Seminars in Cancer Biology 14 (2004) 105–114
Oncogenic Ras and its role in tumor cell invasion and metastasis
Paul M. Campbell∗ , Channing J. Der
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
CB# 7295, Chapel Hill, NC 27599-7295, USA
Abstract
The processes by which cancer cells leave the tumor and enter adjacent tissue is known as invasion, whereas metastasis refers to
secondary tumor colonization of tissue at a distance from the primary lesion. These two events are the most lethal of cancer phenomena
and the signaling mechanisms that govern them are complex. The Ras signaling pathways are well represented in their involvement in
tumor initiation, but considerably less is known about their contribution to invasion and metastasis. In this review, we discuss the current
evidence for mutant Ras proteins as significant players in these aspects of cancer progression.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: Ras; Oncogene; Signaling; Invasion; Metastasis
1. Introduction
As the initiation and progression of solid tumors is in part
dependent on a variety of signaling pathways, so too are the
processes that allows for invasion and metastasis. In metastasis, cells migrate from the primary tumor, cross the tumor
marginal border gaining access to the vascular and/or lymphatic system, and enter distal tissue. At this point, these
“rogue” cells may lie dormant for an indefinite period of
time, but upon stimulation by signals that at the present are
not entirely known, these micrometastases can subsequently
proliferate into solid tumors themselves. Because the process of metastasis can lead to potentially many secondary
tumor sites, and because evidence indicates a more aggressive and lethal phenotype for the metastatic tumor versus the
primary lesion, it is imperative to gain further knowledge of
the biology of metastasis and invasion. Understanding the
signaling mechanisms that lead to such migration will enable discovery of additional targets for therapeutic design.
Ras proteins (of which H-, N-, and K-Ras4A/4B are prototypical) are associated with the inner face of the plasma
membrane where they facilitate signaling initiated by diverse
extracellular stimuli [1]. Ras activity is regulated by cycling
between inactive GDP-bound and active GTP-bound forms
[2] (Fig. 1). Increase in GDP/GTP nucleotide exchange
involves interaction between Ras and guanine exchange factors (GEFs), for example Sos1/2, RasGRP and RasGRF1/2
∗ Corresponding
author. Tel.: +1-919-962-1057; fax: +1-919-966-0162.
E-mail address: paul campbell@med.unc.edu (P.M. Campbell).
1044-579X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcancer.2003.09.015
proteins [3]. When GTP-bound, Ras binds to and activates
a plethora of effector molecules [1,4,5]. Hydrolysis of GTP
by Ras is facilitated by GTPase-activating proteins (GAPs)
such as p120GAP and NF1. Mutated variants of Ras (mutations at residues 12, 13 or 61) are found in 30% of all
human cancers, are insensitive to GAP stimulation, and are
consequently rendered constitutively activated [6,7].
In addition to mutational activation, Ras GTPase signaling
can be upregulated due to increased coupling to cell surface
receptors. In particular, members of the epidermal growth
factor (EGF) family of receptor tyrosine kinases (RTKs; including EGFR/ErbB/HER1 and ErbB2/Her2/Neu) [8–10] or
other tyrosine kinases (e.g. Bcr-Abl) are commonly overexpressed in many cancers, causing persistent activation of Ras
in the absence of mutations in Ras genes. Thus, Ras activation has been shown to be an important mediator of tumor
cell invasion and metastasis caused by these and other tyrosine kinases. However, for the purpose of this review, we
will limit our focus to the effects of mutationally activated
Ras proteins on invasive and metastatic phenotypes.
