Integrin β4 signaling promotes tumor angiogenesis

Article (PDF Available)inCancer Cell 6(5):471-83 · December 2004with72 Reads
DOI: 10.1016/j.ccr.2004.09.029 · Source: PubMed
Abstract
Mice carrying a targeted deletion of the signaling portion of the integrin beta4 subunit display drastically reduced angiogenesis in response to bFGF in the Matrigel plug assay and to hypoxia in the retinal neovascularization model. Molecular cytology indicates that alpha6beta4 signaling promotes branching of beta4+ medium- and small-size vessels into beta4- microvessels without exerting a direct effect on endothelial cell proliferation or survival. Signaling studies reveal that alpha6beta4 signaling induces endothelial cell migration and invasion by promoting nuclear translocation of P-ERK and NF-kappaB. Upon subcutaneous implantation of various cancer cells, the mutant mice develop smaller and significantly less vascularized tumors than wild-type controls. These results provide genetic evidence that alpha6beta4 signaling promotes the onset of the invasive phase of pathological angiogenesis and hence identify a novel target for antiangiogenic therapy.

Figures

Figure
Figure
Figure

Full-text (PDF)

Available from: Sotiris Nikolopoulos, Jan 15, 2015
ARTICLE
Integrin 4 signaling promotes tumor angiogenesis
Sotiris N. Nikolopoulos,
1
Pamela Blaikie,
1
Toshiaki Yoshioka, Wenjun Guo, and Filippo G. Giancotti*
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
1
These authors contributed equally to this work.
*Correspondence: f-giancotti@ski.mskcc.org
Summary
Mice carrying a targeted deletion of the signaling portion of the integrin 4 subunit display drastically reduced angiogenesis
in response to bFGF in the Matrigel plug assay and to hypoxia in the retinal neovascularization model. Molecular cytology
indicates that 64 signaling promotes branching of 4
medium- and small-size vessels into 4
microvessels without
exerting a direct effect on endothelial cell proliferation or survival. Signaling studies reveal that 64 signaling induces
endothelial cell migration and invasion by promoting nuclear translocation of P-ERK and NF-B. Upon subcutaneous
implantation of various cancer cells, the mutant mice develop smaller and significantly less vascularized tumors than wild-
type controls. These results provide genetic evidence that 64 signaling promotes the onset of the invasive phase of
pathological angiogenesis and hence identify a novel target for antiangiogenic therapy.
Introduction the last phase of the process, the endothelial cells acquire a
quiescent, differentiated phenotype: they deposit a basement
The possibility of ameliorating or even suppressing the progres-
membrane and acquire polarity, coincident with the formation
sion of cancer with antiangiogenic drugs has attracted vivid
of a lumen. Pericytes and smooth muscle cells are finally re-
interest (Hanahan and Folkman, 1996). Studies on transgenic
cruited to ensheathe the newly formed vessels. These steps are
mouse models of multistage carcinogenesis have revealed the
repeated in an iterative manner, as mature vessels become
existence of a discrete angiogenic step. In RIP-Tag and K14-
locally destabilized and groups of endothelial cells reacquire an
HPV16 mice, which develop islet cell and epidermal squamous
invasive phenotype to generate a new vascular branch (reviewed
cell carcinoma, respectively (Arbeit et al., 1994; Hanahan, 1985),
in Risau, 1997).
enhanced angiogenesis precedes the transition from carcinoma
Multiple integrins are likely to contribute to tumor angiogen-
in situ to invasive carcinoma (Arbeit et al., 1996; Folkman et al.,
esis. The integrins mediate adhesion to the extracellular matrix
1989), and mutations that impair angiogenesis inhibit disease
and regulate cell survival, proliferation, and migration (Giancotti
progression (Bergers et al., 2000; Coussens et al., 2000; Inoue
and Ruoslahti, 1999; Miranti and Brugge, 2002). Known angio-
et al., 2002). Thus, the angiogenic step precedes and is poten-
genic factors, such as bFGF and VEGF, enhance the expression
and activity of endothelial integrins (Byzova et al., 2000; Kleintially rate limiting for tumor invasion and growth. Small meta-
static lesions co-opt existing host vessels rather than eliciting et al., 1993), whereas negative regulators of angiogenesis, such
as class 3 semaphorins, promote vascular remodeling by inhib-angiogenesis, but these vessels eventually regress, and subse-
quent tumor expansion requires robust neoangiogenesis (Ho- iting integrin function (Serini et al., 2003). Studies with adhesion
blocking reagents and knockout mice have implicated 51lash et al., 1999). These observations suggest that angiogenesis
is required both during initial tumor invasion and growth and and v integrins in angiogenesis (Eliceiri and Cheresh, 1999;
Hynes, 2002). However, the mechanisms by which these andduring metastatic spread.
Tumor cells elicit angiogenesis through both enhanced pro- possibly other integrins function in angiogenesis are not clear
(Sheppard, 2002). In addition to playing an adhesive role, theduction of proangiogenic factors, generally VEGF and bFGF,
and decreased generation of angiogenesis inhibitors (Hanahan integrins may play a signaling role during tumor angiogenesis.
Integrin-specific signals impart a stringent control to the actionand Folkman, 1996). As a result, host vessels in the vicinity of
the tumor are destabilized, and specific endothelial cells acquire of receptor tyrosine kinases (RTKs), determining whether cells
proliferate or undergo growth arrest, migrate or remain station-an invasive phenotype. Upon detaching from adjacent cells and
penetrating the underlying basement membrane, these cells ary, and live or undergo apoptosis when adhering to a specific
matrix (Giancotti and Tarone, 2003). Hence, integrin signals canproliferate and migrate as cords in the interstitial matrix. During
SIGNIFICANCE
To analyze the physiological role of 64 signaling in the absence of the potentially confounding effect of loss of adhesion, we have
generated mice carrying a targeted deletion of the C-terminal signaling portion of the integrin 4 subunit. Our analysis of these
mice provides genetic evidence that 64 signaling controls pathological angiogenesis by promoting the acquisition of an invasive
phenotype by angiogenic endothelial cells. Since it is known that 64 signaling also promotes carcinoma cell invasion, its inhibition
may be especially beneficial for cancer therapy.
CANCER CELL : NOVEMBER 2004 · VOL. 6 · COPYRIGHT 2004 CELL PRESS 471
ARTICLE
potentially affect various phases of angiogenesis. In accordance such a mutation in mice. To construct the vector, we cloned
the sequences encoding the cytoplasmic domain of 4uptowith this hypothesis, studies on signaling molecules that func-
tion downstream of integrins and RTKs, such as focal adhesion amino acid 1355, a stop codon, a SV40 polyadenylation signal,
and a neomycin resistance gene, immediately downstream ofkinase (FAK), Src, Shc, and ILK, have documented a general
role for joint integrin-RTK signaling in angiogenesis (Hood et the exon encoding the transmembrane segment of the protein
(Figure 1A). Southern blotting and PCR analysis indicated suc-al., 2003; Lai and Pawson, 2000; Tan et al., 2004).
The 64 integrin—a receptor for laminin-5—has been stud- cessful introduction of the mutation in mice (Figures 1B and
1C). Analysis of the intercrosses between heterozygous miceied predominantly in the context of epithelial and tumor biology
studies. 64 signaling proceeds through Src family kinase- carrying the targeted deletion revealed that the mutation was
transmitted with the expected Mendelian frequency. Both ho-mediated phosphorylation of the large cytoplasmic tail of 4,
recruitment of Shc, and activation of Ras (Dans et al., 2001; mozygous and heterozygous 4 mutant mice were found to be
viable and fertile and to not manifest skin fragility. HistologicalGagnoux-Palacios et al., 2003; Mainiero et al., 1995) and PI-
3K (Shaw et al., 1997). In stratified and transitional epithelia, analysis of the skin did not reveal any defect in epidermal adhe-
sion to the basement membrane (data not shown). Thus, dele-64 mediates, upon cessation of signaling, assembly of hemi-
desmosomes (Dans et al., 2001; Murgia et al., 1998; Spinardi tion of the signaling domain of 4 has no obvious effect on
embryonic and postnatal development.et al., 1993). Activation of the EGF-R and Ron RTKs enhances
phosphorylation of 4, causing disruption of hemidesmosomes Immunoprecipitation and FACS analysis on primary kera-
tinocytes from wild-type and mutant mice indicated that the 4-and increased epithelial cell migration (Dans et al., 2001; Santoro
et al., 2003; Trusolino et al., 2001), suggesting that these RTKs 1355T subunit associates with 6 and is expressed at the cell
surface as well as wild-type 4 (Figures 1D and 1E). To test thedecrease the ability of 64 to mediate stable adhesion but
increase its signaling function. Deregulation of 64-RTK cosig- adhesive ability of the mutant integrin, wild-type and mutant
keratinocytes were plated on laminin-5 at 4C. At this tempera-naling contributes to carcinoma invasion and growth (Gambaletta
et al., 2000; Trusolino et al., 2001). Although it is conceivable ture, the function of 31, which also binds to laminin-5, is
inactivated, and adhesion proceeds only through 64 (Gag-that similar mechanisms underlie the invasive phase of angio-
genesis, the observation that 4 null embryos do not display noux-Palacios et al., 2003; Xia et al., 1996). The mutant keratino-
cytes attached to laminin-5 at 4C as efficiently as wild-typedefective vasculogenesis or developmental angiogenesis (Dow-
ling et al., 1996; van der Neut et al., 1996) has discouraged an keratinocytes, suggesting that the mutant integrin retains intact
ligand binding capacity (Figure 1F). In accordance with the ab-examination of the role of 64 during angiogenesis.
