Neuropilin-2 regulates a6b1 integrin in the formation of
focal adhesions and signaling
Hira Lal Goel1,*, Bryan Pursell1, Clive Standley2, Kevin Fogarty2and Arthur M. Mercurio1
1Department of Cancer Biology, University of Massachusetts Medical School Worcester, MA 01605, USA
2BioMedical Imaging Group, University of Massachusetts Medical School Worcester, MA 01605, USA
*Author for correspondence (email@example.com)
Accepted 5 August 2011
Journal of Cell Science 125, 497–506
? 2012. Published by The Company of Biologists Ltd
The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in
breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells
with the ECM. Using epithelial cells from breast tumors, we defined NRP2highand NRP2lowpopulations that differed in integrin
expression and adhesion to laminin. Specifically, the NRP2highpopulation adhered more avidly to laminin and expressed high levels of
the a6b1 integrin than the NRP2lowpopulation. The NRP2highpopulation formed numerous focal adhesions on laminin that were not
seen in the NRP2lowpopulation. These results were substantiated using breast carcinoma cell lines that express NRP2 and a6b1 integrin.
Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for
adhesion on laminin. A specific interaction between NRP2 and a6b1 integrin was detected by co-immunoprecipitation. NRP2 is
necessary for focal adhesion formation on laminin and for the association of a6b1 integrin with the cytoskeleton. NRP2 also facilitates
a6b1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin.
The mechanism by which NRP2 regulates the interaction of a6b1 integrin with laminin to form focal adhesions involves PKC
activation. Together, our data reveal a new VEGF–NRP2 signaling pathway that activates the a6b1 integrin and enables it to form focal
adhesions and signal. This pathway is important in the pathogenesis of breast cancer.
Key words: Cancer, Neuropilin, Integrin, Laminin, FAK
An emerging area of importance in cancer biology is the function
of receptors for VEGF on tumor cells. Although most studies on
VEGF receptors have focused on endothelial cells and their role in
angiogenesis, it has become apparent that tumor cells also express
specific VEGF receptors and that these receptors contribute to
tumor initiation, migration, invasion and survival (Bachelder et al.,
2001; Gray et al., 2008; Hu et al., 2007; Lichtenberger et al., 2010;
Matsushita et al., 2007; Miao et al., 2000; Muders et al., 2009;
Sulpice et al., 2008; Wang et al., 2007). The neuropilins (NRPs)
are one class of VEGF receptors that are particularly interesting
with respect to cancer biology. NRP1 and NRP2 were identified
initially as neuronal receptors for semaphorins, which are axon
guidance factors that function primarily in the developing nervous
system (Uniewicz and Fernig, 2008). The seminal finding by
Klagsbrun that neuropilins can also function as VEGF receptors
and that they are expressed on endothelial and tumor cells
launched studies aimed at understanding their function in
angiogenesis and tumor biology (Soker et al., 1998). NRPs have
the ability to interact with and modulate the function of tyrosine
kinase VEGF receptors (VEGFR1 and VEGFR2), as well as other
growth factor receptors (Neufeld et al., 2002; Sulpice et al., 2008).
There is also evidence that NRPs can function independently of
other receptors (Gray et al.,2005)and that theyarevalidtargets for
therapeutic inhibition of angiogenesis and cancer (Caunt et al.,
2008; Gray et al., 2008; Pan et al., 2007). The observation that the
expression of NRP2 is induced is some cancers such as breast
cancer (Yasuoka et al., 2009) and that its expression correlates
with aggressive disease and poor survival suggests that this NRP
has a substantial influence on the behavior of breast carcinoma
The possibility that NRP2 influences the activation and
function of specific integrins that contribute to tumor behavior
merits consideration. Previous studies demonstrated that VEGF
signaling activates specific integrins in endothelial cells (Byzova
et al., 2000) and that integrins can be regulated by NRP1
(Valdembri et al., 2009). In this study we observed that loss of
NRP2 expression in breast carcinoma cells impedes their ability
to interact with laminin matrices. This later observation
suggested that NRP2 regulates the a6 integrins, which function
as laminin receptors. This hypothesis is compelling because the
a6 integrins have been implicated in breast tumor formation and
progression (Chung and Mercurio, 2004; Guo et al., 2006;
Lipscomb et al., 2005), so we pursued this hypothesis in this
study. Unexpectedly, we discovered that NRP2 is located in focal
adhesions, that it associates with the a6b1 integrin specifically
and that it regulates the ability of this integrin to form focal
adhesions and signal. These studies add a new dimension to the
function of the NRPs and their contribution to cell biology.