The aberrant activation of Ras proteins been implicated in
facilitating virtually all aspects of the malignant phenotype
of the cancer cell, including cellular proliferation, transformation, invasion and metastasis (reviewed in [11]). Additionally, the functions of other Ras-related proteins are also
regulated by Ras signaling and also contribute to oncogenesis. While much is known regarding the mechanisms by
which aberrant Ras promotes uncontrolled proliferation by
deregulation of cell cycle progression and promotion of cell
survival, less is known regarding how Ras promotes tumor
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P.M. Campbell, C.J. Der / Seminars in Cancer Biology 14 (2004) 105–114
Expression of exogenous Ras has also been shown to
promote the invasive and metastatic properties of other cell
types. Epithelial and other cell types from a variety of human and murine tissues have been made invasive and/or
metastatic by introduction of mutated Ras genes [16,20–27].
3. Contribution of specific effectors downstream of
oncogenic Ras
Fig. 1. Ras upstream and downstream signaling. Extracellular stimuli
signal through cell surface plasma membrane receptors, for example,
RTKs. Through a variety of adaptor proteins, these signals cause guanine nucleotide exchange factors to replace the GDP-bound to inactive
Ras with GTP. GAPs trigger the hydrolysis of GTP back to the inactive
GDP-bound form. GTP-bound Ras binds to a plethora of downstream
effector molecules to stimulate intracellular signaling of several pathways. Those with established roles in Ras oncogenesis include the Raf
serine/threonine kinases, the PI3K lipid kinases, Ral GEFs, and Tiam1.
Activation of these pathways and others has been shown to cause changes
in many mechanisms leading to transformation, invasion and metastasis.
cell invasion and metastasis. In this review, we summarize
the current understanding of the mechanisms by which oncogenic Ras promotes the malignant phenotype of cancer cells.
2. Mouse and in vitro experimental models
A variety of experimental approaches have been undertaken to ascertain the degree to which Ras GTPases are involved in and/or causative for metastasis and invasion. In
both in vitro and in vivo experimental models, transfection
of mutated, constitutively active forms of Ras into previously
noncancerous cells can lead to invasive and metastatic phenotypes [12,13]. One of the most commonly studied models of Ras activation is the murine NIH 3T3 fibroblast cell
line. Ectopic expression of oncogenic, constitutively active
Ras results in transformation, increased invasion in vitro
and in vivo, and acquisition of metastatic properties. The
hematogenic metastasis model, using tail vein injection of
transformed cells and observing subsequent lung nodules,
has been commonly employed to investigate the molecular
mechanisms for metastasis [14]. Invasive potential is often
quantified by looking at a cell’s ability to migrate through
reconstituted basement membrane (Matrigel) or other extracellular matrix (ECM) components [15,16]. Ras transformation of other rodent fibroblasts also promotes invasive and
metastatic growth [17–19].
Ras interacts with and regulates multiple downstream
effectors that stimulate diverse cytoplasmic signaling activities [1,4,5]. As new effectors continue to be identified, one
of the critical issues concerns the specific role of each effector in Ras-mediated oncogenesis. While some are clearly
important positive mediators of the oncogenic properties of
Ras (e.g. Raf, PI3K, RalGEF, Tiam1), others may serve negative regulatory roles in oncogenesis (e.g. Nore1, RASSF).
One important area of Ras research has been the determination of the role of specific effectors in mediating specific
facets of Ras-mediated oncogenesis [28]. Various experimental approaches have been very useful to dissect effector
function (Fig. 2). One powerful approach has been the use
of H-Ras effector domain mutants [29–32]. These mutants
harbor missense mutations in the core Ras effector domain
(residues 32–40) that is critical for Ras to bind to all effectors. Specific amino acid substitutions result in the differential impairment of effector binding. For example, the T35S
mutation causes loss of PI3K and RalGEF, but not Raf, activation. In contrast, the E37G mutation causes a loss of Raf
and PI3K, but not RalGEF, activation. Finally, the Y40C
substitution does not impair PI3K activation, but causes a
loss of Raf and RalGEF activation. One caution in the use
of these effector domain mutants is that their specificity of
effector activation may differ when expressed in some cell
types. Furthermore, while equivalent mutants of activated
K-Ras4B and N-Ras have been generated, their effector interactions are not equivalent to the H-Ras mutants.