In this study, we have used a genetic approach to examine sence of a skin fragility phenotype, transmission electron mi-
croscopy (EM) revealed that the skin of mutant mice containedthe role of 64 signaling in postnatal angiogenesis. Prior stud-
ies had shown that mice carrying a targeted deletion of the well-structured hemidesmosomes (C. Puri, C. Tacchetti, and
F.G.G., unpublished data). Thus, deletion of the C-terminal sig-entire cytoplasmic domain of 4 lack hemidesmosomes and,
like 4 null mice, die at birth due to extensive blistering of the naling domain of 4 does not affect the ability of 64 to estab-
lish a transmembrane connection between laminin-5 and theskin and upper gastrointestinal tract (Murgia et al., 1998). To
analyze the role of 64 signaling in the absence of the effect hemidesmosomal cytoskeleton and to mediate stable epidermal
adhesion in vivo.of loss of adhesion strengthening, we have generated mice
carrying a deletion of the C-terminal signaling segment of the To examine the effect of deletion of the 4 substrate domain
on signaling, primary keratinocytes isolated from wild-type and4 tail. These mice are viable and fertile and do not display
signs of epidermal fragility. Through an analysis of these mice, mutant mice were plated on laminin-5 or, as a control, on colla-
gen I in the presence of serum and subjected to immunoblottingwe provide evidence that 64 signaling promotes pathological
and tumor angiogenesis. with anti-phospho-ERK and anti-phospho-AKT antibodies. As
shown in Figure 1G, adhesion to laminin-5 induced significant
phosphorylation of ERK in wild-type but not in mutant keratino-Results
cytes, whereas adhesion to collagen I caused similarly high
activation of ERK in both types of cells. This result is consistentTargeted deletion of the integrin 4 substrate domain
impairs signaling to ERK and AKT with the role of the 4 substrate domain recruitment of Shc and
activation of Ras to ERK signaling (Dans et al., 2001; MainieroTwo developments made it possible to address the role of 4
signaling in postnatal life in the absence of potentially confound- et al., 1997). In addition, adhesion to laminin-5 led to significant
phosphorylation of AKT in wild-type keratinocytes, but it in-ing effects of loss of adhesion. First, it became clear that the
N-terminal part of the cytoplasmic domain of 4 to amino acid duced a much more limited effect in mutant keratinocytes (Fig-
1355 is sufficient for interaction with the plakin HD-1/plectin
ure 1G), in agreement with the hypothesis that the 4 substrate
and hence for association with the keratin cytoskeleton (Schaap-
domain activates PI-3K to AKT signaling (Shaw et al., 1997).
veld et al., 1998). Second, mapping studies revealed that the
We concluded that targeted deletion of the C-terminal segment
five major tyrosine phosphorylation sites of 4, including those
of the 4 tail impairs 64-dependent signaling through ERK
involved in the recruitment of Shc and PI-3K, are located in the
and AKT, but it does not affect adhesion to laminin-5 and assem-
C-terminal portion of the 4 tail, downstream of amino acid
bly of hemidesmosomes.
1355 (Dans et al., 2001). We thus reasoned that a deletion of
the C-terminal portion of the 4 cytoplasmic domain (henceforth 64 and its ligand, laminin-5, are expressed
in tumor vasculaturereferred to as “substrate domain”) would suppress 64 signal-
ing without interfering with adhesion strengthening. The mutant mice did not display any macroscopic defect sug-
gestive of defective cardiovascular development, indicating thatWe used homologous recombination in ES cells to introduce
472 CANCER CELL : NOVEMBER 2004
ARTICLE
Figure 1. Targeted deletion of the 4 substrate domain
A: Replacement vector, wild-type locus, and mutant locus are shown above. Solid boxes, exons; TM, exon encoding the transmembrane segment; open
boxes, cDNA sequences; solid asterisk, stop codon; polyA, SV40 polyadenylation signal; neo, neomycin resistance cassette; TK, thymidine kinase; E, EcoRI;
N, NcoI; p5 and p3, probes for Southern blotting. Wild-type protein (4 WT) and truncated mutant (4 1355T) are shown below. White boxes, fibronectin
type-III repeats; open asterisks, tyrosine phosphorylation sites.
B: Southern blotting on genomic DNA from wild-type (/), homozygous (/), and heterozygous mutant (/) mice. Samples were digested with NcoI
and probed with a 500 bp radioactive cDNA probe complementary to sequences in the extracellular domain of 4.
C: PCR analysis on intercrosses between heterozygous mutant (/) mice. The 0.7 kb band originates from the homozygous mutant allele, and the 0.3 kb
band originates from the wild-type allele.
D: Wild-type (/) and homozygous mutant (/) keratinocytes were immunoprecipitated with the anti-6 mAb GoH3 and probed with rabbit anti-4-
exo. Equal amounts of total lysates were directly probed with anti-4-exo. Arrows point to the expected electrophoretic mobilities of wild-type and mu-
tant 4.
E: Wild-type and mutant keratinocytes were subjected to FACS analysis with mAb 346-11A, which binds to the extracellular domain of mouse 4.
F: Wild-type (WT) and mutant (1355T) keratinocytes were plated for 1 hr on microtiter plates coated with the indicated amounts of laminin-5 at 4C. Cell
adhesion to fibronectin at 4C was negligible (data not shown).
G: Wild-type (WT) and mutant (1355T) keratinocytes were deprived of growth factors, detached, and plated for 2 hr on laminin-5 (Ln-5) or collagen I (Col I)
(top), or they were kept in suspension (S) or plated on laminin-5 for the indicated minutes (bottom). Equal amounts of total proteins were probed with
antibodies to activated ERK (p-ERK) and keratin-5 (top) or to activated AKT (p-AKT) and vinculin (bottom).
64 signaling does not play an essential role during embryonic al., 2003). To examine the potential role of 64 in tumor angio-
genesis, we first studied the expression of 64 in paraffin-vasculogenesis and angiogenesis. This conclusion is consistent
with the observation that 64 is expressed in blood vessels embedded sections of human papillary thyroid carcinoma,
breast adenocarcinoma, prostate carcinoma, and glioblastomaonly after completion of developmental angiogenesis (Hiran et
CANCER CELL : NOVEMBER 2004 473
ARTICLE
Figure 2. Expression of 64 in tumor vasculature
A: Consecutive paraffin-embedded sections of
the indicated human tumors were subjected to
immunohistochemistry with goat anti-4 and rat
anti-PECAM-1 or stained with anti-4 alone.
Scale bar, 10 m.
B: Frozen sections of B16F0 melanoma tumors
from wild-type mice were doubly stained with
rat anti-4 (green) and goat anti-PECAM-1,
mouse anti-smooth muscle -actin, or rabbit
anti-Laminin-5 (red) (far left and two center col-
umns) or doubly stained with goat anti-PECAM-1
(green) and rabbit anti-Laminin-5 (red) (far right
column). In the far left column, the arrows point
to 4-positive vessels, and the double arrowhead
points to a presumptive lymphatic vessel. In the
center left column, the arrows point to 4-posi-
tive vessels ensheathed by mural cells, and the
arrowheads point to 4-positive vessels lacking
mural cells. Asterisks indicate 4
peripheral
nerves. Scale bar, 40 m.
multiforme. Significant levels of 64 were detected in medium- 1993) and the increasing evidence that tumors, including mela-
noma, are innervated (Seifert and Spitznas, 2002). Notably, theand small-size vessels in all these tumors (Figure 2A). Since
tumor cells in breast and prostate cancer samples expressed anti-4 antibodies also stained vessel-like structures that re-
acted with anti-PECAM-1 very weakly (Figure 2B, double arrow-high levels of 64, these samples were subjected to anti-
PECAM-1 staining to unequivocally identify tumor vessels (Fig- head). These structures reacted with antibodies to the lymphatic
endothelial hyaluronan receptor (LYVE-1) (data not shown), sug-ure 2A).
To further characterize the expression of 64 during tumor gesting that 64 is also expressed in tumor lymphatics.
To examine if the expression of 4 in endothelial cells corre-angiogenesis, we examined frozen sections of B16F0 melanoma
xenografts. Double staining with antibodies to 4 and PECAM-1 lated with the presence of vascular smooth muscle cells, we
subjected the tumor sections to double staining with antibodiesshowed that 64 is expressed in these tumors in medium-
(arrows) and small-size vessels, but not in microvessels (Figure to 4 and to smooth muscle -actin. As shown in Figure 2B,
approximately half of the 4
vessels were found to be en-2B). The anti-4 antibodies also reacted with structures resem-
bling peripheral nerves (Figure 2B, asterisks). Double staining sheathed by smooth muscle cells (arrows), whereas the remain-
der were not (arrowheads), suggesting that endothelial cells dowith antibodies to 4 and to the neurofilament protein S-100
confirmed the identification of these structures as peripheral not express 4 in response to a signal generated by mural cells.
