Molecular pathology of tumor metastasis III

Abstract

Therapy of tumor progression and the metastatic disease is the biggest challenge of clinical oncology. Discovery of the diverse
molecular pathways behind this complex disease outlined an approach to better treatment strategies. The development of combined
cytotoxic treatment protocols has produced promising results but no breakthrough in the clinical management of metastatic
disease. The multiple-specific and non-specific pathways and cellular targets of tumor progression are outlined in this review.
Such an approach, individually designed for various cancer types, may have a better chance to treat or even cure cancer patients
with progressive disease.

Full text

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Molecular Pathology of Tumor Metastasis III.
Target array and combinatorial therapies
József TÍMÁR,1,2Andrea LADÁNYI,1István PETÁK,2András JENEY,2László KOPPER2
1National Institute of Oncology;21stInstitute of Pathology and Experimental Cancer Research, Semmelweis University,
Budapest, Hungary
SEMINAR
© 2003 Arányi Lajos Foundation
PATHOLOGY ONCOLOGY RESEARCH Vol 9, No 1, 2003
Article is available online at http://www.webio.hu/por/2003/9/1/0049
Introduction
Recently, advances in molecular cancer research have
revealed numerous new therapeutic targets, some of which
have already been tested in clinical settings. However, the
clinical management of cancer patients is still based on sur-
gical and/or radiological eradication of the primary solid
tumor and chemical/radiological anti-proliferative/cyto-
toxic treatment of the disseminated disease. Despite a great
deal of improvement in the control of loco-regional disease,
only slow progress has been seen in systemic disease. As a
result, mortality of cancer patients is still high due almost
exclusively to the development of metastases. One obvious
explanation for this discrepancy could be a difference
between the molecular pathways controlling tumor growth
and tumor progression (as analyzed in detail in the previous
publications of this series).1,2
By definition, antimetastatic therapy covers all those
available approaches which can prevent cancer cell dis-
semination or eradicate already disseminated and arrested
tumor cells or their growing colonies outside the primary
site, irrespective of the size of the cell population. Since
Received: Febr 24, 2003; accepted: March 22, 2003
Correspondence: József TÍMÁR, MD, PhD, DSc, Department of
Tumor Progression, National Institute of Oncology, Budapest, Ráth
György u. 7-9, H-1122, Hungary. Phone: 36-1-224-8786, Fax: 36-
1-224-8706, E-mail: jtimar@oncol.hu
Therapy of tumor progression and the metastatic dis-
ease is the biggest challenge of clinical oncology.
Discovery of the diverse molecular pathways behind
this complex disease outlined an approach to better
treatment strategies. The development of combined
cytotoxic treatment protocols has produced promis-
ing results but no breakthrough in the clinical man-
Keywords: metastasis therapy, molecular target, cancer cell, host cell
agement of metastatic disease. The multiple – specif-
ic and non-specific pathways and cellular targets of
tumor progression are outlined in this review. Such
an approach, individually designed for various can-
cer types, may have a better chance to treat or even
cure cancer patients with progressive disease. (Patho-
logy Oncology Research Vol 9, No 1, 49–72, 2003)
metastatization (or tumor progression) is a complex phe-
nomenon, its effective inhibition requires more than an
anti-proliferative protocol.
Management of primary cancer and its eradication is
primarily based on surgery and/or irradiation. On the other
hand, chemotherapy is applied mostly as “anti-metastatic
therapy”, with the exception of the treatment of hemato-
logical malignancies and the primary chemotherapy of
solid tumors. Since the process of metastasis is a complex
interplay between the disseminating cancer cells and the
host, rational and successful anti-metastatic interventions
may target all the participants of these interactions in
which anti-proliferative/cytotoxic interventions are only
parts of a much more complex approach.
This review intends to describe all the possible targets in
the metastatic cascade, which could be attacked pharmaco-
logically, and to provide experimental and clinical examples
for their therapeutic potentials. On the other hand, we will not
review the currently available radio- and chemotherapeutic
protocols (including endocrine therapy) of advanced cancer
extensively documented in the current literatures.