A second approach has been the use of constitutively activated effectors and their downstream substrates. For example, variants of Raf, p110, and RalGEFs that contain plasma
membrane targeting sequences represent constitutively activated versions of these effectors [32–35]. The use of effector domain mutants or activated effectors can be utilized
to determine if activation of a particular effector pathway is
sufficient to mediate a specific facet of Ras function.
Finally, various pharmacologic (e.g. the U0126 and
PD98059 MEK inhibitors) [36] or genetic inhibitors of
specific effector pathway signaling have been utilized to
determine if a particular signaling cascade is necessary for
Ras function [1] (Fig. 2). Recent reviews had summarized
the role of specific effectors in regulation of cell proliferation, cell survival, and regulation of gene expression
[1,5,37–39]. In this review, we have summarized the evidence for the role of specific effectors in Ras-mediated
tumor cell invasion and metastasis.
P.M. Campbell, C.J. Der / Seminars in Cancer Biology 14 (2004) 105–114
Fig. 2. Experimental approaches to study Ras effector function. H-Ras
effector domain mutants, with single amino acid substitutions in the core
effector domain of Ras, cause differential impairment of effector binding, and result in preferential activation of Raf (T35S), PI3K (E37G),
or Ral GEF (Y40C). An important caution with the interpretation of results with these mutants is that they do bind other effectors. For example, the E37G mutant retains interaction with phospholipase C epsilon
and Rin1. Hence, observations made with this mutant require verification
with activated effectors. Genetically engineered mutants of Raf, the p110
PI3K catalytic subunit, and Ral GEFs terminate with the carboxyl terminal plasma membrane targeting sequence from Ras (designated CAAX),
are constitutively membrane associated and activated. Similarly, constitutively activated variants of effector substrates have also been used to
study the function of each effector pathway. These include constitutively activated MEK1/2 (with charged residue substitutions, for example
SS → ED, at sites of Raf phosphorylation), Akt (e.g. membrane-targeted
by addition of a myristylation (Myr) signal sequence), and Ral GTPases
(GTPase-deficient). These reagents are utilized to determine if a particular
Ras effector pathway is sufficient to mediate a particular cellular consequence. Finally, various pharmacologic approaches can be used to block
MEK1 and MEK2 activation of ERK1 and ERK2 (U0126 and PD98059)
or PI3K (LY294002), or genetic approaches to block PI3K (the PTEN
lipid phosphatase or dominant negative (DN) mutants of PI3K, ERK activation (kinase-dead mutants of MEK, and ERK phosphatases, such as
MKP-1), or dominant negative Ral mutants (e.g. RalA28N), which block
Ral GEF activity. These reagents can be used to determine if a particular
effector signaling pathway is necessary for Ras function.
3.1. Raf activation of the ERK–MAPK protein kinase
cascade
The most widely studied effectors for Ras signaling are
the Raf serine/threonine kinases (c-Raf-1, A-Raf, B-Raf)
[40]. The recent identification of mutated B-Raf in a diverse
spectrum of human cancers provides further validation of
the importance of this effector pathway in Ras oncogenesis
[41]. Ras promotes Raf association with the plasma membrane, where other events facilitate Raf activation. Raf then
phosphorylates and activates the MEK1 and MEK2 dual
specificity kinases. MEK1/2 are kinases for the ERK1 and
ERK2 mitogen-activated protein kinases (MAPKs). Activated MAPKs translocate to the nucleus [42] whereby they
regulate gene expression by modulating transcriptions factors including those of the Ets family [43,44].
