Update on the Molecular Biology of Malignant
Amie Y. Lee
Dan J. Raz, MD
Biao He, PhD
David M. Jablons, MD
Department of Surgery, Division of Cardiothoracic
Surgery, University of California, San Francisco,
Malignant mesothelioma (MM) is a highly aggressive tumor with a very poor
prognosis. The disease is largely unresponsive to conventional chemotherapy or
radiotherapy, and most patients die within 10–17 months of the first symptoms.
Novel, more effective therapeutic strategies are needed for this inexorably fatal
disease. Improvement in our understanding of the molecular biology of MM has
identified promising new candidates for targeted treatments. In this review the
key molecular signaling pathways, including vascular endothelial growth factor
(VEGF), epidermal growth factor (EGF), Wnt, and the cell cycle control genes
p53, pRb, and bcl-2 that appear to play an important role in the pathogenesis of
MM are explored. Cancer 2007;109:1454–61. ? 2007 American Cancer Society.
KEYWORDS: mesothelioma, VEGF, EGFR, p16(INK4A), p14(ARF), Wnt, bcl-2 genes.
arises from the surface serosal cells of the pleura and, less fre-
quently, the peritoneum. The incidence of the disease in the major-
ity of Western nations is expected to steadily rise and peak over the
next 2 decades.1There is no known curative modality for MM and
long-term survival is rare even with aggressive multimodal therapy.
A strong link has been established between exposure to asbestos
and increased risk for MM.2Subsequent laboratory research has
confirmed the carcinogenicity of asbestos in mesothelial cell lines
and in animal models. Despite recent restrictions on the use of
asbestos, the incidence of MM is continuing to rise due to the long
latency period for the development of MM after asbestos exposure.
Simian virus 40 (SV40) has also been implicated in the pathogenesis
of MM. SV40 may have synergistic oncogenic effects when com-
bined with asbestos, although the exact role and importance of this
virus in the development of MM is controversial.3One recent study
demonstrated that significantly lower amounts of asbestos were suf-
ficient to cause MM in animals infected with SV40 compared with
uninfected animals, supporting the possible role of SV40 as a co-
carcinogen in this disease.4Using a novel SV40 large T antigen (Tag)
transgenic model of mesothelioma, Robinson et al.5also demon-
strated that asbestos-induced malignant transformation is SV40
dose-dependent, with higher copy numbers of the transgene
increasing the rate of tumor formation.
Treatment of MM with surgery, chemotherapy, or radiation ther-
apy is rarely curative. Clinical trials of single modality treatment
with extrapleural pneumonectomy or pleurectomy, chemotherapy,
or radiation therapy have not shown significant improvement in
survival compared with supportive treatment. Median survival has
ranged from 10–17 months. A multimodal approach, including
alignant mesothelioma (MM) is a highly aggressive tumor that
Supported by a grant from Supported by the
Larry Hall Memorial Trust and National Institutes
of Health Grant (RO1 CA 093708-01A3) (both to
D.M.J.). A.Y.L. is supported by the UCSF Medical
Student Research Committee.
Address for reprints: David M. Jablons, MD, Depart-
ment of Surgery, Cancer Center, 1600 Divisadero
St., Box 1724, San Francisco, CA 94143-1724; Fax:
(415) 353-9530; E-mail: firstname.lastname@example.org. edu
Received August 10, 2006; revision received
December 31, 2006; accepted January 2, 2007.
ª2007 American Cancer Society
Published online 8 March 2007 in Wiley InterScience (www.interscience.wiley.com).
surgery, chemotherapy, and radiation therapy, is
thought to improve survival in selected patient,
although this remains
et al.6reported a 15% 5-year survival with multimo-
dal therapy and a 25% 5-year survival in patients
who underwent complete surgical resection. Recent
trials of new-generation platinum- and pemetrexed-
based regimens have reported encouraging results. In
particular, a Phase III trial of pemetrexed plus cispla-
tin for MM reported a median survival of 12 months
compared with 9 months after treatment with cispla-
Despite these promising results, long-term sur-
vival with currently available treatments is rare.
Novel therapies for MM are needed. Improvement in
our understanding of the molecular biology of MM
has already identified promising new therapies and
pathways that are candidates for targeted therapies.
