Integrin β4 signaling promotes tumor angiogenesis
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.
Integrin ␤4 signaling promotes tumor angiogenesis
Sotiris N. Nikolopoulos,
Toshiaki Yoshioka, Wenjun Guo, and Filippo G. Giancotti*
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
These authors contributed equally to this work.
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 ␣6␤4 signaling promotes branching of ␤4
medium- and small-size vessels into ␤4
exerting a direct effect on endothelial cell proliferation or survival. Signaling studies reveal that ␣6␤4 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 ␣6␤4 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 ␣5␤1lash 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
To analyze the physiological role of ␣6␤4 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 ␣6␤4 signaling controls pathological angiogenesis by promoting the acquisition of an invasive
phenotype by angiogenic endothelial cells. Since it is known that ␣6␤4 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
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 ␣6␤4 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. ␣6␤4 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-␣6␤4 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 ␣6␤4 to mediate stable adhesion but
increase its signaling function. Deregulation of ␣6␤4-RTK cosig- adhesive ability of the mutant integrin, wild-type and mutant
keratinocytes were plated on laminin-5 at 4⬚C. 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 ␣3␤1, which also binds to laminin-5, is
inactivated, and adhesion proceeds only through ␣6␤4 (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 4⬚C 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 to estab-
lish a transmembrane connection between laminin-5 and theskin and upper gastrointestinal tract (Murgia et al., 1998). To
analyze the role of ␣6␤4 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 and␤4 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 ␣6␤4 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 ␣6␤4-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 ␣6␤4 and its ligand, laminin-5, are expressed
in tumor vasculaturereferred to as “substrate domain”) would suppress ␣6␤4 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
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-
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 4⬚C. Cell
adhesion to ﬁbronectin at 4⬚C 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).
␣6␤4 signaling does not play an essential role during embryonic al., 2003). To examine the potential role of ␣6␤4 in tumor angio-
genesis, we ﬁrst studied the expression of ␣6␤4 in parafﬁn-vasculogenesis and angiogenesis. This conclusion is consistent
with the observation that ␣6␤4 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
Figure 2. Expression of ␣6␤4 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
nerves. Scale bar, 40 m.
multiforme. Signiﬁcant levels of ␣6␤4 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 ␣6␤4, 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 ␣6␤4 during tumor gesting that ␣6␤4 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 ␣6␤4 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 ␣6␤4 in Schwann cells (Einheber et al., membrane of both ␤4
medium- and small-size vessels and
474 CANCER CELL : NOVEMBER 2004
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-
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 ⬍
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).
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 ␣6␤1 (data not
shown). It is possible that ␣6␤1 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 ␣1␤1, 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 ␣6␤4 signaling is required for angiogenesis␣6␤4.
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 ␣6␤4 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 ␣6␤4tree was in mutant plugs much less developed and complex
CANCER CELL : NOVEMBER 2004 475
signaling promotes both bFGF- and VEGF-induced angiogen-
␣6␤4 signaling is not required for endothelial
proliferation or survival
To examine the cellular mechanism by which ␣6␤4 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 ␣6␤4,
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
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
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
compared to the smaller PECAM-1
that ␣6␤4 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 ␣6␤4
Figure 4. The ␤4 substrate domain promotes branching of quiescent small-
during angiogenesis, these results suggest that ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 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
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 ␣6␤4 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 ␣6␤4 (Hiran et al.,
2003). We thus used transient transfection to introduce wild-
type or mutant ␣6␤4 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 ␣6␤4, respectively (Figure 5A).