Vande Woude and coworkers found that activated H-Ras
was able to induce tumor growth of NIH 3T3 cells express-
107
ing mutant forms of the oncogene and subcutaneously implanted in nude mice [45]. All effector domain mutants of
activated H-Ras gave rise to tumorigenesis, regardless of
the specific pathway activated, suggesting that tumorigenesis could occur by Raf-dependent as well as Raf-independent
routes. Similar to the results found by others, cell lines derived from tumor explants showed unchanged levels of Ras
expression as compared to the parental cells. However, when
injected into tail vein of the mice, NIH 3T3 cells expressing
the T35S mutant, specific for activation of the Raf pathway,
showed the same occurrence of lung metastases as seen with
H-RasG12V. In contrast, cells expressing effector domain
mutants specific for PI3K or RalGEF downstream of Ras
(Y40C or E37G, respectively) showed no metastatic development up to 14 weeks post-injection. These data indicate
that metastatic growth of Ras-transformed NIH 3T3 cells
occurs through a Raf-dependent mechanism in these mice.
To confirm the participation of Raf effectors in distal tumor initiation, the authors demonstrated that cells expressing ectopic Mos, an activator of MEK [46], or constitutively
activated MEK [47] produced lung metastases as well. Finally, the authors reintroduced the tumor-derived cells as
secondary xenografts in nude mice, and found that all three
effector mutants were capable of producing lung metastases,
although the 12V/37G and 12V/40C mutant cells produced
fewer nodules than 12V/35S. Cells derived from these PI3Kand Ral-specific secondary metastases showed upregulation
of Met, which, as they had already demonstrated, can lead to
increased invasion in vitro [48], and confirming the hypothesis that invasion and metastasis can be driven by oncogenic
Ras through a variety of signaling pathways.
3.2. Phosphoinositide 3 -OH kinase (PI3K) activation of
Akt and Rac
One of the most commonly studied signal transduction routes downstream of oncogenic Ras activation is
PI3K pathway [32,49]. This kinase phosphorylates the signaling molecule phosphatidylinositol 4,5-bisphosphate to
form phosphatidylinositol 3,4,5-triphosphate (PIP3 ). PIP3
can then activate the serine/threonine kinase Akt/PKB.
Ras-dependent stimulation of Akt activation leads to upregulation of the transcription factor NF-␬B [50], and can
increase cell survival, perhaps by blunting apoptotic signals
[49].
With respect to increases in cellular motility, changes in
the stability of cell–cell adhesion and cell–ECM interaction
appear to be at least in part controlled by PI3K signaling.
Upregulation of the kinase activity in MDA-MB 435 breast
carcinoma cells via ␣6␤4 integrin signaling led to an increase in invasion that was Rac dependent [51]. This increase in migration through Matrigel was found to not be
dependent on MAPK (downstream of the Ras-ERK axis) nor
Akt or p70S6kinase (downstream of the Ras-PI3K axis [52]).
That the intracellular portion of ␤4 lacks the YMXM consensus binding site for the p85 regulatory subunit of PI3K
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suggests that an intermediate must exist between the integrin and PI3K [53]. Since the ␤4 cytoplasmic domain harbors Shc motifs which can subsequently recruit Grb2 and
Sos1/2 adaptor proteins, it is tempting to speculate that ␣6␤4
integrin-regulated PI3K-dependent invasion occurs via Ras
activation.
A crucial requirement for tumor cell metastasis is the ability to transit from the primary tumor, via the blood or lymphatic system, to distant sites to initiate secondary tumor
formation. This requires the ability of the tumor cell to escape matrix deprivation-induced apoptosis, or anoikis [54].
The PI3K–Akt cascade has been implicated in this process,
with oncogenic Ras protecting MDCK canine epithelial cells
from suspension-induced programmed apoptosis [55]. This
inhibition of anoikis affords the detached tumor cell the viability to migrate to a conduit by which it can access distal tissues. That Ras signaling is particular for different cell
types is illustrated by the findings of McFall et al., who describe in rat intestinal epithelial cells anoikis that is downstream of oncogenic Ras, but independent of PI3K and RalGEF pathways [56].