In this review, we focus on the key molecular signal-
ing pathways, including vascular endothelial growth
factor (VEGF), epidermal growth factor (EGF), Wnt,
and cell cycle control genes p53, pRb, and bcl2,
which appear to play an important role in the patho-
genesis of MM.
Vascular Endothelial Growth Factor
VEGF is a potent inducer of angiogenesis and its crit-
ical role in tumor progression is well established.7
VEGF-targeted therapy is a promising approach
aimed at inhibiting tumor growth, invasion, and me-
tastasis. VEGF up-regulation appears to play an im-
portant role in mesothelial cell transformation. High
levels of VEGF have been observed in the serum of
mesothelioma patients compared with the serum of
normal subjects.8,9Also, elevated pleural effusion
VEGF levels are associated with diminished survival
in patients with MM.10Similarly, Demirag et al.11
reported that VEGF overexpression on immunohisto-
chemistry (IHC) independently predicts short sur-
vival in patients with MM (P ¼.0002).
Recent reports have suggested that SV40 may
play a role in inducing VEGF expression in MM cells.
One study demonstrated that the SV40 large tumor
antigen (Tag) is involved in VEGF promoter acti-
vation and that it potently increases VEGF levels in
several MM cell lines.12Moreover, transfection of
normal human mesothelial cells with full-length
SV40 DNA resulted in a significant increase of VEGF
increased VEGF production by mesothelial cells.13
Evidence also suggests that interleukin-6 (IL-6), a
cytokine that is frequently overexpressed in MM,
might play a role in the induction of VEGF produc-
tion. A recent study by Adachi et al.14demonstrated
that IL-6/IL-6R stimulation increased expression of
VEGF in 80% of MM cell lines and that an anti-IL-6R
antibody inhibited this expression.
In addition to stimulating the neovascularization
essential for tumor growth and metastasis, recent
evidence has demonstrated that VEGF may also act
in a functional autocrine loop capable of directly
stimulating the growth of malignant mesothelioma
cells. Strizzi et al.11reported that MM cell lines ex-
press elevated levels of both VEGF and the VEGF
receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR), com-
pared with normal mesothelial cells. In that study
they found that recombinant VEGF phosphorylated
both Flt-1 and KDR and increased proliferation of all
MM cell lines examined. In contrast, neutralizing
antibodies against VEGF, Flt-1, or KDR significantly
reduced MM cell proliferation. Similar autocrine sig-
naling in MM cells was demonstrated with VEGF-C
and its cognate receptor VEGFR-3.15Simultaneous
inhibition of VEGF and VEGF-C displayed a synergis-
tic effect in reducing the viability of mesothelioma
cells in vitro.
Interestingly, significant arterial vascularization is
rarely exhibited in MM. Whereas at least 2 stu-
dies16,17reported a significant correlation between
VEGF expression by IHC and intratumoral microvas-
cular and microlymphatic density, other studies
failed to find a correlation.10,11Strizzi et al.11pro-
posed that competitive binding of VEGF in favor of
rapidly proliferating MM tumor cells might make
VEGF less available to stimulate the endothelial cells
necessary to promote angiogenesis.
These reports suggest that VEGF may play a key
role in MM tumor progression by regulating both
angiogenesis and tumor cell proliferation, thus rais-
ing interest in VEGF as a therapeutic target in MM.