which express almost undetectable levels of endogenous ␣6␤4,
To examine the effect of ␣6␤4 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
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 ␤4or␤4-
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 ␤4or␤4-
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 ␤4or␤4-1355T 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
either wild-type or mutant ␣6␤4 were plated on laminin-5 at Zahir et al., 2003). To examine the mechanism by which ␣6␤4
conﬂuency and subjected to in vitro wound assay. Expression
promotes endothelial cell migration, we compared ERK and NF-
of wild-type ␣6␤4 increased endothelial cell migration in re-
B signaling in HUVECs expressing wild-type or mutant ␣6␤4
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 ␣6␤4 signaling on endothelial cell
the p65 subunit of NF-B. The cells expressing wild-type ␣6␤4
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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4-expressing
HUVECs to levels similar to those displayed by control HUVECs
The ␤4 substrate domain induces nuclear
or HUVECs expressing mutant ␣6␤4 (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 ␣6␤4 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
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
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 ␣6␤4 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 ␣6␤4 signaling plays a sig-
niﬁcant and broad, but perhaps not universal, role in tumor
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, ␣6␤4 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
Figure 7. The ␤4 substrate domain promotes tu-
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 ␣6␤4 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 ␣6␤4 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 allowing␣6␤4 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, ␣5␤1 has attracted considerable interest. Bothphase of angiogenesis. These results suggest the intriguing
possibility that ␣6␤4 performs a similar signaling function in knockout studies and antibody-blocking experiments have indi-
cated that ␣5␤1 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 ␣5␤1genesis, 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 ␣6␤4 signaling speciﬁcally con-adhesion-blocking antibodies and RGD-containing peptides
have led to the hypothesis that ␣v␤3 and av␤5 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
genesis, these results provide a novel potential target for thera-
The role of ␣6␤4 in angiogenesis described here is unex-
pected. Prior studies have shown that neither ␣6␤4 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 ␣6␤4 levels
increase during vessel maturation, La Flamme and colleagues
have proposed that ␣6␤4 limits angiogenesis (Hiran et al., 2003).
In retrospect, it is not surprising that ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 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 ␣6␤4 and its ligand, laminin-5 (Ruzinova et al.,
phenotype. In particular, we have shown that ␤4 signaling pro-
2003), it is possible that ␣6␤4 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 ␣6␤4 signaling controls
are necessary to promote endothelial cell migration in vitro. In
angiogenesis? ␣6␤4 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
coordinately control the expression of genes involved in cell
play a very low proliferative index, making it unlikely that ␣6␤4
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 ␣6␤4
genic compounds has attracted considerable interest. Because
signaling plays a necessary role in endothelial cell survival. The
␣6␤4 signaling is not required during development and normal
severe reduction of PECAM
capillaries observed in mutant
adult life, compounds blocking ␣6␤4 signaling may curb patho-
plugs suggests that ␣6␤4 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, ␣6␤4 promotes endothelial cell migration
cells invade through the extracellular matrix, they are met by
and invasion in vitro, we propose that ␣6␤4 plays a similar role
cords of angiogenic endothelial cells, bringing them nourish-
in vivo (Figure 8). In fact, ␣6␤4 may play a general role during
ment. Since ␣6␤4 signaling appears to play key roles in both
branching morphogenesis, as it has been shown that anti-␣6␤4
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 ␣6␤4
cords by breast epithelial cells embedded in Matrigel (Stahl et
inhibitors for cancer therapy.
Sprouting angiogenesis is thought to commence with the
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-m␤4-
We have observed a defect in nuclear accumulation of ERK and
480 CANCER CELL : NOVEMBER 2004
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-m␤4-ClaI/
conﬂuent, and starved. Monolayers were scratched with a P200 pipette tip
XbaI, as well as pcDNA3-h␤4 (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/Sm␤4-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
(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
For immunoprecipitation and immunoblotting analyses, keratinocytes were
lysed in RIPA buffer with 10 mM EDTA and protease inhibitors. Equivalent
Six-month-old mice were injected s.c. with 10
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
al., 2001), were implanted orthotopically at 5 ⫻ 10
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 ␤4or␤4-1355T (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
Giancotti, F.G., and Ruoslahti, E. (1999). Integrin signaling. Science 285,
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,
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