Significant amounts of laminin-5 were detected in the basementnerves (data not shown). This observation is consistent with the
known expression of 64 in Schwann cells (Einheber et al., membrane of both 4
medium- and small-size vessels and
474 CANCER CELL : NOVEMBER 2004
ARTICLE
Figure 3. The 4 substrate domain promotes an-
giogenesis in response to bFGF in the Matrigel
plug assay and to hypoxia in the retinal neovas-
cularization model
A: Wild-type (WT) and mutant (1355T) mice were
injected s.c. with Matrigel containing PBS or
bFGF. After 7 days, the plugs were removed and
photographed. The picture shows representa-
tive bFGF-containing plugs excised from wild-
type and mutant mice.
B: Confocal images of representative bFGF-con-
taining plugs excised from FITC-Lectin-injected
wild-type and mutant mice.
C: bFGF-containing plugs from FITC-Lectin-
injected wild-type and mutant mice were lysed
and subjected to fluorimetry. The graph shows
the mean (SD) from three experiments (*p
0.003).
D: PBS- and bFGF-containing plugs from wild-
type and mutant mice were lysed and subjected
to immunoprecipitation and immunoblotting
with anti-VEGF-R. Scale bar, 100 m.
E: P7 wild-type (WT) and mutant (1355T) mice
were exposed to hyperoxia and returned to nor-
moxic conditions. Eye cross-sections were stained
with anti-PECAM-1 and counterstained with he-
matoxylin. Scale bar, 50 m.
F: Quantification of vascular glomeruli abutting
the limiting membrane in wild-type (WT) and mu-
tant (1355T) retinas (n 5 mice per genotype)
(*p 0.004).
4
microvessels, suggesting the existence of another laminin-5 than that in wild-type plugs. The medium-size vessels penetrat-
ing into these plugs generated significantly fewer branches thanbinding integrin in these smaller vessels. In fact, the staining
patterns generated by anti-laminin-5 and anti-PECAM-1 anti- expected, and these secondary branches only occasionally
formed tertiary ramifications (Figure 3B). Fluorimetry indicatedbodies were virtually identical (Figure 2B). Antibodies to 6 dec-
orated all PECAM-1
vessels, irrespective of 4 expression, that the mutant plugs had incorporated approximately 5-fold
less FITC-Lectin than wild-type controls (Figure 3C). In addition,indicating that the 4
microvessels express 61 (data not
shown). It is possible that 61 or another laminin binding integ- immunoblotting showed that the mutant plugs contained a
much smaller amount of VEGF-R and, by inference, of angio-rin, such as 11, mediates endothelial cell adhesion to lami-
nin-5 in microvessels. These results indicate that the endothelial genic endothelial cells than wild-type plugs (Figure 3D). These
observations indicate that loss of 4 signaling impairs bFGF-cells of tumor vessels deposit and organize a laminin-5-rich
basement membrane and, as they mature, attach to it through induced angiogenesis to a significant extent.
We examined if 64 signaling is required for angiogenesis64.
in the retinal neovascularization model. In this model, angiogen-
esis is driven by hypoxia-induced production of VEGF (ShweikiThe 4 substrate domain promotes
bFGF- and VEGF-mediated angiogenesis et al., 1992). P7 mice were maintained in 75% oxygen for 5
days to induce central avascularization in the retina and thenTo examine if 64 signaling plays a role in bFGF-induced
angiogenesis, Matrigel plugs containing bFGF were implanted returned to normoxic conditions for 5 additional days. Histologi-
cal analysis indicated that numerous vascular glomeruli pene-in wild-type and mutant mice and recovered 7 days later. Macro-
scopic analysis revealed that the plugs from mutant mice were trated the inner limiting membrane and abutted in the vitreous
in wild-type mice, whereas the development of these abnormalmuch paler than those from control mice (Figure 3A). To visualize
the development of vascular ramifications in the plugs, the mice vessels was significantly blunted in mutant mice (Figure 3F).
Quantification of the results confirmed that mutant mice havewere injected with an endothelial-specific FITC-labeled Lectin
prior to euthanasia. Confocal analysis indicated that the vascular a significantly reduced angiogenic response to retinal hypoxia
(Figure 3G). Taken together, these results indicate that 64tree was in mutant plugs much less developed and complex
CANCER CELL : NOVEMBER 2004 475
ARTICLE
signaling promotes both bFGF- and VEGF-induced angiogen-
esis.
64 signaling is not required for endothelial
proliferation or survival
To examine the cellular mechanism by which 64 signaling
regulates angiogenesis, we conducted immunohistochemical
studies on Matrigel plugs from wild-type and mutant mice. Anti-
PECAM-1 staining of frozen sections showed that the angio-
genic vessels of mutant mice penetrated significantly less into
the bFGF-containing Matrigel plugs than those of wild-type mice
(Figure 4A). The wild-type plugs contained two types of vessels:
small-size vessels, which were detected predominantly at the
periphery of the plug, and microvessels, which penetrated inside
the plug. By contrast, the mutant plugs contained almost exclu-
sively peripheral small-size vessels, and these were somewhat
reduced in number as compared to those of wild-type plugs.
While the endothelial cells of small-size vessels expressed 64,
those of microvessels did not express the integrin (Figure 4A).
These observations suggest that deletion of the 4 substrate
domain interferes with the sprouting of 4
small-size vessels
into 4
microvessels.
We next evaluated endothelial cell survival and proliferation
in Matrigel plugs from wild-type and mutant mice. Anti-BrdU
staining revealed that the number of endothelial cells in S phase
was significantly reduced in the plugs from mutant mice. How-
ever, the number of BrdU
nuclei per PECAM-1
vessel was
similar in wild-type and mutant plugs, suggesting that the overall
reduction of BrdU staining in the plugs of mutant mice was
secondary to reduced sprouting, and it was not due to an intrin-
sic proliferative defect (Figure 4B). In addition, the small-size
vessels, which express 4, displayed very few BrdU
nuclei as
compared to the smaller PECAM-1
4
capillaries, indicating
that 64 is expressed in quiescent vessels (Figure 4C). These
observations suggest that signaling by the 4 substrate domain
is not required for endothelial proliferation during angiogenesis.
TUNEL staining did not reveal endothelial cell apoptosis in either
wild-type or mutant plugs (data not shown), suggesting that
4 signaling is not required for endothelial cell survival during
angiogenesis. Together with the pattern of expression of 64
Figure 4. The 4 substrate domain promotes branching of quiescent small-
during angiogenesis, these results suggest that 64 signaling
size vessels into proliferative microvessels without exerting a direct effect
promotes the onset of the invasive phase of angiogenesis. These
on endothelial cell proliferation
observations are consistent with the hypothesis that 64 func-
A: Sections of bFGF-containing plugs from wild-type and mutant mice were
tions at a step of angiogenesis that precedes overt endothelial
stained with rat anti-PECAM-1 (left panels). Sections of bFGF-containing
plugs from wild-type mice were subjected to double staining with goat anti-
cell proliferation and migration in the interstitial matrix.
PECAM-1 (green) and rat anti-4 (red) (right panels). The margins of the
plugs are marked with a dotted white line. The endothelial cells of small
The 4 substrate domain promotes endothelial
vessels express 64 while those of capillaries do not. Scale bar, 100 m.
migration and invasion
B: Sections of bFGF-containing plugs from wild-type and mutant mice in-
jected with BrdU were subjected to double staining with anti-PECAM-1 (red)
To examine the effect of 64 signaling on endothelial migration
and anti-BrdU (green) (left). The graph shows the mean (SD) number of
and invasion, we isolated endothelial cells from the lungs of
BrdU
cells per 100 vessel cross-sections examined (right).
wild-type and mutant mice. However, both types of cells lost
C: Sections of bFGF-containing plugs from wild-type mice injected with BrdU
expression of 64 upon plating in culture (data not shown).
were subjected to double staining with anti-4 (red) and anti-BrdU (green).
We note that endothelial cells migrating out of human saphe-
nous vein explants also lose expression of 64 (Hiran et al.,
2003). We thus used transient transfection to introduce wild-
type or mutant 64 in human umbilical vein endothelial cells
plates to isolate cells expressing comparable levels of recombi-
(HUVECs), as reported previously (Dans et al., 2001). HUVECs,
nant wild-type or mutant 64, respectively (Figure 5A).
which express almost undetectable levels of endogenous 64,
To examine the effect of 64 signaling on endothelial cell
were electroporated with plasmids encoding 6 and either wild-
type 4 or mutant 4-1355T and panned on anti-4 coated migration, parental HUVECs and their derivatives expressing
476 CANCER CELL : NOVEMBER 2004
ARTICLE
Figure 5. The 4 substrate domain promotes en-
dothelial cell migration and invasion in vitro
A: HUVECs (Ctrl) and HUVECs transfected with
6 in combination with either wild-type 4or4-
1355T were subjected to immunoblotting with
anti-4-exo and anti--actin.
B: HUVECs (Ctrl) and HUVECs transfected with 6
in combination with either wild-type 4or4-
1355T were plated on laminin-5 and induced to
migrate across an artificial wound in response
to bFGF. When indicated, migrating cells were
treated with the NF-B inhibitor BAY 11-7082
(12.5 M) or the MAPK inhibitor PD98059 (50 M).