Bevacizumab (Avastin, Genentech, South San Francisco,
Calif), a monoclonal antibody that targets VEGF, in
combination with cisplatin produces an inhibitory
effect in an in vivo model of human MM. A Phase II
clinical trial of bevacizumab combined with gemcita-
bine-cisplatin chemotherapy in patients with malig-
nant mesothelioma is under way, with encouraging
preliminary results.18A Phase II clinical trial at the
University of Chicago of SU-5416, a selective inhibi-
tor of VEGF-R2, in patients with MM is also under
Epidermal Growth Factor Receptor
The role of epidermal growth factor receptor (EGFR)
signaling in MM has become an active area of re-
search in large part due to the success of EGFR-tar-
geted therapies in lung cancer. EGFR is a member of
the erbB family of tyrosine kinase receptors, which
Molecular Biology of Mesothelioma/Lee et al.1455
also includes erbB-2, -3, and -4. Multiple ligands
bind to and activate EGFR, including amphiregulin,
betacellulin, EGF, and transforming growth factor
(TGF)-a. The latter 2 are believed to be the most im-
portant ligands for EGFR. Ligand binding with EGFR
leads to receptor dimerization and internalization
followed by autophosphorylation of the receptor ty-
rosine kinase domains. This subsequently results in
the activation of signaling pathways that are involved
in cell proliferation, differentiation, and survival.20
Overexpression of EGFR has been recognized to play
an important role in the pathogenesis and progres-
sion of a variety of malignancies, including colon can-
cer, nonsmall-cell lung cancer (NSCLC), and breast
The involvement of EGFR in malignant pleural
mesothelioma was first reported by Dazzi et al.,21
who found expression of the receptor in 68% of fixed
paraffin-embedded MM tissue specimens. A similar
recent study found EGFR immunoreactivity in 55.7%
of MM samples and no immunoreactivity in normal
pleura.22Both studies found that EFGR expression is
not an independent prognostic factor for patient sur-
vival. One recent IHC study of 168 tumor sections
identified EGFR expression in 44% of cases, with
EGFR positivity associated with improved survival
(P ¼.01).23The authors suggest that the favorable
prognosis associated with EGFR positivity may be
partly explained by the greater expression of EGFR in
the epithelioid cell type, which is known to have a
better prognosis than the sarcomatoid cell type.
Thus, the correlation between EGFR expression with
improved survival does not negate EGFR as a poten-
tial therapeutic target in MM.
Asbestos fibers are involved in the activation of
EGFR in mesothelioma. Pache et al.24demonstrated
that asbestos fibers cause aggregation and increased
immunoreactivity of the EGFR protein in human
mesothelial cells in vivo and that asbestos-induced
EGFR autophosphorylation may lead to the induction
of the AP-1 family members, c-fos and c-jun.25,26
Crocidolite and erionite asbestos fibers were found
to induce EGFR expression in rat pleural mesothelial
cells.27Intense patterns of EGFR protein expression
were linked to mesothelial cells phagocytosing longer
asbestos fibers. Previous studies have shown a rela-
tion between asbestos fiber length and carcinogeni-
It is uncertain whether there is a role for EGFR
inhibitors in the treatment of MM. A recent in vitro
study by Cole et al.29demonstrated that inhibition
of EGFR signaling with the small molecule tyrosine
kinase inhibitor (TKI) PD153035 suppresses MM cell
motility and invasion. Similarly, Janne et al.30found
that gefitinib (Iressa, AstraZeneca, Wilmington, Del),
another EGFR-TKI, significantly inhibits EGFR-de-
pendent cell signaling including AKT phosphoryla-
tion and extracellular signal-regulated kinases 1 and
2 in all MM cell lines examined. This resulted in
marked antiproliferative effects of these MM cells
in vitro. In another study, gefitinib increased tumor
responsiveness to radiation therapy in an animal
model of MM.31Despite the promising in vitro activ-
ity of gefitinib in MM cell lines, the recently pub-
lished Cancer and Leukemia Group B (CALGB) Phase
II clinical trial of gefitinib treatment in 43 previously
untreated mesothelioma patients found that gefitinib
was not effective in this disease.32This was in spite
of the fact that 97% of patients displayed EGFR over-
expression. Interestingly, a recent study found that
common EGFR tyrosine kinase domain mutations
conferring sensitivity to gefitinib in lung cancer are
not prevalent in MM.33
p53 and pRB Pathways
Functional inactivation of the p53 and retinoblas-
toma protein (pRB) pathways appears to be a critical
requirement in the development of several human
cancers, including lung cancer. These pathways play
key roles in apoptosis and cell cycle regulation.34The
tumor suppressor protein pRB limits cell prolifera-
tion by inhibiting entry into the S-phase of the cell
cycle. Another key tumor suppressor protein, p53,
accumulates and is activated in response to cellular
stresses such as hypoxia, DNA damage, and onco-
gene activation. Activated p53 initiates transcription
of genes that can lead to cell cycle arrest and apo-
ptosis. Mutations and deletions the p53 and pRb
tumor suppressor genes occur frequently in many
human cancers. Such mutations, however, are extre-
mely rare in MM, and several studies have demon-
strated that pRb35and p5336remain genetically intact
in all MM specimens examined. In fact, 1 recent IHC
study37found overexpression p53 in 81% of patient
In contrast, 1 of the predominant genetic ab-
normalities in human MM is homozygous deletion of
the 9p21 region, occurring at a frequency of greater
than 70% in MM cell lines.38The INK4a/ARF locus
within the 9p21 chromosome band plays a critical
role in the regulation of both the pRb and p53 tumor
suppressor pathways (Fig. 1). This locus encodes 2
distinct proteins, p16INK4a and p14ARF, translated
from alternatively spliced mRNA. p16INK4a exerts its
tumor suppressive effect by inhibiting the cyclin D-
dependent kinases (CDKs), thus preventing CDK-
mediated hyperphosphorylation and inactivation of
pRB and leading to G1-phase cell cycle arrest.
