Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
THE ability of cancer cells to proliferate in the absence of adhesion to extracellular matrix (ECM)1
proteins, termed anchorage independence of growth,
correlates closely with tumorigenicity in animal models
(14). This property of cancer cells presumably reflects the
tendency of tumor cells to survive and grow in inappropriate locations in vivo. Such incorrect localization, as occurs
in invasion and metastasis, is the characteristic that distinguishes malignant from benign tumors (31).
Great progress has been made in the last 20 years toward understanding how growth is controlled in normal
cells and how oncogenes usurp these controls. Yet studies
on how oncogenes (or loss of tumor suppressors) overcome the mechanisms that govern cellular location have
lagged considerably. The finding that integrins transduce
signals that influence intracellular growth regulatory pathways provided some insight into anchorage dependence.
Available evidence indicates that integrin-dependent signals mediate the growth requirement for cell adhesion to
ECM proteins.
Our understanding of integrin signaling has now reached a
stage that connections to oncogenesis are becoming clear,
enabling us to place a number of proto-oncogenes and oncogenes with respect to their adhesion dependence or independence. While many details of molecular mechanisms remain to be elucidated, sufficient information is now available
to propose a general framework for how oncogenes lead
to anchorage-independent growth.
Integrin Signaling
Integrins transduce a great many signals that impinge
upon growth regulatory pathways (for review see 3, 37, 44). These include activation of tyrosine kinases such as
focal adhesion kinase (FAK), pp60src, and c-Abl; serine-threonine kinases such as MAP kinases, jun kinase (JNK),
and protein kinase C (PKC); intracellular ions such as protons (pH) and calcium; the small GTPase Rho; and lipid
mediators such as phosphoinositides, diacylglycerol, and
arachidonic acid metabolites. Integrin-mediated adhesion
also regulates expression of immediate-early genes such as
c-fos and key cell cycle events such as kinase activity of cyclin-cdk complexes and phosphorylation of the retinoblastoma protein (Rb).
It is striking that extensive investigations into integrin-dependent pathways have revealed no novel signaling
pathways. Integrins appear to regulate the same pathways
that have been identified in studies of oncogenes and
growth factors. Mediators such as c-src, phosphoinositides,
protein kinase C, and so on were well established as participants in cytokine or growth factor-dependent signaling.
Even FAK, p130cas, and paxillin, which localize to focal
adhesions and mediate integrin signaling, connect downstream to known growth factor-regulated pathways such
as phosphatidylinositol (PI) 3-kinase (for FAK) and MAP
kinase (for FAK, paxillin, and p130cas). Thus, integrins and
growth factors regulate the same pathways. This fact then
raises the question of how these pathways are jointly controlled by both cell adhesion to ECM proteins and soluble
factors.
Convergence of Integrin and Growth Factor Pathways
In many if not most instances where the combined effects
of soluble factors and integrins have been examined, synergistic activation has been observed. Cell adhesion has
been shown to greatly enhance autophosphorylation of
the EGF and PDGF receptors in response to their cognate
ligands (10, 23). In cells where growth factor receptor
function is not affected by ECM, activation of PKC via hydrolysis of phosphoinositides depends on cell adhesion (22, 34). Cell adhesion regulates transmission of signals to
MAP kinase by altering the activation of MEK or Raf (20, 30). There is also evidence that activation of PI 3-kinase
and downstream components such as AKT and p70RSK in
response to growth factors depends on cell adhesion (17, 18). Thus, at least three major signaling pathways controlled by growth factors also require cell adhesion (Fig. 1).
In the cases listed above, the combined output from integrins and growth factors is synergistic. Thus, the response to either cell adhesion or growth factors alone is
quite low in most cases, while both stimuli together give a
strong response. These results imply that integrins and
growth factor receptors act upon different points in the
pathway. For example, in the case of inositol lipid hydrolysis, integrins control the synthesis and supply of phosphatidylinositol 4,5-bisphosphate, whereas growth factor receptors control the activity of phospholipase C (22).
Many other instances of synergism have been observed.
Integrin Expression of early cell cycle genes such as c-fos and
c-myc is also stimulated by both cell adhesion and growth
factors (12). Gene expression driven by the fos promoter
shows strongly synergistic activation by integrin-mediated
adhesion and growth factors (41). Later cell cycle events
such as activation of G1 cyclin-cdk complexes and Rb
phosphorylation require both cell adhesion to ECM and
growth factors (for review see 3). There are also numerous
examples in which complex cellular functions such as migration, proliferation, gene expression, or differentiation
require stimulation by both integrin-mediated adhesion
and soluble factors (for review see 1, 6, 13).