The graph shows the mean percentage of
wound closure at 8 hr (SD) from three experi-
ments (*p 0.001 versus 4-1355T; p 0.002 ver-
sus Ctrl or BAY 11-7082 inhibitor; and p 0.003
versus PD98059 inhibitor).
C: HUVECs and the indicated derivatives were
grown on Cytodex-3 beads and placed in colla-
gen gels containing bFGF for 72 hr. The graph
indicates the average number (SD) of cord-
like structures emanating from each bead (*p
0.002 versus Ctrl, and p 0.001 versus 4-1355).
D: HUVECs transfected with 6 in combination
with either wild-type 4or4-1355T were in-
duced to migrate across an artificial wound in
response to bFGF for 30 min, fixed, and stained
with antibodies to p65 or P-ERK. Nuclei were
stained with DAPI. Arrows point to nuclei showing
significant nuclear accumulation of P-ERK or
NF-B.
either wild-type or mutant 64 were plated on laminin-5 at Zahir et al., 2003). To examine the mechanism by which 64
confluency and subjected to in vitro wound assay. Expression
promotes endothelial cell migration, we compared ERK and NF-
of wild-type 64 increased endothelial cell migration in re-
B signaling in HUVECs expressing wild-type or mutant 64
sponse to bFGF. By contrast, the mutant integrin did not cause
during migration on laminin-5. The cells were plated at conflu-
this effect (Figure 5B), indicating that the 4 substrate domain
ency on laminin-5 and, 30 min after wounding, were subjected
promotes endothelial cell migration.
to immunofluorescent staining with antibodies to P-ERK and
To evaluate the effect of 64 signaling on endothelial cell
the p65 subunit of NF-B. The cells expressing wild-type 64
invasion, HUVECs expressing wild-type or mutant 4 were
displayed significant nuclear accumulation of P-ERK and NF-
grown on Cytodex-3 beads and then incubated in collagen gels
B as they entered into the wound. In contrast, those expressing
containing bFGF. Over a 3 day period, the endothelial cells
mutant 64 did not show significant nuclear accumulation of
expressing wild-type 4 migrated radially out of the beads and
P-ERK or NF-B under the same conditions (Figure 5D). These
assembled into cords invading the collagen gel. By contrast,
results suggest that 64 signaling promotes both ERK and
both control cells and cells expressing mutant 4 invaded the
NF-B signaling in migrating endothelial cells.
collagen gel only to a limited extent (Figure 5C). Taken together,
To examine the role of ERK and NF-B signaling in endothe-
these observations suggest that signaling by the 4 substrate
lial cell migration, HUVECs expressing wild-type 64 were
domain promotes endothelial cell migration and invasion, in
subjected to in vitro wound closure assay in the presence of
accordance with the hypothesis that it controls the onset of the
the MEK inhibitor PD98059 or the NF-B inhibitor BAY11-072.
invasive phase of angiogenesis.
These compounds reduced the migration of 64-expressing
HUVECs to levels similar to those displayed by control HUVECs
The 4 substrate domain induces nuclear
or HUVECs expressing mutant 64 (Figure 5B), suggesting that
accumulation of ERK and NF-B during endothelial cell
the 4 substrate domain promotes endothelial cell migration by
migration in vitro and angiogenesis in vivo
inducing NF-B and ERK signaling.
Prior studies had provided evidence that 64 controls ERK
and NF-B signaling (Mainiero et al., 1997; Santoro et al., 2003; To examine if deletion of the 4 substrate domain impairs
CANCER CELL : NOVEMBER 2004 477
ARTICLE
signaling in endothelial cells in vivo, sections of Matrigel plugs
from wild-type and mutant mice were subjected to double stain-
ing with antibodies to PECAM-1 and to P-ERK or the p65 subunit
of NF-B. We observed significant levels of P-ERK and p65 in
the nuclei of many endothelial cells of small- and intermediate-
size vessels from wild-type plugs. By contrast, both signaling
molecules were predominantly confined to the cytoplasm in
endothelial cells of similar vessels from mutant plugs (Figure
6A). To confirm this observation, the samples were subjected
to double staining with antibodies to 4 and to P-ERK or p65
and to counterstaining with DAPI. Both P-ERK and p65 accumu-
lated in the nuclei of endothelial cells expressing wild-type 4
but remained largely confined to the cytoplasm in endothelial
cells expressing the mutant integrin (Figure 6B). Taken together,
these results suggest that the 4 substrate domain promotes
nuclear translocation of ERK and NF-B during angiogenesis.
The 4 substrate domain promotes
tumor angiogenesis
To test the role of the 4 substrate domain in tumor angiogen-
esis, we injected B16F0 melanoma cells, LLC1 Lewis lung carci-
noma cells, B6RV2 lymphoma cells, and 60.5 fibrosarcoma cells
s.c. in wild-type and mutant mice. The B16F0, LLC1, and B6RV2
tumors grew in mutant mice to a size significantly smaller than
they did in wild-type mice. Although the 60.5 tumors also ex-
panded less rapidly in mutant mice, the reduction in tumor
growth was in this case smaller (Figure 7A). To compare the
density of microvessels in the tumors grown in wild-type and
mutant mice, we used anti-PECAM-1 staining. The density of
microvessels in each of the four tumors grown subcutaneously
in mutant mice was significantly reduced as compared to that
of tumors grown under identical conditions in wild-type mice
(Figure 7B). This was also true for the 60.5 tumors, which grew
relatively well in mutant mice, suggesting that these tumors are
somewhat less dependent on angiogenesis for growth. Finally,
in the context of other studies, we also examined the effect of
loss of 4 signaling on angiogenesis in an orthotopic model
of mammary carcinogenesis. In this case, the tumors became
vascularized and grew to a similar extent in wild-type and mutant
mice (Figures 7A and 7B), suggesting that 64 signaling does
not contribute to tumor angiogenesis in this specific system.
Four major parameters—tumor cell type, transformation mecha-
nism, injection protocol, and specific genetic background of
mice—may have influenced the outcome of this specific experi-
ment. Since loss of 4 signaling inhibited tumor angiogenesis
to a significant extent in four out of five xenotransplantation
models tested, we concluded that 64 signaling plays a sig-
nificant and broad, but perhaps not universal, role in tumor
angiogenesis.
Figure 6. The 4 substrate domain promotes nuclear translocation of ERK
and NF-B in endothelial cells
To visualize the effect of loss of 4 signaling on tumor vascu-
A: Confocal analysis of nuclear translocation of P-ERK and NF-B in vivo.
lature, we injected wild-type and mutant mice bearing B16F0
Sections of bFGF-containing plugs from wild-type and mutant mice were
xenografts with FITC-Lectin. Confocal analysis and 3D recon-
stained with anti-PECAM-1 (green) and anti-P-ERK (red) (left) or anti-
struction confirmed that the defective angiogenic response of
PECAM-1 (green) and anti-p65 (red) (right). Nuclei were stained with DAPI
mutant mice to tumors was due to reduced branching (Figure
(blue). Arrowheads point to endothelial cells of wild-type vessels containing
7C). Two arguments rule out the possibility that immunological
nuclear P-ERK or NF-B or to endothelial cells of mutant vessels containing
cytoplasmic P-ERK or NF-B. Scale bar, 4 m.
factors contribute to the tumor angiogenesis defect of mutant
B: Sections from the same plugs were subjected to double staining with
mice. First, 64 is not expressed in the immune system. Sec-
anti-4 (green) and anti-P-ERK (red) or anti-p65 (red) and counterstaining
ond, the 60.5 fibrosarcoma, which are derived from 129 Sv mice,
with DAPI. Arrowheads are as in A. Scale bar, 10 m.
were injected in wild-type and mutant mice of pure syngeneic
background, making an immunological response unlikely. In
addition, the reduced angiogenesis in tumors of mutant mice
478 CANCER CELL : NOVEMBER 2004
ARTICLE
Figure 7. The 4 substrate domain promotes tu-
mor angiogenesis
A: The B16F0 melanoma, LLC1 Lewis Lung Carci-
noma, B6RV2 lymphoma, and 60.5 fibrosarcoma
cells were injected s.c. in wild-type or 4 mutant
mice. The YD-Neu mammary carcinoma cells
were injected orthotopically in MMTV-Neu mice
expressing wild-type or mutant 4 to avoid an
immune response to rat Neu. The graphs show
mean tumor volumes (SD) after 10 days (60.5),
12 days (B16F0 and LLC1), 13 days (B6RV2), or 20
days (YD-Neu) (*p 0.004 in B16F0; p 0.09 in
LLC1; p 0.01 in B6RV2).
B: Sections of the indicated tumor xenografts
from wild-type and mutant mice were stained
with anti-PECAM-1 antibodies. The graphs show
the average microvessel densities (SD) in each
tumor. Ten random high-power fields per tumor
section were evaluated. Scale bar, 200 m (*p
0.01 in B16F0; p 0.004 in LLC1; p 0.02 in B6RV2;
and p 0.005 in 60.5).