Pathomechanism
Since chemical compounds with pharmacologically
exploitable properties may act on pathobiological events
and interfere with one of the relevant molecular mechanisms
it appears highly important to analyze the series of steps in

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the metastatic process and to attempt to select their appro-
priate inhibitors. It is conceivable that antimetastatic therapy
might not be limited to a priviledged drug but hopefully will
cover multiple agents acting on molecular mechanisms
implicated in the sequence of the metastatic cascade.1Cer-
tainly these pathobiological events represent only those
sequential steps that are strictly related to the passage of the
tumor cells from the primary tumor to the secondary site. For
the full completion of metastasis the tumor cells must have
high viability to survive the circulation and have the poten-
tial for growth and vascularization. These latter events how-
ever occur in the primary lesion as well and their biological
consequences do not necessarily lead to metastasis. There
are overlaps in the molecular mechanisms in the metastatic
cascade (i.e. shedding and proteolysis, which occur at sever-
al steps), in addition, some features may be present in both
the primary tumor and the metastatic lesions.
There are two unique features in the metastatic process
that have come to light recently, namely its inefficiency
(i.e., only a small proportion of the tumor cells escaped
from the primary tumor can form metastatic lesions) and
the transition of the carcinoma cells between the epithelial
and mesenchymal pheno- or genotype.3Until now, it was
assumed that most tumor cells entering circulation are
killed by monocytes, lymphocytes or leukocytes. Howev-
er, in experimental models it was shown that a great pro-
portion of tumor cells can survive the hostile circum-
stances of the circulation and their viability and growth
potential are controlled by the secondary site. This implies
that in hematogenous dissemination the initial steps
(intravasation and survival in the circulation) could be
much more efficient processes than the subsequent ones
(extravasation and survival at the secondary site).4Conse-
quently, the fate of disseminated tumor cells is determined
by the new environment which controls growth to allow
persistence and the formation of micro- and macroscopic
lesions, or death of the tumor cells.
Another principal question concerns the uniformity in the
mechanism of tumor dissemination, which must be consid-
ered seriously when designing antimetastatic therapy. The
prevailing model of metastasis holds that few tumor cells
with metastatic potential (one in ten million) within the
large primary lesions acquire metastatic capacity through
somatic mutations.5The genotypic heterogeneity of the
tumors characterized by identical histology indicates the
existence of diverse mechanisms involved in the progression
of the individual tumors. Therefore it came as a surprise that
metastatic solid tumors of diverse histological types have
been found to have a common gene expression profile.6
Since in lung-, breast-, prostate adenocarcinomas and
medulloblastoma (but not in lymphoma) a similar tendency
of association between gene expression profiles and metas-
tasis has been observed, a generic gene expression program
related to metastasis rather than distinct mechanisms of
metastasis in different tumor types has been proposed. The
characteristic set of genes expressed are designated as a mol-
ecular signature of metastasis, and show a close relationship
with overall survival of the patients. Perhaps more important
is the finding that this gene expression profile can be identi-
fied in a subset of primary tumors with higher metastatic
capacity, suggesting that propensity to metastasize is a con-
sequence of the predominant genetic state of the primary
tumor and making less plausible the emergence of rare cells
with metastatic phenotype in large tumors. These results
argue for the development of metastatic phenotype during
malignant transformation and indicate that the metastatic
potential may be encoded in the entire tumor, consequently
providing guidelines to identify tumors with high propensity
to metastasize before surgery. Nevertheless, it must be men-
tioned that the above gene expression profile associated with
metastasis includes many less characterized genes not listed
among those known to contribute to the invasive/metastatic
potential of tumor cells. Rather, these genes are expressed by
stromal components of the tumor tissue, supporting the
important contribution of the host to the entire process.7
There is a vast amount of clinical data indicating that the inci-
dence of metastasis is not simply related to the size of the
primary tumors. Micrometastasis can appear in patients with
small, low stage tumors and also in the absence of clinically
detectable primary tumors.8Thus the chronic and systemic
features of malignant disease indicate the importance of
planned patient control after surgery and the availability of an
arsenal of pharmaceutical interventions for the
prevention/treatment of disease progression.
