(Received for publication, November 21, 1995)
From the
The effects of insulin-like growth factor I (IGF-I) on the
migration of two human breast cancer cell lines, MCF-7 and MDA-231,
were examined using a modified Boyden chamber. 10 ng/ml was the optimal
IGF-I concentration for stimulation of migration. The majority of
IGF-I-stimulated migration in both cell types was due to chemotaxis.
MCF-7 cells failed to migrate on membranes coated with gelatin or
fibronectin and migrated only in small numbers on laminin. In contrast,
when vitronectin- or type IV collagen-coated membranes were used, the
MCF-7 cells migrated in large numbers specifically in response to IGF-I
but not to 10% fetal calf serum, epidermal growth factor, fibroblast
growth factor, or platelet derived growth factor-BB. An IGF-I
receptor-blocking antibody inhibited IGF-I-stimulated migration in both
cell types. In addition, a blocking antibody to the v
5
integrin (a vitronectin receptor) inhibited migration of MCF-7 cells in
response to IGF-I through vitronectin but not through type IV collagen.
Similarly, blocking antibodies specific for
2 and
1 integrins
significantly inhibited migration of both cell types through type IV
collagen-coated membranes but not through vitronectin-coated membranes.
We conclude that: 1) IGF-I stimulates migration of these two cell types
through the IGF-I receptor; 2) interaction of vitronectin with the
v
5 integrin or collagen with the
2
1 integrin is
necessary for the complete IGF-I response in MCF-7 cells, and 3)
because migration represents an in vitro model for metastatic
spread, integrins, extracellular matrix proteins, and IGF-I may play
coordinated roles in the metastasis of breast cancer in vivo.
Insulin-like growth factor I (IGF-I) ()has been shown
to be a mitogen for human breast cancer cells including the MDA-231 and
MCF-7 cultured cell lines(1, 2, 3) . The
increased growth of these cells in the presence of IGF-I requires the
normal function of the IGF-I receptor and can be blocked by a
monoclonal antibody to this receptor,
IR-3(4) . Terranova et al.(5) first reported on the correlation between
malignant cells capable of traversing a porous membrane coated with
extracellular matrix components and the ability of these cells to cause
metastases. Using modified Boyden chambers or monolayer wounding
migration assays, other authors have found that IGF-I can stimulate
migration in human melanoma cells(6) , FG pancreatic carcinoma
cells(7) , arterial smooth muscle
cells(8, 9) , and endothelial cells(10) .
Other chemoattractants, such as interleukin-1 and -6 for breast cancer
cells (11) and epidermal growth factor (EGF) for FG
cells(7) , have also been demonstrated to stimulate cellular
migration in these assays.
The integrins are a superfamily of
heterodimer cell surface glycoprotein receptors composed of distinct
and
subunits(12) . The integrins were originally
described as cell adhesion receptors, but their functions in cell
behavior including motility and invasion and their interactions with
classical growth factor receptor signaling pathways have been
increasingly recognized in the past few years(13) . Although
integrins themselves do not possess tyrosine kinase activity,
activation of integrins by cell adhesion (14) or anti-integrin
antibodies (15) causes tyrosine phosphorylation of p125
and other cytoplasmic proteins. The
v
3 integrin, a
vitronectin receptor, was recently shown to associate with insulin
receptor substrate-1 in fibroblasts after the cells were stimulated
with 100 nM insulin(16) , a dose capable of activating
IGF-I receptors(17) . A connection between EGF stimulation and
the activation of the
v
5 integrin (another vitronectin
receptor) with cell migration has also been described in pancreatic
cancer cells(7) . Our work began with the hypothesis that IGF-I
would stimulate migration of MDA-231 and MCF-7 cells, which we have
demonstrated utilizing microwell Boyden chambers. We have characterized
the conditions required for optimal migration to IGF-I in these two
cell lines. This has led to the conclusion that the integrins expressed
by the cells play a key role in mediating the effect of IGF-I to
stimulate migration.
Figure 1: Migration of MDA-231 and MCF-7 cells: effects of different extracellular matrix proteins. The stimulatory effect of IGF-I (10 ng/ml) on migration of these two cell lines is illustrated. The number of cells indicated is the mean ± S.D. counted per well in multiple assays with either no IGF-I (open bars) or with IGF-I (filled bars) in the lower wells only. The membranes were coated with gelatin (G), fibronectin (Fn), vitronectin (Vn), or type IV collagen (Col). Importantly, the MCF-7 cells migrated only when the membrane was coated with vitronectin or collagen and only when IGF-I was present.
Figure 2:
Immunoprecipitation of breast carcinoma
cell integrins. To determine the presence and identity of integrin
receptors on the surface of the MCF-7 (A) and MDA-231 (B) cells, proteins on the surfaces of intact cells were
labeled with NaI, and the labeled cells were lysed and
immunoprecipitated with specific antibodies directed against integrin
or
subunits, as indicated beneath each lane. The
immunoprecipitates were resolved under nonreducing conditions on 6%
SDS-polyacrylamide gels. Autoradiographs of the dried gels are shown.