1456 CANCERApril 15, 2007 / Volume 109 / Number 8
p14ARF acts by directly binding to and promoting
the degradation of Mdm2, thus leading to the stabili-
zation of p53.39Therefore, a single mutational event
at the INK4a/ARF locus has the potential to disrupt
both of these 2 key growth control pathways.
Xio et al.40found deletion of this locus, by fluo-
rescence in situ hybridization, in greater than 70% of
MM. Homozygous deletion of the p16INK4a gene
has been reported in 85% of MM cell lines and in
22% of primary tumor specimens examined.41,42It
has been suggested that the greater frequency of p16
deletions in MM cell lines than in tumor samples
may be either because 1) this alteration occurs in the
culturing process and is an in vitro phenomenon or
2) the tumor specimens may be contaminated with a
small population of nonmalignant cells making it dif-
ficult to detect genetic abnormalities.43At the protein
level it has been demonstrated that p16INK4A is
abnormally expressed in all cell lines and all primary
MM specimens examined.35These findings also sug-
gest that epigenetic mechanisms might play a role in
silencing gene expression in cells with an absence of
mutations in the INK4a/ARF locus. Indeed, Wong
et al.44detected methylation of p16INK4a in 10% of
cell lines and 27% of primary tumors. Treatment of
these methylated MM cell lines with an inhibitor of
DNA methylation (50-aza-20deoxycytidine) resulted in
re-expression of the p16INK4a protein. Wong et al.44
hypothesize that methylation of p16INK4a may be a
possible therapeutic target in MM and suggest that
responses to treatment of mesothelioma with the
demethylating agent DHAC in clinical trials may in
part be due to re-expression of p16INK4a. However,
due to the nonspecific actions of demethylating
agents it is difficult to link any specific gene to the
Recent studies have shown loss of p16 to be
associated with poor survival in MM patients. A micro-
array analysis of 99 MM specimens found homozy-
gous deletion of p16 to be a significant independent
adverse prognostic factor in pleural mesotheliomas,
with a median survival of 10 months for p16-deleted
cases vs 34 months for nondeleted cases (P ¼.001).45
This is consistent with a prior study by Borczuk
et al.46that identified loss of p16 immunoreactivity
to be an independent predictor of poor survival in
The high frequency of abnormalities in the
INK4A/ARF locus in MM presents an attractive diag-
nostic marker or target for therapeutic intervention.
It has been proposed that detection of homozygous
deletion of this locus by FISH might be an efficient
approach for improving the cytologic diagnosis of
MM from body cavity effusions.47Another study48
reported that mice treated with p16INK4a-based ade-
noviral vector gene therapy demonstrated prolonged
survival and even a potential cure. In MM cell lines
and mouse xenografts, re-expression of p16INK4a
led to cell cycle arrest, inhibition of cell growth, and
Similarly, we recently evaluated the potential
therapeutic efficacy of p14ARF expressed in an ade-
noviral vector (Adp14ARF) in human mesothelioma
cell lines that lack p14ARF expression.50Overexpres-
sion of p14ARF resulted in increased levels of p53
and the p21WAF protein as well as dephosphoryl-
ation of the retinoblastoma protein. Infection with
the Adp14ARF also led to cell cycle arrest, cell growth
inhibition, and apoptotic cell death. p21WAF, a p53
transcriptional target, is the universal cyclin-depend-
ent kinase inhibitor. p21WAF inhibits cdk-mediated
phosphorylation of pRB, thus leading to cell cycle
arrest at the G1-phase checkpoint. In this way,
p14ARF can affect both the p53 and pRb pathways.
p14ARF gene therapy, by restoring and increasing
p53 activity, might also sensitize mesothelioma cells
to ionized irradiation and chemotherapeutic agents.