C: Confocal images of B16F0 melanoma tumors
excised from FITC-Lectin-injected wild-type and
mutant mice. The graph shows the average
number of branches (SD) per high-power field
for each tumor (*p 0.02). Scale bar, 100 m.
does not appear to be a consequence of reduced tumor growth, 1999). However, genetic studies suggest more complex roles
(Hynes, 2002). In particular, it is possible that the v integrinsbecause the 60.5 tumors grew relatively well but evoked re-
duced angiogenesis in mutant mice. Taken together, these re- may have both positive and negative signaling roles during tu-
mor angiogenesis. Perhaps they stimulate endothelial cell prolif-sults identify a role for 64 signaling in tumor angiogenesis.
eration and migration by binding to components of the interstitial
matrix during the invasive phase of tumor angiogenesis, butDiscussion
they induce an active, negative signal at the end of the process,
either upon becoming unligated or upon binding to a knownOur results provide genetic evidence that the 64 integrin
promotes tumor angiogenesis—and, presumably, other forms negative regulator of angiogenesis, such as Thrombospondin,
Tumstatin (a fragment of the 3 chain of type IV collagen), orof pathological angiogenesis—by a signaling mechanism. Im-
munohistochemical and cell biological experiments suggest that PEX (a fragment of MMP2) (Sheppard, 2002). In this model, the
blocking agents interfere with positive signaling while allowing64 promotes nuclear translocation of P-ERK and NF-B and
acquisition of an invasive phenotype at the onset of the invasive negative signaling to occur. Among other integrins involved in
angiogenesis, 51 has attracted considerable interest. Bothphase of angiogenesis. These results suggest the intriguing
possibility that 64 performs a similar signaling function in knockout studies and antibody-blocking experiments have indi-
cated that 51 and its ligand, fibronectin, are required forcancer cells and in angiogenic endothelial cells.
In order to design effective anti-integrin drugs for antiangio- developmental and pathological angiogenesis (Hynes, 2002).
However, it is not known at what step of angiogenesis 51genesis, it is important to understand the mechanisms by which
specific integrins participate in this process. Prior studies with functions and whether it acts by an adhesive or signaling mecha-
nism. Our results indicate that 64 signaling specifically con-adhesion-blocking antibodies and RGD-containing peptides
have led to the hypothesis that v3 and av5 promote tumor trols the invasive phase of pathological angiogenesis. In addition
to adding to our understanding of integrin function during angio-angiogenesis by a signaling mechanism (Eliceiri and Cheresh,
CANCER CELL : NOVEMBER 2004 479
ARTICLE
genesis, these results provide a novel potential target for thera-
peutic intervention.
The role of 64 in angiogenesis described here is unex-
pected. Prior studies have shown that neither 64 nor its sig-
naling functions are required during developmental angiogen-
esis (Dowling et al., 1996; Murgia et al., 1998; van der Neut et
al., 1996). In addition, based on the observation that 64 levels
increase during vessel maturation, La Flamme and colleagues
have proposed that 64 limits angiogenesis (Hiran et al., 2003).
In retrospect, it is not surprising that 64 does not play a
role during developmental angiogenesis, as it is expressed in
endothelial cells only after completion of this process (Hiran et
al., 2003). In addition, our studies do not rule out the possibility
that 64 also contributes to the maturation of adult vessels.
They simply show that that its signaling function contributes to
initiate the invasive phase of angiogenesis. We have demon-
strated this role of 64 signaling in several systems: the Matri-
gel plug assay, the retinal neovascularization model, and four
xenograft models of tumor angiogenesis. This said, increasing
Figure 8. Hypothetical model of 64 function in angiogenesis
evidence indicates that angiogenesis is driven by different
growth factors and cytokines and, hence, proceeds by partially
distinct mechanisms, depending on developmental stage, tis-
sue, and disease state (LeCouter et al., 2002; Risau, 1997). In
NF-B in the endothelial cells of small vessels in mutant plugs.
particular, the two major angiogenic growth factors, bFGF and
This finding suggests that the 4 substrate domain regulates
VEGF, cooperate with distinct v integrins to induce angiogen-
sprouting angiogenesis by promoting nuclear translocation of
esis (Friedlander et al., 1995; Hood et al., 2003). We have ob-
key transcription factors and that this event precedes and may
served that loss of 4 signaling does not suppress angiogenesis
indeed be necessary for the acquisition of an invasive phenotype
in a specific orthotopic model of mammary carcinogenesis.
by sprouting endothelial cells. In agreement with this model,
Thus, although our results suggest that 64 signaling partici-
prior studies have shown that angiogenesis requires integrin
pates in both bFGF- and VEGF-induced angiogenesis, future
signaling to both ERK and NF-B (Hood et al., 2003; Klein et
studies will be necessary to examine how general the require-
al., 2002). Although these transcriptional regulators may play
ment for 64 signaling is during tumor angiogenesis. Since
multiple distinct roles in angiogenesis, our observations suggest
the angiogenic Id transcription factors induce expression of the
that they play specific roles in the acquisition of the invasive
genes encoding 64 and its ligand, laminin-5 (Ruzinova et al.,
phenotype. In particular, we have shown that 4 signaling pro-
2003), it is possible that 64 signaling is especially important
motes nuclear translocation of P-ERK and NF-B as endothelial
when angiogenesis is driven by Id.
cells commence to migrate on laminin-5 and that these signals
What is the mechanism by which 64 signaling controls
are necessary to promote endothelial cell migration in vitro. In
angiogenesis? 64 is expressed during angiogenesis in rela-
agreement with this model, it is known that AP-1 and NF-B
tively mature vessels. The endothelial cells of 4
vessels dis-
coordinately control the expression of genes involved in cell
play a very low proliferative index, making it unlikely that 64
migration and invasion (Vincenti and Brinckerhoff, 2002).
signaling promotes endothelial proliferation. In addition, the an-
The possibility of treating chronic diseases, such as diabetic
giogenic endothelium of mutant mice does not display evidence
retinopathy, rheumatoid arthritis, and cancer, with antiangio-
of increased apoptosis, excluding the hypothesis that 64
genic compounds has attracted considerable interest. Because
signaling plays a necessary role in endothelial cell survival. The
64 signaling is not required during development and normal
severe reduction of PECAM
4
capillaries observed in mutant
adult life, compounds blocking 64 signaling may curb patho-
plugs suggests that 64 signaling is necessary for the genera-
logical angiogenesis without exerting significant toxic effects.
tion of 4
sprouts from 4
vessels, i.e., during the initial step
In addition, it is clear that neoangiogenesis is an integral compo-
of the invasive phase of angiogenesis. As expression of wild-
nent of tumor invasion (Hanahan and Folkman, 1996). As cancer
type, but not mutant, 64 promotes endothelial cell migration
cells invade through the extracellular matrix, they are met by
and invasion in vitro, we propose that 64 plays a similar role
cords of angiogenic endothelial cells, bringing them nourish-
in vivo (Figure 8). In fact, 64 may play a general role during
ment. Since 64 signaling appears to play key roles in both
branching morphogenesis, as it has been shown that anti-64
tumor invasion and tumor angiogenesis, its inhibition may be
antibodies suppress branching of the ureteric bud in the devel-
especially beneficial for cancer therapy. If validated, this model
oping kidney (Zent et al., 2001) and the formation of epithelial
would provide a rational basis to future efforts to develop 64
cords by breast epithelial cells embedded in Matrigel (Stahl et
inhibitors for cancer therapy.
al., 1997).
Sprouting angiogenesis is thought to commence with the
Experimental procedures
acquisition of an invasive phenotype by specific endothelial
cells. The basement membrane underlying these cells is de-
Targeted deletion of the 4 substrate domain
graded as they migrate into the underlying interstitial matrix.
The ClaI/XbaI fragment of mouse 4 gene was isolated from a 129 Sv library
(Murgia et al., 1998) and subcloned in pBluescript to generate pB/S-m4-
We have observed a defect in nuclear accumulation of ERK and
480 CANCER CELL : NOVEMBER 2004
ARTICLE
ClaI/Xba. Site-directed mutagenesis was used to introduce a NheI site within mutant 4-1355T were plated on dishes coated with laminin-5, grown until
the sequences encoding the transmembrane domain of pB/S-m4-ClaI/
confluent, and starved. Monolayers were scratched with a P200 pipette tip
XbaI, as well as pcDNA3-h4 (Dans et al., 2001), without altering their reading
and incubated in the presence of serum and 20 ng/ml bFGF for 18 hr. Wound
frames. PCR was used to introduce a stop codon followed by an XbaI and
closure was monitored by digital photography. To monitor ERK and NF-B
an EcoRI site in pcDNA3-4, thereby generating pcDNA3-4Cyto-1355T. To
signaling during migration, control and transfected HUVECs were subjected
insert the cDNA fragment encoding the N-terminal portion of the cytoplasmic
to in vitro wounding for 30 min, fixed with 3.7% formaldehyde, and subjected
domain of 4 (amino acids 741–1355) downstream of and in frame with
to immunofluorescent staining with anti-P-ERK and -p65, as described (Klein
the exon encoding the transmembrane domain of the protein, a NheI/XbaI
et al., 2002). To examine the effect of 4 signaling on endothelial cell invasion,
fragment of pcDNA3-4Cyto-1355T was subcloned in pB/Sm4-ClaI/XbaI,
control and transfected HUVECs were grown on Biosolin Cytodex-3 micro-
and a ClaI/EcoRI fragment of the resulting plasmid was inserted in the
carrier beads (NUNC) until confluent. The beads were then placed in collagen
targeting vector previously used to delete the entire the cytoplasmic domain
gels (3D Collagen Cell Culture Kit; Chemicon). The gels were overlaid with
of 4 (Murgia et al., 1998). The resulting replacement vector, which carried
DMEM with 10% fetal bovine serum, 2 mmol glutamine, and 10 ng/ml bFGF.