PI3K can also activate Rac GEFs (e.g. Sos, Vav) to promote activation of the Rac small GTPase [57,58]. Rac regulation of actin reorganization and membrane ruffling can
promote increased cell motility and contribute to tumor cell
invasion and metastasis [59]. The involvement of Rac and
other Rho family small GTPases in promotion of malignant tumor growth has been summarized in recent reviews
[60,61].
3.3. RalGEF activation of Ral small GTPases
Another signaling molecule downstream of Ras and
within the small GTPase family is Ral [62]. The proteins
RalA and RalB are activated by Ras binding and activation
of RalGEFs (RalGDS, RGL, RGL2/Rlf, RGL3). Recent
studies support the importance of RalGEF signaling in promoting oncogenic Ras induction of anchorage-independent
growth in human cells [63]. This pathway has also been
implicated in progression from neoplasia to adenoma or
carcinoma and then metastasis. Kelly and coworkers found
that ectopic expression oncogenic Ras of many forms could
lead to distal implantation and growth of lung nodules following tail vein injections of transformed NIH 3T3 cells
[64]. They discovered that mutant Ras12V was able to
cause this metastasis. In addition, they found that while
Ras12V/37G, specific for the sole activation of Ral by
RalGEFs [5], could also lead to metastasis, the degree of
secondary tumors was reduced by reversion of the ERK
pathway by the specific phosphatase PAC1. These results
were interesting given that ERK activation is not a downstream effector of Ras12V/37G. Their study examined
the nature of the metastases caused by the oncogenic Ras
signaling, and found that while a variety of mutant Ras
and Raf forms caused distal tumors in the lungs of mice,
those cells expressing Ras12V/37G were more invasive of
lung and neighboring tissue, with less encapsulation of the
tumor. Secondary metastases were also evident, and this
increase in invasiveness was decreased in those cells concurrently expressing PAC1. The model indicates that ERK
activation is necessary for the implantation and progression of lung metastases from transformed cells expressing
activators of Ral, and in vitro studies showed that migration and invasion through Matrigel by these cells was
dependent on ERK activation. Similar in vitro results were
seen with Ras12V/37G-expressing human MCF-10A and
murine NMuMg mammary epithelial cells, indicating that
the invasion caused by the oncogene is not restricted to fibroblasts. The RalGEF–Ral pathway may also be important
for Ras-mediated invasion of bladder carcinoma cells [65].
3.4. Tiam1 activation of Rac and cell motility
Tiam1 was recently determined to be an effector of Ras
[66]. Though the activation of Rac by oncogenic Ras has
been shown to occur via PI3K stimulation, here it was found
that GTP loading of Rac can be produced synergistically by
Tiam1 and activated H-Ras in a PI3K-independent manner.
Originally identified as a T-lymphoma invasion and
metastasis protein (Tiam1), it was discovered to be a
GEF for Rac [67]. T-lymphoma cells expressing constitutively active Rac1V12 become invasive, indicating that the
Tiam1–Rac signaling pathway could be involved in the invasion and metastasis of tumor cells [68]. This hypothesis was
confirmed by the recent studies in Tiam1 knockout mice,
which show decreased Rac activation compared to wild-type
mice, and developed fewer and smaller skin tumors following application of both 7,12-diaminobenzylanthracene,
a known carcinogen that creates mutations in H-Ras, and
12-O-tetradecanoyl-13-phosphate [69]. Interestingly, the
fewer tumors in the Tiam1 mice had a greater propensity
for dermal invasion, even though Rac-GTP content was
decreased.
4. Mediators of oncogenic Ras induction of invasion
and metastasis
4.1. Met
The protooncogene Met is a RTK that is activated by
its ligand hepatocyte growth factor/scatter factor (HGF/SF)
[70]. Met and HGF/SF are overexpressed in metastases, and
aberrant Met–HGF/SF signaling increased motility and invasion of cells in vitro and in vivo in part by augmenting the
activity of urokinase plasminogen activator (uPa) [71]. uPa
is known to be involved in the destruction of ECM/basement
membrane, a necessary event in the migration of cells from
the solid tumor.