In addition to abnormalities in the INK4a/ARF
locus, SV40 is also a key candidate for the disruption
of the p53 and pRb tumor suppressor pathways.
SV40 is a powerful carcinogen that has been shown
to transform human mesothelial cells in vitro and to
induce mesothelioma development in animal mod-
FIGURE 1. The role of the INK4A/ARF locus in the regulation of the pRb
and p53 tumor suppressor pathways.
Molecular Biology of Mesothelioma/Lee et al.1457
els.3The SV40 large T antigen (Tag) has been shown
to bind and inactivate both p53 and pRB in human
The Wnt signal transduction pathway plays an im-
portant role in the pathogenesis of MM. The Wnt
pathway plays a critical role in cell fate determina-
tion, proliferation, and patterning during embryogen-
esis. Although this signaling is essential for normal
developmental processes, aberrant activation of the
Wnt pathway has been closely associated with tu-
morigenesis. Activation of the canonical Wnt path-
way, via binding of the Wnt ligands to the frizzled
transmembrane receptors, leads to the stabilization
and accumulation of b-catenin in the cytoplasm. b-
Catenin subsequently becomes translocated into the
cell nucleus, where it interacts with TCF/LEF tran-
scription factors to promote the expression of Wnt-
responsive genes including the oncogenes c-myc and
cyclin D (Fig. 2).
In a recent IHC study using serous effusions and
pleural biopsies, Dai et al.53found staining of b-cate-
nin confined to the cell membrane in all normal and
reactive nonneoplastic mesothelium. In contrast,
they found reduced membranous staining and mark-
edly increased nuclear and cytoplasmic staining of
b-catenin in 26 of 33 advanced stage and 7 of 9 early
stage MM. These findings are consistent with a simi-
lar study54that found all MM tumors examined to be
positive for cytoplasmic b-catenin and 19% to also
have nuclear b-catenin, whereas no reactive meso-
thelial hyperplasia samples stained with either cyto-
plasmic or nuclear b-catenin. These findings strongly
suggest that the Wnt-b-catenin pathway is abnor-
mally activated in MM.
Dysregulation of b-catenin signaling by activat-
ing mutations in exon-3 of the b-catenin gene is an
important event in the genesis of several human can-
cers but this has not been found in MM.54,55Instead,
it appears that genes more upstream in the Wnt
pathway, including extracellular signaling compo-
nents, may play a more important role. Our group
previously demonstrated that the Wnt signaling path-
way is activated in MM through the overexpression
of disheveled proteins. Additionally, we have recently
found that endogenous extracellular Wnt antagonists,
such as the secreted frizzled related proteins (sFRPs)
and Wnt inhibitory factor-1 (Wif-1), are frequently
tumors.56,57A monoclonal antibody or siRNA direc-
ted against Wnt-1 and Wnt-2 induced apoptosis in
cancer cells overexpressing Wnt. Furthermore, MM
cells treated with these antibodies displayed down-
cell linesand primary
regulation of key downstream Wnt signaling effectors
such as disheveled and b-catenin.58,59These findings
suggest that Wnt-targeted therapies may be useful in
the treatment of MM.