a left arm of 5 kb and a right arm of 3.8 kb, was linearized and electroporated
HUVEC invasion was quantified 72 hr later by counting the average number
in ES cells. Positively transfected cells that had undergone homologous
of capillary-like structures per microcarrier bead.
recombination were selected in 0.5 mg/ml G418 and 0.2 mM Gancyclovir
and identified by Southern blotting. Two distinct ES cell lines were found to
Immunofluorescence microscopy and immunohistochemistry
carry the expected mutation, and both were injected into blastocyst-stage
Tissues and plugs were embedded in paraffin or snap frozen in OCT com-
C57BL/6 mouse embryos. The embryos were then transplanted into the
pound (Tissue-Tek). Paraffin-embedded sections were stained with hema-
uteri of pseudopregnant C57BL/6 mice. Extensively chimeric mice derived
toxylin and eosin or subjected to immunoperoxidase staining with the indi-
from both lines were crossed to C57BL/6 females. Heterozygous offspring
cated antibodies using the ABC Staining Kit (Vector Laboratories). Frozen
were used to generate mice homozygous for the targeted deletion. Mice
sections were subjected to immunofluorescent staining with the indicated
were genotyped by PCR using tail genomic DNA. The following primers were
antibodies. To measure cell proliferation in vivo, mice were injected i.v. with
used for amplification: 5-GGAAATAGCAGAGCAGGATAC-3 (wild-type),
5 M BrdU/100 g body weight and sacrificed 1 hr later. Cryostat as well
5-CTCGTGCTTTACGGTATCGC-3 (recombinant), 5-CTCGGTTGCAGCA
AGGAAG-3 (common). For Southern blotting, tail genomic DNA was di-
as paraffin-embedded sections of Matrigel or tumors were subjected to
gested with NcoI and, after agarose gel electrophoresis and transfer to
immunofluorescent or immunohistochemical staining with anti-BrdU anti-
a nylon membrane, was hybridized to a 500 bp radioactive cDNA probe
bodies (BrdU Labeling and Detection Kit I; Roche). To estimate cell death
complementary to sequences in the extracellular domain of 4, as described
in vivo, TUNEL assays were performed on paraffin-embedded sections (In
previously (Murgia et al., 1998). Except when indicated, the experiments
Situ Cell Death Detection Kit; Roche).
were conducted on mice of mixed genetic background.
Matrigel plug assay
Cells, antibodies, and other reagents
Eight-week-old mice were injected s.c. with 400 l of growth factor-depleted
Keratinocytes were isolated from the skin of newborn mice and grown on
Matrigel (BD Biosciences) supplemented with 400 ng/ml bFGF and 1 g/ml
collagen I-coated plates in EMEM.06 with 8% Chelex-treated FBS, 2 ng/ml
heparin sulfate and sacrificed 7 days later (Passaniti et al., 1992). To visualize
EGF, and 0.06 mM CaCl
2
(Hager et al., 1999). Primary HUVECs were cultured
angiogenesis, the mice were injected intravenously with 20 gofFITC-
on gelatin-coated dishes (Klein et al., 2002). Rat mAbs to 4 (346-11A), 6
Isolectin B4 (Vector Labs) 30 min before harvesting the plugs. Fluorescently
(GoH3), and PECAM-1 (MEC 13.3) were from Pharmingen. Goat anti-
labeled vessels were examined by confocal microscopy. To quantify angio-
PECAM-1 (M-20) and -4 (C-20) and rabbit anti-VEGF-R2 (C-20) and -NF-
genesis, FITC-Lectin-containing plugs were homogenized in RIPA buffer
B p65 (C-20) were from Santa Cruz. Rabbit anti-P-ERK and -P-AKT were
containing protease and phosphatase inhibitors and subjected to fluorimetric
from Cell Signaling, and anti-keratin-5 (AF 138) was from Babco. Mouse
analysis. Alternatively, plug lysates were immunoprecipitated and subjected
mAbs to smooth muscle -actin (clone 1A4) and to -actin (clone AC-74)
to immunoblotting with anti-VEGF-R2 antibodies. Each experimental group
were from Sigma, and those to NF-B p65 (clone 2A12A7) were from Zymed.
consisted of five mice. Each mouse was injected with Matrigel alone or
Affinity-purified rabbit antibodies to the LE4-6 modules of the mouse laminin
Matrigel supplemented with bFGF and heparin sulfate. Experiments were
2 chain (Sasaki et al., 2001), mouse mAb 3E1 to 4, and rabbit anti-4-
repeated three times.
exo serum to a GST fusion protein comprising the N-terminal domain of 4
(Mainiero et al., 1997) were described previously. FITC- and Cy3-conjugated
affinity-purified secondary antibodies were from Jackson Laboratories. Lam-
Retinal hypoxia model
inin-5 matrices were prepared as previously described (Spinardi et al., 1995).
P7 mice were exposed to 75% oxygen for 5 days and then returned to
Purified laminin-5 was from Chemicon. Human fibronectin and rat tail colla-
normoxic conditions for 5 days. Mice of the same age kept in normal air
gen type I were from Collaborative Research. FITC-Lectin (isolectin B4)
were used as controls. Eyes were fixed in 4% paraformaldehyde, embedded
was from Vector Laboratories. The MEK inhibitor PD98059 and the NF-B
in paraffin, sectioned, and subjected to staining. Angiogenesis was quantified
inhibitor BAY 11-7082 were from Calbiochem.
by counting the number of PECAM-1-positive glomeruli penetrating the inner
limiting membrane.
Keratinocyte studies
For immunoprecipitation and immunoblotting analyses, keratinocytes were
Tumor xenografts
lysed in RIPA buffer with 10 mM EDTA and protease inhibitors. Equivalent
Six-month-old mice were injected s.c. with 10
6
tumor cells per flank. B6RV2
amounts of total proteins were immunoprecipitated with mAb GoH3 and
human lymphoma cells, B16F0 mouse melanoma cells, and LLC1 Lewis
subjected to immunoblotting with anti-4-exo or directly subjected to immu-
Lung carcinoma cells were injected in wild-type and mutant mice of mixed
noblotting. FACS analysis and adhesion assays were performed as pre-
genetic background. The 60.5 fibrosarcoma cells, which are derived from
viously described (Murgia et al., 1998). For signaling studies, the keratino-
129 Sv mice (Pozzi et al., 2000), were injected in syngeneic wild-type and
cytes were plated on laminin-5 or collagen I for the indicated times, lysed,
mutant mice of pure background. The YD-Neu mouse mammary carcinoma
and subjected to immunoblotting with anti-phospho-ERK and anti-phos-
cells, which were generated by introducing rat Neu in YD cells (Dankort et
pho-AKT.
al., 2001), were implanted orthotopically at 5 10
6
in Matrigel diluted 1:1
in PBS. To avoid an immune response to rat Neu, the cells were injected in
Endothelial cell studies
MMTV-Neu transgenic mice expressing either wild-type or mutant 4, as
HUVECs were electroporated with equimolar amounts of plasmids encoding
these mice are tolerant to rat Neu. These mice had been backcrossed into
6 and either 4or4-1355T (Dans et al., 2001), deprived of growth factors
an FVB/n background (W.G. and F.G.G., unpublished data). The tumors
for 18 hr, and then panned on plates coated with the anti-4 mAb 3E1. Bound
were excised after the indicated number of days. Final tumor dimensions
cells were washed with PBS and recovered by trypsin-EDTA treatment. For
in vitro wound assays, equal numbers of cells expressing wild-type 4 or were measured by caliper.
CANCER CELL : NOVEMBER 2004 481
ARTICLE
Giancotti, F.G., and Ruoslahti, E. (1999). Integrin signaling. Science 285,
Acknowledgments
1028–1032.
We thank K. Owaribe, R. Timple, and H. Gardner for reagents; the Transgenic
Giancotti, F.G., and Tarone, G. (2003). Positional control of cell fate through
and Knockout Mouse Facility and the Molecular Cytology Facility of the
joint integrin-receptor protein tyrosine kinase signaling. Annu. Rev. Cell Dev.
Memorial Sloan-Kettering Cancer Center (MSKCC) for help; and M. Dans,
Biol. 19, 173–206.
S. Klein, D. Lyden, and A. Petridis for help and discussions. This work was
Hager, B., Bickenbach, J.R., and Fleckman, P. (1999). Long-term culture of
supported by NIH awards F32 CA97886 (to S.N.N.), R37 CA58976 (to F.G.G.),
murine epidermal keratinocytes. J. Invest. Dermatol. 112, 971–976.
and P30 CA08748 (to MSKCC).
Hanahan, D. (1985). Heritable formation of pancreatic -cell tumours in
transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.