Vande Woude and coworkers have demonstrated that
oncogenic H-Ras transforms mouse NIH 3T3 fibroblasts
and C127 epithelial cells, and increases the expression of
P.M. Campbell, C.J. Der / Seminars in Cancer Biology 14 (2004) 105–114
the Met receptor (RNA and protein) [48]. Ras-transformed
C127 cells showed higher migration indices through Matrigel in response to HGF treatment. Later research by
this same group illustrated blockade of mutant Ras-driven
metastasis in vivo by using dominant negative forms of Met
[72].
While a direct link between constitutive Ras and increased
Met protein has not been made, previous work indicated that
Met expression is induced by the transcription factor Ets-1
[73]. Since Ets-1 is one of the transcription factors known
to be downstream of mutant Ras activation [74], this may
be one possible route by which oncogenic Ras expression
triggers the steps of invasion and metastasis.
4.2. Rho GTPases
Key steps in invasion and metastasis include alterations
in cell adhesion, cell–matrix and cell–cell interactions, and
the acquisition of an increased migratory phenotype. These
cellular properties are regulated, in part, by Rho family GTPases and their control of actin organization. The aberrant activities of Rho GTPases (including RhoA, Rac1, and Cdc42)
have been implicated in contributing to a metastatic and invasive phenotype (reviewed in [60,61]). The oncogenic properties of Ras have been shown to be critically dependent on
Rho GTPase function [75]. Consequently, Ras regulation of
Rho GTPase function may contribute to tumor cell invasion
and metastasis.
Like Ras proteins, Rho and Rho-like proteins signal by cycling between GDP and GTP-bound forms. However, unlike
Ras oncogenes, few activating mutations have been found
in Rho GTPases in cancer, and aberrant regulation of expression and/or GTP/GDP-bound ratios appear to be critical
in Rho members’ roles in invasion [76]. There is evidence
that these three Rho members have distinct actions on the
cytoskeleton, and may all play distinct roles in the plasticity
necessary for increased cell motility of migration and invasion [77]. RhoA promotes stress fiber and focal adhesion
formation, Rac stimulates membrane ruffling, and Cdc42 induces actin microspike formation and the induction of filopodia. As described earlier, Ras can cause Rac activation via
PI3K or Tiam1. How Ras regulates RhoA and Cdc42 function is not clearly understood. Possible mechanisms include
Ral regulation of its effector, RalBP1, which acts as a GAP
for Rac and Cdc42 [62], or the ERK MAPK cascade [78].
Like Ras, Rho GTPases also interact with multiple downstream effectors. For RhoA, the effector implicated in promoting invasion is Rho kinase [79].
Rho GTPases can also regulate epithelial cell morphogenesis [80]. Some [81–85] have suggested that in cancer
cells, a feedback loop exists between Rho proteins and PI3K
signaling such that when epithelial–mesenchymal transition
(the critical step toward an invasive and/or metastatic phenotype, for review see [86]) occurs, PI3K can induce the
activation of Rho, Rac and Cdc42, which in turn lead to
the generation of phosphatidylinositol (3,4,5)-trisphosphate
109
(PtdInsP3 ). Finally, Rho GTPases can also regulate changes
in the expression of genes involved in tumor cell invasion.
5. Consequences of oncogenic Ras signaling
5.1. Actin cytoskeleton
Gelsolin is a protein able to disrupt the actin cytoskeleton by cleaving F-actin subunits. It has been proposed that
the upregulation of gelsolin may account for the transition
from benign to invasive cell growth in some but not all tumors [87,88]. Kwiatkowski and coworkers demonstrated a
connection between gelsolin activity and Rac in fibroblast
motility [89], and Gettemans and coworkers demonstrate
that gelsolin activity is affected by the oncogenic Ras pathway [90], and that invasion of gelsolin-expressing MDCK
cells is dependent on Rac activation through a PI3K-, but
not Raf/MEK-dependent pathway.