Implications for Oncogenes
The pathways in Fig. 1 can be represented in a general way
as shown in Fig. 2 A. This figure displays a basic conceptual framework for considering effects of integrins and
growth factors on cell functions. Because oncogenes are
points on normal growth regulatory pathways that are
constitutively activated by mutation or overexpression, one can make predictions about the effects of oncogenes
on cell growth based on the placement of the corresponding proto-oncogenes with respect to the integrin and
growth factor receptor pathways. For example, constitutive activation of a step after convergence of integrin and
growth factor pathways should bypass the requirements for both adhesion and serum, i.e., it should induce both serum- and anchorage-independent proliferation. Oncogenes
such as Ras, src, or SV40 large T antigen appear to fit this
description.
On the other hand, constitutive activation of a step on
the integrin arm of the pathway before convergence should
give rise to anchorage-independent but serum-dependent
growth. A number of oncogenes have recently been found
to fit this description. Activation of Rho leads to anchorage-independent but serum-dependent growth (38), consistent with results suggesting that Rho mediates integrin-dependent signaling (4, 8, 29). The Rho family protein
Cdc42 gives similar effects (26), and recent work suggests that Cdc42 is activated by integrins and plays an important
role in cell spreading and cytoskeletal organization (Price,
L., J. Leng, M. Schwartz, and G. Bokoch, manuscript submitted for publication; Clark, E., W. King, J. Brugge, M. Symons, and R. Hynes, manuscript in preparation). An
activated variant of FAK was also shown to induce anchorage-dependent survival and growth of MDCK cells
without altering their dependence on serum (15). Overexpression of the 70-kD integrin-linked kinase, a protein that
was found to bind directly to integrin cytoplasmic domains,
also induces anchorage-independent but serum-dependent
growth (27).
The Abl tyrosine kinase provides a particularly interesting example. Chronic myelogenous leukemia (CML) is
caused by the Philadelphia chromosomal translocation,
which fuses Bcr to the NH2 terminus of c-Abl to produce
the Bcr-Abl oncogene (for review see 40). CML cells exit
the bone marrow and enter the circulation prematurely, where they proliferate excessively. Thus the behavior of
CML cells is reminiscent of anchorage-independent growth
in vitro. Indeed, expression of Bcr-Abl in 3T3 cells induced
anchorage-independent but serum-dependent growth (28).
Consistent with these results, c-Abl localization and tyrosine kinase activity are regulated by integrin-mediated
cell adhesion (19). Thus, at least some of the behavior of
Bcr-Abl can be understood as constitutive activation of
c-Abl's adhesion-dependent functions.
Conversely, one would predict that constitutive stimulation of growth factor pathways would be mitogenic but not
necessarily oncogenic. Such mutations might give rise to
benign tumors, where cells show accelerated growth but
their structure and behavior remain relatively normal (31).
Production of autocrine growth factors is a prime candidate for effects of this sort. In support of this model, ectopic expression of growth factors in vivo induces benign
hyperplasia in several animal models (7, 21, 33); in some
cases neoplasia results but occurs at a later stage and arises
focally, indicating a requirement for additional mutations (33). In vitro, autocrine growth factor expression can also be associated with accelerated or serum-independent growth
of otherwise normal cells (24). Circumstances where autocrine expression of growth factors lead to anchorage independence are discussed below.
The Plot Thickens
The conceptual scheme shown in Fig. 2 is simple, but signaling pathways may not be. The proto-oncogene c-src, for
example, associates both with growth factor receptors and
with FAK (2, 9, 43). Oncogenic variants of src induce tyrosine phosphorylation both of focal adhesion proteins
and proteins involved in growth factor receptor signaling
(16). Thus, a multifunctional tyrosine kinase like src might
phosphorylate substrates that independently induce anchorage and serum independence.
Second, incorrect targeting or compartmentalization of
a signaling protein may result in novel functions that do
not occur under normal conditions. For example, attachment of a membrane localization sequence to c-Abl (as in
v-Abl or mutant forms of Bcr-Abl) creates a much more potent oncogene that strongly induces both anchorage- and
serum-independent growth (11, 28), most likely by phosphorylating substrates that are normally inaccessible.
Third, very strong activation of a pathway may overcome a partial blockade. For example, loss of integrin-
mediated adhesion inhibits the activation of MAP kinase
by serum or active forms of Ras or Raf by 75-90% (20,
30). However, oncogenic Ras or Raf activate the pathway
two to three times more strongly than serum. Hence, ERK
activation in suspended Ras- or Raf-transformed cells is
~40% of that obtained in adherent cells treated with serum. These oncogenes can therefore induce a significant
degree of anchorage independence. It should be noted,
however, that the rate of growth of suspended transformed
cells is still much slower than when they are adherent.