The arrows indicate the locations in the gels of the integrin
subunits. Because
and
integrin subunits are tightly
associated until SDS-polyacrylamide gel electrophoresis, an antibody
against a given
or
subunit will precipitate both subunits.
In both cell lines the
2
1 and
3
1 collagen/laminin
receptors are abundant. The abundance of the
V
5
vitronectin-binding integrins is greatest in the MCF-7 cells. No
V
3 integrins were detected in either cell
line.
Figure 3: MDA-231 and MCF-7 cells vary in their responses to chemoattractants. The effect of IGF-I on stimulation of migration through vitronectin-coated membranes was compared with that of other chemoattractants. Each is compared with the response of the cells to IGF-I (10 ng/ml) in the same assay, which is assigned an arbitrary value of 100%. EGF (10 ng/ml) elicited a response similar to that of IGF-I in the MDA-231 cells. The effects of bFGF (20 ng/ml) and PDGF-BB (10 ng/ml) were less pronounced. In contrast, the MCF-7 cells (hatched bars) had a significant response only to IGF-I.
Figure 4: Directional migration of MCF-7 and MDA-231 cells. The graph depicts the effects on MDA-231 (open bars) and MCF-7 (hatched bars) cell migration of adding IGF-I (10 ng/ml) to either the upper chamber, lower chamber, neither chamber, or both chambers and was performed as described under ``Materials and Methods.'' The number of migrated cells when IGF-I was present in the lower well only is assigned a relative value of 100% and is a measure of chemotaxis. Migration in response to the addition of IGF-I to only the upper wells or to both wells is a measure of chemokinesis, stimulation of migration in the absence of a concentration gradient. For both cell lines, IGF-I is more potent in stimulating migration when there is a positive concentration gradient toward which the cells migrate.
Figure 5:
Inhibition of chemotaxis by an IGF-I
receptor antibody. Chemotaxis in response to IGF-I was determined in
the presence and the absence of the IGF-I receptor blocking antibody
-IR3 or control mouse IgG at 25 µg/ml. Taking the total number
of migrated cells (in response to IGF-I, 10 ng/ml) with no added
antibody as 100% migration,
-IR3 reduced migration of MDA-231
cells (open bars) to 36 ± 12%, whereas nonspecific
mouse IgG had a minimal effect. This effect was more pronounced with
the MCF-7 cells (hatched bars), where migration was reduced to
9 ± 5% by
-IR3.
Figure 6:
IGF-I-stimulated migration of MCF-7 cells:
effects of anti-integrin antibodies. IGF-I-stimulated (10 ng/ml)
chemotaxis was determined in the presence and the absence of monoclonal
anti-integrin blocking antibodies (25 µg/ml) against V
3
(clone LM609, Chemicon),
V
5 (clone P1F6, Becton Dickinson),
2 (clone P1E6, Becton Dickinson), and
1 (clone mAb13, Becton
Dickinson) and control nonimmune mouse IgG. Experiments were performed
utilizing membranes coated with either vitronectin (A) or type
IV collagen (B). Migration in the absence of antibodies was
taken as 100%. Through vitronectin-coated membranes (A),
anti-
V
5 inhibited migration by 70 ± 18%, whereas the
other antibodies and IgG had small effects. In contrast, through
collagen-coated membranes, both anti-
2 and anti-
1 had major
effects, reducing migration by 63 ± 5% and 96 ± 6%,
respectively, whereas anti-
V
5 and IgG had minimal
effects.
This is the first report to our knowledge on the effects of IGF-I on the chemotaxis of human breast cancer cells. Although the potency of IGF-I to stimulate mitosis of human breast cancer cells was established over a decade ago(2) , its ability to induce chemotaxis in these cells has not previously been studied. The initial process in the metastatic spread of breast carcinomas involves the invasion of malignant cells through the extracellular matrix of a basement membrane followed by their migration into lymphatic or vascular channels. IGF, synthesized by surrounding stromal cells, has been implicated in the development of tumors from breast cancer cells seeded into nude mice(4) . The modified Boyden chamber assays allow us to examine the cellular processes of invasion and migration and provide a valid in vitro model of the in vivo process of metastatic spread(5) , which is responsible for nearly all of the mortality from breast cancer(23) . In addition, the Boyden chamber assays avoid any confusion of migration with proliferation even in these rapidly proliferating transformed cells by virtue of its 4-h duration. It also allows for precise definition of the extracellular matrix proteins over which the cells migrate.
We selected the MCF-7 and MDA-231 cell lines because their increased growth in response to IGF-I has been well characterized (1, 2, 3) and because they were both isolated from metastatic sites. Other authors have used the MDA-231 cells for study based on their high degree of invasiveness(24) . Our results are in agreement with this, because we found that the MDA-231 traversed every membrane coating used, including gelatin, fibronectin, laminin, vitronectin, and type IV collagen. Furthermore, the MDA-231 cells migrated in the absence of growth factors and demonstrated chemotaxis toward bFGF, EGF, PDGF-BB, and 10% fetal bovine serum as well as toward IGF-I.