The poor response of MM to conventional therapeu-
tic agents is partly due to this tumor’s resistance to
apoptosis. The bcl-2 family of genes is known to play
a major role in the intrinsic apoptotic pathway. Its
members include both proapoptotic proteins such as
Bax, Bak, and Bad and antiapoptotic proteins such as
Bcl-2, Bcl-xL, and Mcl-1. The proapoptotic Bcl-2
family members are thought to induce apoptosis by
promoting permeability of the mitochondrial mem-
branes. This increased permeability results in the
release of caspase activators such as cytochrome c
and subsequently leads to the activation of down-
stream caspases that cause cellular demolition and
apoptotic morphology.60In contrast, antiapoptotic
Bcl-2 and Bcl-xL are thought to block this mitochon-
drial permeabilization. Bcl-2, in particular, has been
demonstrated to protect neoplastic cells from chem-
otherapy and radiation-induced apoptosis.61Addi-
tionally, a low bax expression has been linked to
higher resistance to chemotherapy and a poor prog-
nosis in breast carcinoma.62It has been hypothesized
that by altering the ratio of expression of various
proapoptotic and antiapoptotic bcl-2 family genes,
apoptosis can either be induced or inhibited.63
Expression of bcl-2 is rare in MM, unlike in
tumors such as breast and endometrial adenocarci-
nomas, which frequently display up-regulation of
bcl-2.64–67In contrast, elevated levels of Bcl-xL mRNA
FIGURE 2. The canonical Wnt signal transduction pathway in its (A) inac-
tive and (B) active states.
1458 CANCERApril 15, 2007 / Volume 109 / Number 8
and protein have been detected in all mesothelioma
cell lines and tumor samples examined.66,68Interest-
ingly, Narasimhan et al.65also found expression of
proapoptotic Bax in all 14 of 14 mesothelioma cell
lines, including those that were highly resistant to
proapoptotic stimuli. This suggests that overexpression
of Bcl-xL may be necessary to counteract the proapop-
totic effect of Bax. Because uniform Bcl-xL overexpres-
sion in malignant mesothelioma could account for
resistance to apoptosis even in the presence of wild-
type p53, Bcl-xL may be a possible oncogenic candi-
date in this tumor type.
Pharmacologic inhibition of Bcl-xL expression by
exposure to a histone deacetylase inhibitor, sodium
butyrate (NaB), has been shown to lead to apoptotic
cell death in malignant mesothelioma.69Antisense
oligonucleotides targeting the bcl-xL gene product
have been shown to similarly facilitate apoptosis in
mesothelioma.68,70Additionally, a recombinant ade-
noviral vector that can transduce human cells with
the proapoptotic bax gene has been shown to effec-
tively induce apoptosis in mesothelioma.71Inhibition
of bcl-xL displayed a synergistic effect when com-
bined with the adenoviral proapoptotic gene therapy
vectors AdBak and AdBax.63
Of interest, 1 recent study found that long-term
recurrent exposure to asbestos affects human immune
cells, leading to Bcl-2 enhancement in CD4þ lympho-
cytes in vitro.72The same study found Bcl-2 expres-
sion in CD4þ peripheral blood lymphocytes to be
significantly increased in MM patients compared with
healthy volunteers (P ¼.0153) and asbestosis patients
with no signs of malignancy (P ¼.129). The clinical
significance of this increased Bcl-2 expression remains
to be addressed.
Further investigation on the role of the bcl-2
family members in the pathogenesis of MM, espe-
cially in relation to improved therapeutic strategies,
Over the past decade there have been significant
advances in our understanding of the molecular
pathways involved in MM carcinogenesis. It is likely
that the implementation of high-throughput proteo-
mic and genomic technologies such as microarray
analysis will further accelerate research in this field.
Despite this continuing progress in our understand-
ing of the biology of MM, progress in treatment of
this fatal tumor has been slow and challenging, with
most treatments having no survival advantage over
Perhaps the greatest and most crucial challenge
now is to translate our growing knowledge of MM
pathogenesis into more effective ways to treat this
devastating disease. MM is a complex tumor that
results from accumulation over many years of multi-
ple genetic alterations, many of which remain to be
uncovered. Thus, it is overly simplistic to expect that
any single ‘‘magic bullet’’ may reverse the malignant
phenotype. Rather, elucidating the several key path-
ways involved will hopefully result in a rational treat-
ment in which several molecularly targeted agents
will be combined with effective chemotherapeutic
regimes. Early diagnosis in high-risk populations
might also be a desirable goal as treatment effective-
ness has been shown to be much greater the earlier
the clinical stage of the tumor at diagnosis. Timely
integration of laboratory-based studies into standard
clinical practice will require continued cooperation
between basic scientists and clinical investigators
working toward improved survival and quality of life
in patients with this aggressive and fatal disease.
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