An obvious question stemming from this model is why
do oncogenes that derive from growth factors or receptors
sometimes induce complete transformation of fibroblast cell
lines. For example, expression of v-sis, which codes for
PDGF-B, promotes growth in soft agar, even though addition
of PDGF to the medium does not (25, 42). This question
may be resolved by the observation that v-sis stimulation
of the PDGF receptor in an intracellular compartment is
crucial to its transforming activity (5). This result makes
the prediction that intracellular receptors might evade some
of the adhesion-dependent controls discussed above, thereby
enabling v-sis to stimulate growth of nonadherent cells.
Summary and Conclusions
Some of the earliest experiments identifying signals from
integrins showed that oncogenes were able to activate
these pathways in suspended cells (16, 35, 36). Thus, anchorage-independent growth of tumor cells could be seen
as a consequence of anchorage-independent activation of
specific pathways. Recent advances have shown that this
view is basically correct but have considerably enriched our understanding. Integrins and growth factor receptors
regulate the same pathways, in many instances in such a
way that ligation of both is required for activation of
downstream events. Oncogenes that constitutively activate
integrin-dependent events before convergence with growth
factor pathways should induce anchorage-independent growth without affecting serum dependence. Conversely,
activation of growth factor-dependent events before convergence should induce accelerated proliferation without
causing anchorage independence. And constitutive activation of events after convergence should result in both anchorage and serum independence.
These predictions have been tested in several instances.
Rho, FAK, Cdc42, ILK, and c-Abl have been implicated
in integrin signaling, and activation or overexpression of
these proteins induces anchorage-independent but serum-dependent growth. Activation of MAP kinase, PI 3-kinase,
expression from the c-fos promoter, kinase activity of cyclin D- and cyclin E-cdk complexes, and Rb phosphorylation all depend on both adhesion and growth factors, and
oncogenes such as v-fos, v-Ras , v-src, and SV40 large T
that constitutively activate these pathways induce both anchorage and serum independence. That these oncogenes
are potent transforming agents may be due in part to their
ability to overcome cellular requirements for both anchorage and growth factors.
The majority of deaths from cancer are due not to primary tumors but to secondary tumors that arise via invasion and metastasis. The ability of tumor cells to survive
and grow in inappropriate environments therefore lies
very much at the core of the problem. This behavior is reflected in vitro by anchorage-independent growth. Constitutive activation of integrin-dependent signaling events by
oncogenes provides a molecular explanation for the link
between growth and adhesion.
Fig. 1.
Convergence of integrin and growth factor receptor
pathways. Integrin-mediated adhesion regulates transmission of
growth factor receptor signals in at least four steps. These steps
are: autophosphorylation and activation of the receptors themselves; hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2)
to produce diacylglycerol and inositol triphosphate, leading to activation of protein kinase C (PKC); activation of Raf and/or
MEK in the MAP kinase pathway; and activation of PI 3-kinase,
leading to activation of p70RSK and Akt protein kinases.
[View Larger Version of this Image (19K GIF file)]
v
3 coprecipitates with IRS-1 after stimulation
with insulin, and though the mechanism of the cooperation
is unclear, this coprecipitation correlates with enhanced
mitogenesis in response to insulin (39). Leukocyte activation in response to cytokines and antigens is also enhanced
by cell adhesion, and in several cases, cell activation correlates with synergistic effects on protein tyrosine phosphorylation (for review see 32).
Fig. 2.
Oncogenes and signaling pathways. (A) General
scheme for convergence of integrin and growth factor-dependent
pathways in which both are required for activation of gene expression and cell growth. (B) Constitutive activation is indicated
by the boldface arrow. Activation of a step on the integrin arm of
a pathway should lead to anchorage-independent growth, as illustrated by the behavior of Rho, Bcr-Abl, and ILK. Activation of a
step on the growth factor receptor arm of the pathway should
lead to serum independence. Activation of a step after convergence should induce both anchorage- and serum-independent
growth.
[View Larger Version of this Image (21K GIF file)]
Received for publication 25 June 1997 and in revised form 13 August 1997.
Address all correspondence to Martin A. Schwartz, Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-7140. Fax: (619) 784-7360. E-mail: schwartz{at}scripps.eduThis work was supported by National Institutes of Health grants RO1 GM47214 and PO1 HL48728.
CML, chronic myelongenous leukemia; ECM, extracellular matrix; FAK, focal adhesion kinase; PI, phosphatidylinositol; Rb, retinoblastoma protein.
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