Moreover, the fact that both NRP2 (Yasuoka et al., 2009) and the
a6b1 integrin (Friedrichs et al., 1995; Wewer et al., 1997)
have been implicated in aggressive breast cancer underscores
the importance of NRP2-mediated regulation of a6b1 integrin
Journal of Cell Science
Characterization of NRP2highand NRP2lowpopulations of
epithelial cells isolated from human breast tumors
We isolated epithelial cells (EpCAM+) from human breast tumors
to characterize the properties of NRP2-expressing cells. This
approach is based on the report that NRP2 is expressed at low
levels in normal breast epithelium and that its expression
increases in breast cancer and correlates with aggressive
disease (Yasuoka et al., 2009). Epithelial cells were isolated
from four separate, invasive breast tumors and analyzed for
NRP2 expression. We observed that a small but significant
proportion of these cells expressed high levels of NRP2, which
ranged from 12–33% of the total population (Fig. 1A). These
cells, as well as the population of cells expressing low levels
of NRP2, were pooled to generate NRP2highand NRP2low
populations, respectively (Fig. 1B). Given our interest in laminin
interactions, we observed that the NRP2highpopulation adhered
much more robustly to laminin than did the NRP2lowpopulation,
and that this adhesion was inhibited by an a6 integrin function-
the expression of the a6-, b1- and b4-integrin subunits in
the NRP2highand NRP2lowpopulations. Indeed, the NRP2high
population expressed considerably more a6 and ab1 integrin than
did the NRP2lowpopulation (Fig. 1D). Interestingly, b4 integrin
expression was very low in the NRP2highpopulation (Fig. 1D)
indicating that a6b1 integrin is the predominant laminin receptor
in these cells.
NRP2 regulates the interaction of breast carcinoma cells
with laminin matrices and the function of the a6b1 integrin
To explore the relationship between NRP2 and a6b1 integrin more
rigorously, we made use of MDA-MBA-435 cells, a breast
carcinoma cell line (Chambers, 2009; Montel et al., 2009) that
expresses NRP2and a6b1 integrinbut nota6b4 integrin. Depletion
of NRP2 expression in these cells using short hairpin RNA
(shRNAs; Fig. 2A) diminished their adhesive strength on laminin
but not on collagen (Fig. 2B). Specifically, NRP2-depleted cells
adhered less avidly to low concentrations of laminin than did
control cells (Fig. 2B). NRP2-depleted cells also exhibited reduced
spreading on laminin compared with control cells (Fig. 2C). Loss
of NRP2, however, did not affect cell proliferation or morphology
on tissue culture plastic (data not shown) or on collagen (Fig. 2C).
Adhesion to laminin is also dependent on VEGF because depletion
of VEGF using siRNA impeded adhesion to laminin but not to
collagen (Fig. 2D). NRP2-depleted cells also invaded Matrigel
poorly compared with control cells (Fig. 2E).
The effect of NRP2 on laminin interactions suggested that this
receptor influences the function of a6b1 integrin. Given that the
Fig. 1. Characterization of NRP2high
and NRP2lowpopulations of epithelial
cells isolated from human breast
tumors. (A) Epithelial cells (EpCAM+)
were isolated from breast tumors and
analyzed for NRP2 expression by flow
cytometry. Approximately 33% of cells
express high levels of NRP2.
(B) Epithelial cells isolated from four
different breast tumors were sorted into
These populations were stained with
either an NRP2 antibody or goat IgG to
confirm the relative expression of NRP2.
Histograms in the left panels show NRP2
expression relative to control IgG in each
population; pseudocolored dot plot in the
right panel show NRP2 expression in the
NRP2highand NRP2lowcell populations.
(C) NRP2highand NRP2lowpopulations
were incubated with either an a6 antibody
(GoH3) or rat IgG for 1 hour, and assayed
for adhesion to laminin. NRP2highcells
adhere much more avidly to laminin and
this adhesion is blocked by GoH3.
(D) The relative expression of a6, b1 and
b4 integrins in the NRP2highand NRP2low
populations was quantified by flow
cytometry. The NRP2highpopulation
expressed relatively high levels of a6 and
b1 integrins, but low levels of the b4
Journal of Cell Science 125 (2) 498
Journal of Cell Science
NRPs are known to interact with other receptors including
integrins (Fukasawa et al., 2007; Robinson et al., 2009;
Valdembri et al., 2009), we initially assessed the possibility of a
specific interaction between NRP2 and a6b1 integrin using co-
immunoprecipitation experiments. These data revealed that NRP2
interacts specifically with a6b1 integrin and not with a3b1
integrin, which can also function as a laminin receptor (Delwel
et al., 1994) (Fig. 3A). This interaction was detected by NRP2
immunoprecipitation (IP) and a6 integrin immunoblotting assays,
and by a6 integrin IP and NRP2 immunoblotting. This interaction
was also detected in SUM-1315 cells, another breast carcinoma
cell line that expresses NRP2 and a6b1 integrin but not a6b4
integrin (supplementary material Fig. S1A,B). These biochemical
microscopy, which revealed substantial colocalization of NRP2
and a6 integrin (Fig. 3B). No colocalization of NRP2 and
a3b1 integrin was observed (supplementary material Fig. S3A).