In contrast, migration of the MCF-7 cells was found to have more specific requirements. The MCF-7 cells migrated only in response to IGF-I not toward a negative control, 10% fetal bovine serum, or three other growth factors. These other growth factors, EGF, bFGF, and PDGF-BB, have each been shown to be capable of inducing increased migration in other cell types(7, 8, 25) . Furthermore, the MCF-7 cells showed a brisk response to IGF-I only when migrating through membranes coated with vitronectin or type IV collagen. While not causing the complete arrest of MCF-7 cell migration reported by other investigators using laminin-coated glass coverslips(18) , the use of membranes coated with laminin in our assays led to dramatic reductions in cell migration compared with vitronectin or type IV collagen. Furthermore, there was no detectable migration of MCF-7 cells on membranes coated with gelatin or fibronectin. The marked differences in conditions required for migration between these two cell lines may be related to their differences in estrogen receptor status. Studies using estradiol and anti-estrogens to modify the migration response of MCF-7 cells will be required to determine the dependence of this response on estrogen receptor activation. Alternatively, the observed differences in growth factor responses between the two cell lines may be due to the highly undifferentiated state of the MDA-231 cells compared with the MCF-7 line, which retains some epithelial characteristics. The two cell lines also have differences in IGF-binding protein expression(26) . Because IGF-binding proteins can have direct, integrin-mediated effects on cell migration (27) as well as effects on IGF interactions with the IGF-I receptor(17) , further studies into the effects of IGF-binding proteins on IGF-stimulated cell migration are warranted.
The results
of the checkerboard analyses (Fig. 4) established that the
majority of increased migration we observed in response to IGF-I was
due to chemotaxis, directional migration toward a higher concentration
of a solubilized chemoattractant. The finding that a positive
concentration gradient of IGF-I, rather than its mere presence, is
necessary for optimal migration may have important physiologic
implications. IGF synthesis in tumor specimens has been shown to
originate from surrounding stromal cells, suggesting the presence of
IGF concentration gradients in breast carcinomas in vivo. The
blocking antibody to the IGF-I receptor, IR-3, has previously been
shown to be capable of inhibiting the increased growth of the MDA-231
and MCF-7 cells associated with their exposure to IGF-I(28) .
Therefore, it was not surprising that preincubation of the cells with
this antibody would also markedly limit their migratory response to
IGF-I. We performed these experiments to be certain that the chemotaxis
induced by IGF-I was an IGF-I receptor-dependent phenomenon.
It has
recently become clear that the integrins function as true receptors,
transmitting signals to the cell interior upon ligation by
extracellular matrix components(29) . Our results demonstrate
that both integrin receptors and IGF-I receptors are involved in
IGF-I-stimulated migration of MCF-7 cells. The immunoprecipitation
studies (Fig. 2) demonstrated that the MCF-7 cell line has a
proportionately higher amount of the v
5 integrin, a
vitronectin receptor, compared with the MDA-231 cells. The predominant
vitronectin receptor expressed by the MCF-7 cells is the
v
5
integrin, and preincubation of these cells with a blocking antibody to
v
5 markedly reduced their migratory response to IGF-I through
vitronectin-coated membranes (Fig. 6A). As predicted,
cells preincubated with the same anti-
v
5 antibody were not
significantly affected when migrating toward IGF-I through type IV
collagen, confirming the specificity of the requirement for
v
5 for migration on vitronectin (Fig. 6B).
The 2
1 integrin can serve to mediate IGF-I-stimulated
migration when the cells migrate through collagen. The
2
1 and
3
1 integrins are both capable of binding to type IV collagen
but not to vitronectin. The antibody to the
1 subunit, common to
both receptors, produced the most significant inhibition of migration
when assays were conducted with membranes coated with type IV collagen (Fig. 6B), almost eliminating the response to IGF-I. In
addition, the anti-
2 antibody inhibited migration on type IV
collagen by 63%. Neither antibody had a significant effect on the MCF-7
cells migrating through vitronectin (Fig. 6A),
confirming that the requirement for
2
1 (and possibly
3
1) functionality is specific for migration on type IV
collagen in these cells.
We therefore conclude that integrins are activated during the process of IGF-I-stimulated chemotaxis in breast cancer cells. The identity of the integrin that is essential for cell migration is specific to the particular extracellular matrix substance used. This is the first description of this association between IGF-I receptor stimulation and integrin function in breast cancer cells. Breast cancer tissues have been shown to have altered patterns of integrin expression (30) and higher numbers of IGF-I receptors (31) when compared with normal breast tissue. It is likely that integrins are involved in the response of these malignant cells to IGF-I, mediating their ability to traverse basement membranes and metastasize.
The role of IGF-I in the stimulation of migration is an important area of study that heretofore has been relatively neglected compared with the role of IGF-I in cellular growth and proliferation. Although the precise mechanisms by which the IGF-I receptor and integrin receptors interact are not yet known, this area of study warrants continued investigation. If the nature of the cross-talk between these two signaling systems can be elucidated, our understanding of malignant behavior will be greatly enhanced.