Below, we will summarize all the possible pharmaco-
logical targets and approaches developed to date, which
are tested at least in experimental metastasis models or in
the clinic for the treatment of advanced metastatic dis-
ease. These approaches can be divided into metastasis-
specific modalities targeting those molecular events that
are specific for the metastatic cascade, and nonspecific
modalities targeting the primary and the metastatic tumor
tissue as well as the dissemination process itself. Fur-
thermore, pharmacological approaches are also divided
according to their targets, i.e. tumor cells or host cells
(tumor-host interactions).
SPECIFIC ANTI-METASTATIC THERAPY
Target: tumor cells
Membrane receptors
Integrins
Tumor cell-extracellular matrix (ECM) interactions are
key events of various repetitive steps in the metastatic cas-
cade involving ECM recognition, concentration of pro-
teases to the invadopodia and migration on matrix sheets.7
In various cancer types a well-defined integrin receptor
50TÍMÁR et al
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pattern develops, which promotes the emergence of the
invasive/metastatic subpopulation. In special circum-
stances individual integrins can even become predominant
over other matrix receptors offering at least a marker of
these invasive cells.1Once a predominant integrin is iden-
tified on the surface of invasive cancer cells it offers a tar-
get for therapeutical intervention.9
Since most ECM proteins share the consensus sequence,
RGD, for integrin binding, small peptide inhibitors of inva-
siveness and metastasis have been primary options. In vitro
data provided ample of evidences that RGD-ligand peptides
are powerful inhibitors of cancer cell adhesion, migration on
matrices and in experimental metastasis models.10Such pep-
tides showed also anti-invasive, anti-metastatic activity,
especially in the case of hematogenous dissemination.11
Unfortunately none of these peptides have yet entered clini-
cal testing. Recently, following the same concept, larger
RGD-containing peptides of fibronectin with fibril-forming
potential have been designed and tested in experimetal mod-
els of various human cancers.12On the other hand, natural
RGD-containing inhibitors, called disintegrins have also
been identified in the venoms of various snake species.
These natural peptide inhibitors of integrins are powerful
inhibitors of cancer cell ahesion to various matrices includ-
ing basement membrane as well as of cell migration, and are
active in vivo in experimental metastasis models, especially
in the case of hematogenous dissemination.13-16
Neutralizing/inhibitory antibodies against integrin recep-
tors have pharmacological potentials too. However, unlike
anti-growth factor antibodies, only a few such antibodies
have been applied as anti-cancer agents. Vitaxinis an αvβ3
integrin inhibitor humanized antibody which was primarily
designed as angiogenesis inhibitor.17However, several can-
cer types are characterized by prominent αvβ3 integrin
expression and in these cases this integrin has been shown
to be involved in the complex process of invasion and
metastasis.1 Meanwhile, to date there are no experimental
data available concerning the direct anti-metastatic poten-
tial of Vitaxin. Another humanized anti-integrin antibody,
abciximab(ReoPro) was introduced to clinic as anti-throm-
botic agent targeting the platelet integrin αIIbβ3and the
endothelial αvβ3.18Abciximab not only inhibits platelet
aggregation with or without the presence of tumor cells but
also angiogenesis.19Ectopic expression of the αIIbβ3inte-
grin has been observed in various cancer types including
melanoma (also known to express αvβ3).20Using experi-
mental human melanoma metastasis models, abciximab has
demonstrated anti-tumoral and anti-metastatic effects,
encouraging further investigation of this subject.19
There is another rationale to target surface integrins on
invasive/metastatic cancer cells. In vitrodata have indicat-
ed that matrix adhesion induces drug resistance through
overexpression of certain integrins. Such a phenomenon
can be observed both in the case of hematological malig-
nancies as well as in solid tumors.21An anti-integrin
approach to the treatment of metastatic tumors may have
the long-awaited „side effect” of reducing the drug-resis-
tance of cancer cells.