Experimental evidence indicates that oncogenic Ras
modulates the effects of other signaling pathways. In normal mammary epithelial cells, stimulation of the TGF␤
pathways led to growth inhibition, but in cells transformed with H-Ras, exogenous TGF␤ signaling resulted
in epithelial–mesenchymal transition, including changes in
morphology to a fibroblastoid phenotype [91]. In addition,
these cells became invasive and showed autocrine secretion
of TGF␤ as well as extracellular signaling that induced the
EMT of other cells.
How Ras cooperates with TGF␤ is unclear, but Oft et al.
have shown that in a variety of tumor cell types, oncogenic
Ras requires TGF␤ signaling to affect metastasis or invasion [92]. Raf or PI3K, both downstream of constitutive Ras
activation, may inhibit the growth arrest or apoptosis that
TGF␤ typically triggers in normal cells [93,94].
5.2. Extracellular matrix degradation
Matrix metalloproteinases (MMPs) comprise a family of
at least 20 members that have been implicated in the progression of transformed cells to an invasive phenotype [95].
These proteinases are critical for degrading the ECM to allow tumor cell migration. Regulation of expression of these
enzymes has been shown to be downstream of constitutively active Ras signaling cascades [96–98], and varies with
the particular MMP and/or cell type [95,99]. The transcription factors, AP-1 and Ets-1, effectors of oncogenic Ras via
MAPK pathways, can induce the expression of MMPs, and
in an H-Ras-transformed human embryonic fibroblast line,
Kähäri and coworkers show that transcription of MMP-1
is ERK-dependent [100]. Oncogenic H-Ras-mediated stimulation of the transcription factor NF-␬B simultaneously
increased expression of MMP-9 and decreased expression
of tissue inhibitor of metallomatrix protein 1 (TIMP-1, a
MMP-9 inhibitor) [101].
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In addition, it has been revealed that urokinase-type plasminogen activator (uPa), another matrix degradation protease, is stimulated by Ras via ERK activation [102,103]. At
the same time, induction of uPa receptor expression is downstream of oncogenic H-Ras activation and RhoA activation,
but not by other members of the Rho family (Cdc42 and
Rac1) [104]. The Jones group continued with this research
to show that the H-Ras effects were due to Ral activation
through increased AP-1 dependent transcription [105]. RalA
was also implicated in uPa expression in Ras-transformed
NIH 3T3 cells by another group, who then found increased
activity, but not expression of MMP-2 and MMP-9 [106].
Finally, Ras-transformed cells show diminished expression of reversion-inducing cysteine-rich protein with Kazal
motifs (RECK) [107]. RECK was initially discovered in a
screen of genes that caused morphologic reversion of oncogenic Ras-mediated transformation, and is a glycoprotein
that inhibits MMP secretion and activity, resulting in decreased invasion.
Ras transformation of NIH 3T3 fibroblasts, MCF-10A
human breast epithelial and other cells is associated with
upregulated expression and secretion of the cathepsin B
lysosomal cysteine proteinase [108,109] which may contribute to increased invasiveness and development of the
malignant cell phenotype [110,111].
Thus, mutant Ras family members are able to increase the
expression of ECM proteases and the systems that activate
them to allow basement membrane degradation, and at the
same time, decrease the expression of protease inhibitors.
5.3. Cell adhesion
The transformation of a tumor cell to a metastatic phenotype necessitates changes in cell–cell adhesion [112]. The
stability of these cell interactions is accomplished through a
variety of proteins and structures, including tight junctions,
adherens junctions, and desmosomes [113]. Adherens junctions between neighboring cells are strengthened by cadherins in a calcium-dependent manner [113] and nectins
in a calcium-independent manner [114]. Inhibition of cell
contact-dependent proliferation and migratory signals are
overcome in cancer cells, allowing for invasion beyond the
tumor border and metastasis in other tissues of the body.