Nature 315, 115–122.
Received: April 16, 2004
Hanahan, D., and Folkman, J. (1996). Patterns and emerging mechanisms
Revised: July 14, 2004
of the angiogenic switch during tumorigenesis. Cell 86, 353–364.
Accepted: September 10, 2004
Published: November 15, 2004
Hiran, T.S., Mazurkiewicz, J.E., Kreienberg, P., Rice, F.L., and LaFlamme,
S.E. (2003). Endothelial expression of the 64 integrin is negatively regu-
lated during angiogenesis. J. Cell Sci. 116, 3771–3781.References
Holash, J., Maisonpierre, P.C., Compton, D., Boland, P., Alexander, C.R.,
Arbeit, J.M., Munger, K., Howley, P.M., and Hanahan, D. (1994). Progressive
Zagzag, D., Yancopoulos, G.D., and Wiegand, S.J. (1999). Vessel cooption,
squamous epithelial neoplasia in K14–HPV16 transgenic mice. J. Virol. 68,
regression, and growth in tumors mediated by angiopoietins and VEGF.
4358–4368.
Science 284, 1994–1998.
Arbeit, J.M., Olson, D.C., and Hanahan, D. (1996). Upregulation of FGFs
Hood, J.D., Frausto, R., Kiosses, W.B., Schwartz, M.A., and Cheresh, D.A.
and their receptors during multi-stage epidermal carcinogenesis in K14-
(2003). Differential v integrin-mediated Ras-ERK signaling during two path-
HPV16 transgenic mice. Oncogene 13, 1847–1857.
ways of angiogenesis. J. Cell Biol. 162, 933–943.
Bergers, G., Brekken, R., McMahon, G., Vu, T.H., Itoh, T., Tamaki, K., Tan-
Hynes, R.O. (2002). A reevaluation of integrins as regulators of angiogenesis.
zawa, K., Thorpe, P., Itohara, S., Werb, Z., and Hanahan, D. (2000). MMP-9
Nat. Med. 8, 918–921.
triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2, 737–
744.
Inoue, M., Hager, J.H., Ferrara, N., Gerber, H.P., and Hanahan, D. (2002).
VEGF-A has a critical, nonredundant role in angiogenic switching and pan-
Byzova, T.V., Goldman, C.K., Pampori, N., Thomas, K.A., Bett, A., Shattil,
creatic cell carcinogenesis. Cancer Cell 1, 193–202.
S.J., and Plow, E.F. (2000). A mechanism for modulation of cellular responses
to VEGF: activation of the integrins. Mol. Cell 6, 851–860.
Klein, S., Giancotti, F.G., Presta, M., Albelda, S.M., Buck, C.A., and Rifkin,
D.B. (1993). Basic fibroblast growth factor modulates integrin expression in
Coussens, L.M., Tinkle, C.L., Hanahan, D., and Werb, Z. (2000). MMP-9
microvascular endothelial cells. Mol. Biol. Cell 4, 973–982.
supplied by bone marrow-derived cells contributes to skin carcinogenesis.
Cell 103, 481–490.
Klein, S., de Fougerolles, A.R., Blaikie, P., Khan, L., Pepe, A., Green, C.D.,
Koteliansky, V., and Giancotti, F.G. (2002). 51 integrin activates an NF-
Dankort, D., Maslikowski, B., Warner, N., Kanno, N., Kim, H., Wang, Z.,
B-dependent program of gene expression important for angiogenesis and
Moran, M.F., Oshima, R.G., Cardiff, R.D., and Muller, W.J. (2001). Grb2 and
inflammation. Mol. Cell. Biol. 22, 5912–5922.
Shc adapter proteins play distinct roles in Neu (ErbB2)-induced mammary
tumorigenesis: implications for human breast cancer. Mol. Cell. Biol. 21,
Lai, K.M., and Pawson, T. (2000). The ShcA phosphotyrosine docking protein
1540–1551.
sensitizes cardiovascular signaling in the mouse embryo. Genes Dev. 14,
1132–1145.
Dans, M., Gagnoux-Palacios, L., Blaikie, P., Klein, S., Mariotti, A., and Gian-
cotti, F.G. (2001). Tyrosine phosphorylation of the 4 integrin cytoplasmic
LeCouter, J., Lin, R., and Ferrara, N. (2002). Endocrine gland-derived VEGF
domain mediates Shc signaling to ERK and antagonizes formation of hemi-
and the emerging hypothesis of organ-specific regulation of angiogenesis.
desmosomes. J. Biol. Chem. 276, 1494–1502.
Nat. Med. 8, 913–917.
Dowling, J., Yu, Q.C., and Fuchs, E. (1996). 4 integrin is required for hemi-
Mainiero, F., Pepe, A., Wary, K.K., Spinardi, L., Mohammadi, M., Schles-
desmosome formation, cell adhesion and cell survival. J. Cell Biol. 134,
singer, J., and Giancotti, F.G. (1995). Signal transduction by the 64 integ-
559–572.
rin: distinct 4 subunit sites mediate recruitment of Shc/Grb2 and association
with the cytoskeleton of hemidesmosomes. EMBO J. 14, 4470–4481.
Einheber, S., Milner, T.A., Giancotti, F., and Salzer, J.L. (1993). Axonal regula-
tion of Schwann cell integrin expression suggests a role for 64 in myelina-
Mainiero, F., Murgia, C., Wary, K.K., Curatola, A.M., Pepe, A., Blumemberg,
tion. J. Cell Biol. 123, 1223–1236.
M., Westwick, J.K., Der, C.J., and Giancotti, F.G. (1997). The coupling of
64 integrin to Ras-MAP kinase pathways mediated by Shc controls kera-
Eliceiri, B.P., and Cheresh, D.A. (1999). The role of v integrins during angio-
tinocyte proliferation. EMBO J. 16, 2365–2375.
genesis: insights into potential mechanisms of action and clinical develop-
ment. J. Clin. Invest. 103, 1227–1230.
Miranti, C.K., and Brugge, J.S. (2002). Sensing the environment: a historical
perspective on integrin signal transduction. Nat. Cell Biol. 4, E83–E90.
Folkman, J., Watson, K., Ingber, D., and Hanahan, D. (1989). Induction of
angiogenesis during the transition from hyperplasia to neoplasia. Nature
Murgia, C., Blaikie, P., Kim, N., Dans, M., Petrie, H.T., and Giancotti, F.G.
339, 58–61.
(1998). Cell cycle and adhesion defects in mice carrying a targeted deletion
of the integrin 4 cytoplasmic domain. EMBO J. 17, 3940–3951.
Friedlander, M., Brooks, P.C., Shaffer, R.W., Kincaid, C.M., Varner, J.A., and
Cheresh, D.A. (1995). Definition of two angiogenic pathways by distinct v
Passaniti, A., Taylor, R.M., Pili, R., Guo, Y., Long, P.V., Haney, J.A., Pauly,
integrins. Science 270, 1500–1502.
R.R., Grant, D.S., and Martin, G.R. (1992). A simple, quantitative method
for assessing angiogenesis and antiangiogenic agents using reconstituted
Gagnoux-Palacios, L., Dans, M., van’t Hof, W., Mariotti, A., Pepe, A., Mene-
basement membrane, heparin, and fibroblast growth factor. Lab. Invest. 67,
guzzi, G., Resh, M.D., and Giancotti, F.G. (2003). Compartmentalization of
519–528.
integrin 64 signaling in lipid rafts. J. Cell Biol. 162, 1189–1196.
Gambaletta, D., Marchetti, A., Benedetti, L., Mercurio, A.M., Sacchi, A., and Pozzi, A., Moberg, P.E., Miles, L.A., Wagner, S., Soloway, P., and Gardner,
H.A. (2000). Elevated matrix metalloprotease and angiostatin levels in integrinFalcioni, R. (2000). Cooperative signaling between 64 integrin and ErbB-2
receptor is required to promote PI-3K-dependent invasion. J. Biol. Chem. 1 knockout mice cause reduced tumor vascularization. Proc. Natl. Acad.
Sci. USA 97, 2202–2207.275, 10604–10610.
482 CANCER CELL : NOVEMBER 2004
ARTICLE
Risau, W. (1997). Mechanisms of angiogenesis. Nature 386, 671–674. Spinardi, L., Ren, Y.L., Sanders, R., and Giancotti, F.G. (1993). The 4
subunit cytoplasmic domain mediates the interaction of 64 integrin with
Ruzinova, M.B., Schoer, R.A., Gerald, W., Egan, J.E., Pandolfi, P.P., Rafii,
the cytoskeleton of hemidesmosomes. Mol. Biol. Cell 4, 871–884.
S., Manova, K., Mittal, V., and Benezra, R. (2003). Effect of angiogenesis
Spinardi, L., Einheber, S., Cullen, T., Milner, T.A., and Giancotti, F.G. (1995).
inhibition by Id loss and the contribution of bone-marrow-derived endothelial
A recombinant tail-less integrin 4 subunit disrupts hemidesmosomes but
cells in spontaneous murine tumors. Cancer Cell 4, 277–289.
does not suppress 64-mediated cell adhesion to laminins. J. Cell Biol.
