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
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
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.
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 αvβ3 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).
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.