Extracellular cues, including cytokines, growth factors, and
ECM proteins, initiate the disruption of cell–cell interactions, and activated Ras family proteins are often the transducers of such signals [113].
Friedman and coworkers found that exogenous mutant KRas, but not H-Ras, could disrupt adhesive qualities and organization of HD6-4 colon epithelial cells, and that this was
due to the oncogene’s ability to interfere with the maturation
of cell surface integrins [115]. Since integrins are thought to
regulate Ras activation via focal adhesion kinase [116], this
represents yet another potential feedback loop in which mutant Ras leads to its own activation. Ras deregulation of Rho
GTPase function, which are important regulators of cell–cell
and cell–substratum, may also cause significant changes in
cellular adhesion [80].
5.4. Angiogenesis
As mentioned earlier, metastatic cells often migrate to secondary sites by way of the vasculature. Ras signaling may
facilitate this migration via stimulation of angiogenesis by
upregulation of vascular endothelial growth factor (VEGF)
[117–119]. Folkman’s group demonstrated that oncogenic
Ras expression in endothelial cells changed their in vivo phenotype from largely benign to highly proliferative and invasive [120]. They indicated that this phenotypic switch was a
result of upregulation of VEGF transcription and MMP activity, mediated through PI3K-dependent pathways, concurrent with decreases in TIMP activity. Al-Mulla et al. similarly demonstrated an increase in VEGF production in Rat1 fibroblasts expressing K-Ras mutants and also noted an
increase in Matrigel invasion of the K-Ras12V mutant as
compared to K-Ras12D [121]. This experimental difference
of K-Ras mutants parallels descriptions of more aggressive
and invasive clinical phenotypes associated with K-Ras12V
[121–123], and further investigation into the mechanisms of
the various oncogenic forms is warranted.
Another of the downstream targets of Ras is the metalloprotease CD13/aminopeptidase (CD13/APN), which is
expressed on angiogenic but not static vascular endothelial
cells [124]. CD13/APN facilitates endothelial migration and
is Ras dependent, through both the PI3K and MEK pathways, with Shapiro and coworkers illustrating that constitutive activation of CD13/APN could overcome Ras effector
blockade and result in capillary network construction from
human umbilical cord endothelial cells plated in Matrigel
[125].
A positive feedback loop between Ras proteins and HER
RTKs has been suggested, as Jorcano and coworkers indicate that in a mouse model of chemically induced skin carcinogenesis, activation of H-Ras induces an increased expression of EGFR, which can then in turn stimulate angiogenesis [126].
6. Conclusions
Much work has been accomplished in discovering the
many facets of oncogenic Ras signaling evident in the
growth transformation of cells. These data have revealed
that the mechanisms downstream of Ras are much more
complex than originally thought. Similar conclusions are
being formed about mutant Ras and its contribution to
increased motility, invasiveness, and metastatic potential.
Clearly, crosstalk and feedback with a multiplicity of signaling networks are in evidence, and the pathways utilized
by oncogenic Ras vary by cell and tumor type. Understanding the variability of this signaling is critical for the future
of target-based therapeutics against metastasis, for it is this
P.M. Campbell, C.J. Der / Seminars in Cancer Biology 14 (2004) 105–114
aspect of cancer that is the most prominent for morbidity
and mortality. Clinical trials are currently underway to assess the efficacy of farnesyltransferase inhibitors (initially
developed to blunt Ras activation) against breast and pancreatic cell metastasis [127,128]. Similarly, pharmacologic
inhibitors of the Raf–ERK cascade are also under clinical
evaluation [129]. It is hoped that by gaining a better understanding of the complexity of oncogenic Ras signaling,
and its role in inducing invasion and metastasis, we will be
better equipped to bring additional therapies to the bedside.
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