Santoro, M.M., Gaudino, G., and Marchisio, P.C. (2003). The MSP receptor
129, 473–487.
regulates 64 and 31 integrins via 14-3-3 proteins in keratinocyte migra-
Stahl, S., Weitzman, S., and Jones, J.C. (1997). The role of laminin-5 and
tion. Dev. Cell 5, 257–271.
its receptors in mammary epithelial cell branching morphogenesis. J. Cell
Sasaki, T., Gohring, W., Mann, K., Brakebusch, C., Yamada, Y., Fassler, R.,
Sci. 110, 55–63.
and Timpl, R. (2001). Short arm region of laminin-5 2 chain: structure,
Tan, C., Cruet-Hennequart, S., Troussard, A., Fazli, L., Costello, P., Sutton,
mechanism of processing and binding to heparin and proteins. J. Mol. Biol.
K., Wheeler, J., Gleave, M., Sanghera, J., and Dedhar, S. (2004). Regulation
314, 751–763.
of tumor angiogenesis by integrin-linked kinase (ILK). Cancer Cell 5, 79–90.
Schaapveld, R.Q., Borradori, L., Geerts, D., van Leusden, M.R., Kuikman,
Trusolino, L., Bertotti, A., and Comoglio, P.M. (2001). A signaling adapter
I., Nievers, M.G., Niessen, C.M., Steenbergen, R.D., Snijders, P.J., and Son-
function for 64 integrin in the control of HGF-dependent invasive growth.
nenberg, A. (1998). Hemidesmosome formation is initiated by the 4 integrin
Cell 107, 643–654.
subunit, requires complex formation of 4 and HD1/plectin, and involves a
direct interaction between 4 and the bullous pemphigoid antigen 180. J.
van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C.M., and Sonnenberg,
Cell Biol. 142, 271–284.
A. (1996). Epithelial detachment due to absence of hemidesmosomes in
integrin 4 null mice. Nat. Genet. 13, 366–369.
Seifert, P., and Spitznas, M. (2002). Axons in human choroidal melanoma
suggest the participation of nerves in the control of these tumors. Am. J.
Vincenti, M.P., and Brinckerhoff, C.E. (2002). Transcriptional regulation of
Ophthalmol. 133, 711–713.
collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex
signaling pathways for the recruitment of gene-specific transcription factors.
Serini, G., Valdembri, D., Zanivan, S., Morterra, G., Burkhardt, C., Caccavari,
Arthritis Res. 4, 157–164.
F., Zammataro, L., Primo, L., Tamagnone, L., Logan, M., et al. (2003). Class 3
semaphorins control vascular morphogenesis by inhibiting integrin function.
Xia, Y., Gil, S.G., and Carter, W.G. (1996). Anchorage mediated by integrin
64 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a mem-
Nature 424, 391–397.
brane-associated 80-kD protein. J. Cell Biol. 132, 727–740.
Shaw, L.M., Rabinovitz, I., Wang, H.H., Toker, A., and Mercurio, A.M. (1997).
Zahir, N., Lakins, J.N., Russell, A., Ming, W., Chatterjee, C., Rozenberg, G.I.,
Activation of PI-3K by the 64 integrin promotes carcinoma invasion. Cell
Marinkovich, M.P., and Weaver, V.M. (2003). Autocrine laminin-5 ligates
91, 949–960.
64 integrin and activates RAC and NFB to mediate anchorage-indepen-
Sheppard, D. (2002). Endothelial integrins and angiogenesis: not so simple
dent survival of mammary tumors. J. Cell Biol. 163, 1397–1407.
anymore. J. Clin. Invest. 110, 913–914.
Zent, R., Bush, K.T., Pohl, M.L., Quaranta, V., Koshikawa, N., Wang, Z.,
Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992). Vascular endothelial
Kreidberg, J.A., Sakurai, H., Stuart, R.O., and Nigam, S.K. (2001). Involve-
growth factor induced by hypoxia may mediate hypoxia-initiated angiogen-
ment of laminin binding integrins and laminin-5 in branching morphogenesis
of the ureteric bud during kidney development. Dev. Biol. 238, 289–302.esis. Nature 359, 843–845.
CANCER CELL : NOVEMBER 2004 483
    • "Because IGFBP-3 can inhibit cancer progression by inhibiting angiogenesis and the metastatic activities of cancer cells [9], IGFBP-3 was expected to affect cancer and/or vascular endothelial cell-matrix adhesion. In support of this notion, the overexpression of IGFBP-3 via adenoviral infection significantly suppresses NSCLC cell adhesion to ECM components, including collagen, fibronectin, and laminin [19]. Consistent with this previous finding, in the current study, modulation of IGFBP-3 via treatment with recombinant protein or transfection with expression vectors affected the matrix adhesion of HNSCC cells and HUVECs. "
    [Show abstract] [Hide abstract] ABSTRACT: We previously reported that IGF binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates angiogenic activities of human head and neck squamous cell carcinoma (HNSCC) cells and human umbilical vein endothelial cells (HUVECs) through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in cell adhesion is largely unknown. We demonstrate here that IGFBP-3 inhibits the adhesion of HNSCC cells and HUVECs to the extracellular matrix (ECM). IGFBP-3 reduced transcription of a variety of integrins, especially integrin β4, and suppressed phosphorylation of focal adhesion kinase (FAK) and Src in these cells through both IGF-dependent and IGF-independent pathways. IGFBP-3 was found to suppress the transcription of c-fos and c-jun and the activity of AP1 transcription factor. The regulatory effect of IGFBP-3 on integrin β4 transcription was attenuated by blocking c-jun and c-fos gene expression via siRNA transfection. Taken together, our data show that IGFBP-3 has IGF-independent and -independent inhibitory effects on intracellular adhesion signaling in HNSCC and HUVECs through its ability to block c-jun and c-fos transcription and thus AP-1-mediated integrin β4 transcription. Collectively, our data suggest that IGFPB-3 may be an effective cancer therapeutic agent by blocking integrin-mediated adhesive activity of tumor and vascular endothelial cells.
    Article · Apr 2015
    • "Screening more than 60 different extracellular proteins revealed that nearly all can occur in phosphorylated states [30]. Most compelling was the finding that the integrin subunits a4 and b1, two key players in cancer progression and signaling, were found in tissue samples to be phosphorylated in their extracellular domains [30,424344. Since fibronectin454647 which is a key component of the ECM is known to be highly upregulated in cancer484950515253, we further analyzed published proteomic data and found that fibronectin is indeed heavily phosphorylated in clinical cancer tissue samples (Fig. 2,Table 2). "
    [Show abstract] [Hide abstract] ABSTRACT: While small-molecule kinase inhibitors became the most prominent anticancer drugs, novel combinatorial strategies need to be developed as the fight against cancer is not yet won. We review emerging literature showing that the release of several ectokinases is significantly upregulated in body fluids from cancer patients and that they leave behind their unique signatures on extracellular matrix (ECM) proteins. Our analysis of proteomic data reveals that fibronectin is heavily phosphorylated in cancer tissues particularly within its growth factor binding sites and on domains that regulate fibrillogenesis. We are thus making the case that cancer is not only a disease of cells but also of the ECM. Targeting extracellular kinases or the extracellular signatures they leave behind might thus create novel opportunities in cancer diagnosis as well as new avenues to interfere with cancer progression and malignancy.
    Full-text · Article · Dec 2014
    • "Integrin b4 promotes the assembly of distinctive adhesive junctions, the hemidesmosomes[9] . Most of the previous researches concentrated on the role of integrin b4 in cancer and cancer therapy[10,11]. Recently, accumulating data reveal that integrin b4 participates in cell differentiation, multiplication[12], adhesion, migration[13,14], macroautophagy[15], apoptosis and signal transduction[16] in various cell types, implying the key roles of integrin b4 in the physiological function of mammalian cells. Furthermore, the cytoplasmic domain of integrin b4 is different from that of other integrin subunits in both size and structure [17,18]. "
    [Show abstract] [Hide abstract] ABSTRACT: Accumulated research has suggested the importance of the adhesion molecules modulation as therapeutic approach for bronchial asthma. Adhesion molecules expression alteration contributes to the pathogenesis of asthma. In order to probe the roles of expression imbalance of adhesion molecules in asthma pathogenesis, expression profiling of adhesion molecules was performed using cDNA microarray assay. The results showed that the expression pattern of adhesion molecules was altered in peripheral blood leucocytes of asthma patients. In this study, we focused on one of the abnormally expressed molecule, integrin β4, which was down-regulated in all asthma patients, to analyze the relevance of asthma susceptibility with the alteration of integrin β4 expressions. Real time PCR was used to verify the down-regulation of integrin β4 in additional 38 asthma patients. Next, the 5'flanking region of integrin β4 DNA were amplified, sequenced and site-directed mutagenesis technology in correspondent variation sites were carried out. Among 4 variation sites found in 5' flanking region of integrin β4, 3 were related to asthma susceptibility: -nt1029 G/A, -nt 1051 G/A, and -nt 1164 G/C. A reduction of human integrin β4 promoter activity was observed at mutants of these sites. This study demonstrates that various adhesion molecules in asthma patients are abnormally expressed. Mutations in 5' flanking region result in reduced integrin β4 expression, which is related to increased risk of asthma.
    Full-text · Article · Apr 2014
Show more