Growth factor (GF) inhibition
One of the earliest pharmacological agents discovered
as a GF competitor was suramin, a polysulfonated naftyl-
urea.22The polyanionic molecule was able to sequester
several growth factors with heparin binding domains
including EGF, IGF, TGF-β, bFGF and PDGF. Several of
these GFs are involved in the invasion and metastasis of
various cancer types, and therefore it was expected that
suramin would be an antimetastatic agent. However,
suramin was too toxic and its non-specific binding to
serum proteins limited its biological effect in vivo.23
Accordingly, pentosan sulfate derivatives of distamycin A
have been designed (heparinoids) and tested in experi-
mental models. These compounds have anti-mitotic and
anti-angiogenic effects and are able to compete with at
least bFGF and IGF, suggesting that they could be devel-
oped as anti-metastatic agents too.
An alternative approach would be to use neutralizing anti-
GF antibodies to suspend auto- or paracrine stimuli to can-
cer cell invasion. The feasibility of such approach is demon-
strated in angiogenesis, where anti-VEGF antibodies have
been demonstrated to be active in vitro and in vivo in pre-
clinical models.24However, development of other anti-GF
antibodies have not yet been reported in the literature.
Growth factor receptor (GFR) inhibition
The majority of growth factors involved in the malignant
phenotype of cancer are also motogenic factors: frequently,
the same mitogen regulates the migration of cancer cells
through partially degraded matrix. Accordingly, targeting
the receptors for these GFs can provide an approach to tar-
get the regulation of cancer cell migration too. Cancer cells
frequently overexpress GFRs at their surface due to genetic
alterations in their genome, i.e. amplification. Most fre-
quently such genes are members of the EGFR family.25
EGFR2 (c-erbB2) is amplified and overexpressed in a sub-
set of breast cancer characterized by a more agressive, more
metastatic phenotype26,27but it has also been demonstrated to
be expressed and functioning in ovarian- lung-, prostate- and
GI-tract cancers (adenocarcinoma) as well as in head and
neck cancers (squamous cell cancers).25EGFR1 (c-erbB1),
on the other hand, is overexpressed mostly in squamous cell
cancers, and in some adenocarcinomas too (such as colon
cancer).25Another reason to target GFR on invasive cancer
cells is that there is a cooperation between the signaling
pathways of integrins and growth factors, and parallel inhi-
bition of the two initiators of cell migration promises more
51Molecular Pathology of Tumor Metastasis III.
Vol 9, No 1, 2003

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success in shutting down the motogenic activity crucial for
cancer cells. Although there are several alternative options
for targeting EGFR (specific ligand-mimetics, and inhibito-
ry antibodies) only the latter approach has proved to be suc-
cessful clinically. The best example is Herceptin, a human-
ized anti-c-erbB2 antibody, which is active clinically in
advanced breast cancer overexpressing c-erbB2. The molec-
ular consequences of the application of anti-c-erbB2 anti-
bodies indicated that they downmodulate the surface recep-
tor, thereby inhibiting the signaling cascade, but also initiate
complement-mediated cytotoxicity as well as antigen-
dependent cytotoxicity against their target.26,27Its success
could well be followed by IMC-C225, an anti-c-erbB1 anti-
body that already has a humanized version. This antibody
proved to be very effective against cancer progression in
preclinical models.28,29
It is a novel approach to inhibit GFR expression at the
level of transcription. The c-met oncogene and its ligand
HGF have been demonstrated to be important regulators of
the invasive/metastatic phenotype of various cancer types
including colon cancer and melanoma.1,30Furthermore,
this receptor system has also been implicated in the liver
metastatic potential of various tumors thereby offering
another rationale for anti-receptor anti-metastatic interven-
tion. There are two tested pharmacological approaches in
the literature, one targets the ligand, HGF, and the other
the expression of the receptor.
