role of VEGF in MM, fumagillin, endogenous angiostatic substances,
such as endostatin and angiostatin, and synthetic angio-genesis inhibitors, such
as thalidomide, were used against MM cell growth (42). All these substances
inhibit neovascularization and angio-genesis in organ cultures as well as
tumor-induced neovascularization in vivo. However, the mechanisms responsible
for these effects are different among antiangiogenic drugs. For example,
angiostatin induces cell growth inhibition by inhibiting HGF-induced
phosphorylation of c-met, Akt, and extracellular signal-regulated kinase
(ERK)1/2 (43). Only fumagillin seems to inhibit endothelial proliferation
through MetAP2 inactivation, which is an enzyme involved in the removal of the
N-terminal methionine from proteins and peptides and is an inhibitor of
phosphorylation of initiation factor eIF-2-associated 67-kd protein, p67 (44).

We hypothesized a direct inhibitory effect of these molecules on tumor cell
proliferation. Our results showed that only the angiostatic agent, fumagillin,
at concentrations comparable to those used for endothelial cell inhibition,
arrested the growth of MM cells which expressed high levels of MetAP2. In fact,
normal mesothelial (NM) cells treated with fumagillin that had poor MetAP2
expression did not show any significant alteration of proliferation (Fig. 9.3).
We have also demonstrated that this growth inhibition effect of fumagillin was
asso¬ciated with downregulation of bcl-2 expression and cell death by apo-ptosis.
Interestingly, a decrease of telomerase activity in a time frame required for
fumagillin to induce downregulation of bcl-2 expression was observed. Several
groups have now reported that overexpression of bcl-2 or bcl-XL confers
protection upon mitochondria, making it more difficult for many stimuli to
induce pore opening and release of AIF and cytochrome c, inducing apoptosis
(45). In our MM cells, MetAP2-positive, stable overexpression of bcl-2 inhibited
the reduc¬tion of telomerase activity and reverted the induction of apoptosis by
fumagillin. Our finding indicates a close relationship among MetAP2, survival
factor bcl-2, and telomerase activity in neoplastic cells com¬pared to normal
mesothelial cells. It remains to be resolved whether the observed modulation of
telomerase activity by fumagillin is medi-ated via changes in bcl-2 expression,
or both bcl-2 expression and telomerase activity are regulated via a fumagillin-responsive
common pathway(s). It also remains to be seen in these cells whether the
regu¬lation of telomerase activity is a phenomenon restricted to bcl-2, or is a
general event associated with other antiapoptotic gene products such as bcl-XL.

Since the activation of telomerase activity and bcl-2 deregulation had been
shown to be associated with the development of human cancer (46), our finding of
potential involvement of MetAP2 in the deregula¬tion of telomerase activity
through a bcl-2–dependent mechanism may provide an important insight into the
role of MetAP2 activity during cell growth and also suggest the potentially
clinical use of fumagillin, or its derivatives, in therapy for MM. Recently,
TNP-470, belonging to the fumagillin family, was shown to inhibit growth
factor–induced DNA synthesis of vascular smooth muscle cells and induced apopto-sis
and senescence in human hepatoma cells (50). These data suggest a broad range of
effects of these compounds and involve this parent compound of fumagillin in
apoptotic and senescence pathway(s). In our study, fumagillin did not inhibit
the production of MM cell growth factors, such as FGF-2 and VEGF. In contrast,
it inhibited growth factor-induced DNA synthesis of both MM and NM cells.
Recently, growth

factors, like VEGF, have been shown to increase bcl-2 expression, and it was
shown that FGF-2 inhibited cell apoptosis by bcl-2-dependent and -independent
mechanisms in endothelial cells (47). In our study, fumagillin antagonized VEGF
proliferative effects. Thus, bcl-2 appeared to be one of many potentially
downregulated proteins byfumagillin. In recent years, approaches such as
identification of agent(s) that can modulate bcl-2 have become the subject of
active investigation to control cancer cell growth. In addition, telomerase has
also attracted a great deal of interest as a possible target in cancer biology.
The appar¬ent lower levels of measurable telomerase activity in normal
mesothe-lial cells and its detection in human mesothelioma cells have raised the
possibility that telomerase may also serve an important target to con¬trol the
growth of this tumor.

In spite of wide occurrence of deregulation of bcl-2 and telomerase activity in
cancer cells, to date, to the best of our knowledge, no close linkage between
MetAP2 and these two phenotypes has been reported. Our findings of the
modulation of telomerase activity by inhibition of MetAP2 activity via a widely
deregulated survival factor, bcl-2, and their implication in an apoptotic
pathway, could open new possibili¬ties to develop novel strategies for MM by
co-targeting angiogenic and apoptotic pathways.

Acknowledgments

This work was supported by grants from the Associazione Italiana per la Ricerca
sul Cancro (AIRC) and from the Italian Ministero dell’ Università e della
Ricerca Scientifica (ex 60%) to A. Procopio. We thank the laboratory staff of
the Institute of Experimental Pathology at the University of Ancona. A. Catalano
is recipient of a Fondazione Italiana Ricerca sul Cancro (FIRC) fellowship.

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