The HGF ligand competition approach has used two
strategies, a „splice variant” recombinant HGF with recep-
tor-inhibitory potential31and the exploitation of the heparin-
binding potential of the ligand and its importance in the
conformational activation. Both the inhibitory ligand pep-
tide NK-4(either recombinant or incorporated in adenovirus
vector 31,32), as well as a peptidomimetic ligand of the
heparin binding domain of HGF33exhibited anti-inva-
sive/anti-metastatic activity in preclinical models including
effects on liver metastasis. Furthermore, since HGF is also
an angiogenic factor, both approaches resulted in the inhi-
bition of tumor-induced angiogenesis,31,34where NK-4
seemed to be more potent.
The approach to transcriptional regulation exploited the
unique potential of geldanamycin (a member of the fami-
ly of anisamycin antibiotics). This drug exhibited very
selective inhibitory activity on the expression of c-met in
cancer cells resulting in the down-regulation of c-met sig-
naling and loss of the invasive/motile phenotype in exper-
imental systems.35
Transmembrane proteoglycans
Proteoglycans at the surface of cancer cells have been
demonstrated to be involved in tumor progression in a Janus-
faced manner.36In certain tumors downmodulation of their
expression could be observed during carcinogenesis, while
in other tumors overexpression occured when the tumor
became invasive and metastatic.36,37The major players in
this respect are the transmembrane type heparan sulfate pro-
teoglycans (HSPGs: syndecans and CD44v3) and GPI
anchored HSPGs of the glypican family. Their function is
regulated by the glycanation process, which adds either
heparan sulfate (HS) or chondroitin sulfate (CS) chains to
the core protein. These proteoglycans, mostly through the
HS-chains, are involved in cytokine/growth factor recruit-
ment as well as in matrix adhesion processes, serving as co-
receptors.36Furthermore, the transmembrane forms (synde-
cans and CD44) have been involved in important signaling
processes such as vnt (syndecan-1), PKCα (syndecan-4) or
motility signaling (CD44v3). These diverse functions of
transmembrane HSPGs in the invasion process make them
potential targets for therapeutic interventions.
It is well documented that in invasive/metastatic cancer
cells glycanation of membrane proteoglycans is frequently
shifted toward heparan sulfates from chondroitin sulfates
offering a target for pharmacological interventions.36,37It was
shown early on that using nonspecific glycosaminoglycan
(GAG) biosynthesis inhibitors (β-xyloside, 2-deoxyglucose)
it is not possible to alter invasive phenotype of cancer cells
unless only the heparan sulfates are preferentially affected.38
5’hexyl-2’-deoxyuridine (HUdR) preferentially inhibits HS
biosynthesis in cancer cells.39-41Such treatment does not affect
cell proliferation, but rather inhibits tumor cell-ECM interac-
tions, thereby down-regulating the metastatic potential.
A unique approach for the inventional use of GAGs has
been developed when neoglycans were prepared from
albumin and CS or HS GAGs.42Among these new glycans
neoCS demonstrated impressive anti-tumoral pro-apoptot-
ic effects in vitroand in vivo in experimental breast cancer
and myeloma. Further studies are required to explore the
pharmacological potentials of these new GAG agents.
Cell adhesion molecules (CAM)
Local invasion (previously defined as shedding), repre-
senting the first step in metastasis, may occur if the intercel-
lular links and – in the case of the epithelial cells – firm con-
tact with the basement membrane have been drastically
altered. Subsequently the detached tumor cells are not con-
fined by their neighbours and are ready for invasive growth.
Assuming that more detailed knowledge of these cell con-
tacts could be utilized in drug development, the question has
been raised whether loss or gain is the dominant feature in
the molecular mechanisms implicated in shedding.
At present cadherins, through the formation of adherent
junctions, are regarded as the critical molecules both in
homophylic and heterophylic intercellular contacts.43It is
noteworthy that MDA-MB-231 human tumor cells, after
transfection with E-cadherin cDNA, lose their capacity to
form osteolytic metastases.44
52TÍMÁR et al
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Several clinical studies have concluded that E-cadherin
expression is reduced in various malignant tumors
(prostate, stomach, kidney, head and neck, etc.). This cor-
relates with the metastatic potential of the tumors, there-
fore cadherins are considered as invasion/metastasis sup-
pressor molecules.44Based on these observations it was
suggested that administering agents that up-regulate E-
cadherin expression may be a potential therapeutic
approach to inhibit tumor spread.
Defects in E-cadherin functions can occur despite high
levels of expression by steric hindrance of cell surface pro-
teoglycans.45In this latter case experimental reduction of
the glycanation of cell associated proteoglycans with β-D-
xyloside resulted in the abrogation of tumor cell invasive-
ness without influencing E-cadherin expression.
Signal transduction
Tumor cell invasiveness and metastatic potential
depend critically on the signaling events generated
through the interactions with ECM and the auto-
paracrine regulatory factors of motogenicity (Figure 1).
These signaling pathways are highly similar to those
functioning in normal cells as far as their final down-
stream targets are concerned, but the upstream pathways
are highly diverse in the various types of cancer depend-
ing on the specific genetic background (such as RAS
mutations, oncogenic splice variants, deletions, fusions
etc). Furthermore, these signaling pathways are different-
ly controlled at receptor level since both the expression
of adhesion receptors and cytokine receptors can be
altered in cancer cells. Accordingly, signal transduction
inhibitors could be potentially useful in controlling inva-
siveness and the metastatic potential of cancer cells. This
idea is further corroborated by the recent findings on
metastasis suppressor genes where a subset of these
genes controls those signaling pathways that are critical
for metastasis and does not affect the mitotic signaling,
arguing for the existence of invasion-specific signaling
pathways.46
Receptor tyrosine kinase (PTK) inhibitors
Although several PTK inhibitors have been recently
developed to target the tyrosine kinase activity of EGFR,47
VEGFR48and ABL/c-kit/PDGFR,49their effects on the
regulation of tumor cell motility is largely unknown. Today
several small molecular EGFR inhibitors are in clinical tri-
als50and some of them have already demonstrated activity
against head and neck-, ovarian- and non-small cell lung
cancers.50,51It is interesting that c-ABL is one of the many
downstream kinases of the integrin-linked signaling,52
which already has a clinically successful inhibitor
(Gleevec, imatinib mesylate, in chronic myloid
leukemia49). Future studies will reveal if this drug has any
anti-invasive potential in solid tumors. On the other hand,
53 Molecular Pathology of Tumor Metastasis III.
Vol 9, No 1, 2003
PLCγ γ
TK -TK*
GREB2
SRC
PI3K
PLCγ γ
FAK
GREB2
SOS
RAS
RAS*
RAF
RAF*
SRC
PI3K
Gia
GREB2
SOS
RAS
RAS*
RAF
RAF*
SOS
PLCγ γ
RAS
RAS*
RAF
RAF*
PI3KPLCγ γ
PLCγ γ
PI3KPLA2
PI3KPLCγ γ
PLCγ γ
(DAG)RAF-
GEF
(DAG)RAF-
GEF
12-
LOX
RAF-
GEF
(DAG)
PK CMEKRho/RACCa++
PK CMEKRho/RACPK CRho/RACMEKPK CCa++
MAPKMAPKMAPK
MLCKRhoKMLCKRhoKRhoKMLCK
MLCMLCMLCMLCMLCMLC MLCMLCMLCMLC
GFR INT* MFR
PM
CYTOSKELETON
(receptor translocation, protease secretion, migration)
Figure 1. Signaling pathways in tumor cell invasion. GFR: growth factor receptor; INT: integrin receptor, MFR: motility factor
receptor; MLC: myosin light chain; PM: plasma membrane; TK: tyrosine kinase; INT*/TK*/RAS*/RAF*: activating mutations.
Red: pharmacologically targeted signal elements