Moreover, NRP2 does not appear to regulate a6 integrin or b4
integrin surface expression as evidenced by flow cytometry
(Fig. 3C; supplementary material Fig. S1A), in contrast to the
reported regulation of a5b1 integrin surface trafficking by NRP1
(Valdembri et al., 2009). The NRP2 antibody used is highly
specific (supplementary material Fig. S1C) and it does not cross-
react with NRP1 (Bae et al., 2008).
Focal adhesion formation mediated by the a6b1 integrin is
dependent on NRP2
The association of NRP2 with a6b1 integrin and its effect on cell
morphology caused us to examine the location of NRP2 in cells
adherent to laminin. MDA-MB-435 cells form well-defined focal
adhesions when plated on either laminin or collagen as evidenced
by phosphorylated FAK (FAK-P; at Y397; Fig. 4A) or vinculin
(supplementary material Fig. S2) immunofluorescence. Depletion
of NRP2 expression in these cells diminished focal adhesions
formation on laminin significantly (P50.01)butithadno effect on
collagen (Fig. 4A). The possibility that the reduction in focal
adhesions was caused by the inability of NRP2-depleted cells to
spread was discounted by the observation that those NRP2-
depleted cells that did spread on laminin lacked focal adhesions
(Fig. 4A). The localization of a6b1 integrin to focal adhesions is
also dependent on NRP2 expression. In cells plated on laminin,
a6b1 integrin was located in focal adhesions at the leading edge
(Fig. 4B). The distribution of a6 was more diffuse in NRP2-
depleted cells, which were more ‘rounded’. These observations are
consistent with our finding that loss of NRP2 increased the
solubilityofa6 in Triton X-100considerably(Fig. 4C), suggesting
that NRP2 facilitates a6 integrin association with the cytoskeleton.
No change in a3b1 integrin localization was observed upon NRP2
downregulation (supplementary material Fig. S3B).
Fig. 2. Neuropilin-2 regulates the interaction of breast
carcinoma cells with laminin. (A,B) MDA-MB-435
transfectants (shGFP, shNRP2-1 or shNRP2-2) were serum-
deprived overnight, detached and plated on either laminin (1,
2.5, 5 or 10 mg/ml) or collagen (1, 2.5, 5 or 10 mg/ml). Cells
were incubated for 30 minutes, fixed, and relative adhesion
was quantified using a colorimetric assay (B).
Immunoblotting verified shRNA-mediated NRP2 depletion in
these cells (A). (C) MDA-MB-435 transfectants (shGFP,
shNRP2-1 or shNRP2-2) were plated on either laminin,
collagen or fibronectin (5 mg/ml) for 2 hours and cells were
imaged using phase contrast microscopy (206 objective).
The number of spread, spindle-shaped cells was counted in 20
random fields and plotted as a percentage of total cells.
(D) MDA-MB-435 cells were transiently transfected with
either scrambled (control) siRNA or VEGF siRNA. Cells
were plated on either collagen- or laminin-coated plates
(2.5 mg/ml) 48 hours after transfection. Cells were incubated
for 30 minutes, fixed, and relative adhesion was quantified
using a colorimetric assay. Immunoblotting verified siRNA-
mediated VEGF depletion in cells. (E) MDA-MB-435
transfectants (shGFP, shNRP2-1 or shNRP2-2) were plated in
the upper chamber of Matrigel-coated Transwell plates. After
12 hours, cells that had migrated through the membranes were
stained with DAPI and counted in 20 random fields.
Neuropilin-2 regulates a6b1 integrin499
Journal of Cell Science
Unexpectedly, we observed that NRP2 itself was located in
focal adhesions on laminin and that it colocalized with F-actin
(Fig. 5A). NRP2 and a6b1 integrin also colocalized in focal
adhesions (Fig. 3B). To substantiate the NRP2 localization to
focal adhesions, we obtained more definitive data using TIRF
microscopy. Using this technique, we detected NRP2 in focal
adhesions whereitcolocalizedwithactiveFAK(FAK-P; Fig. 5B).
An ,60% colocalization of NRP2 and FAK-P in these structures
was revealed by quantification of these TIRF images. Together,
these data suggest that NRP2 is located in focal adhesions on
laminin and that it is necessary for such focal adhesions to form.
To validate this hypothesis, we compared the ability of the
NRP2highand NRP2lowpopulations of freshly isolated tumor cells
described in Fig. 1 to form focal adhesions on laminin. Consistent
with our hypothesis, the NRP2highcells formed numerous, well-
defined focal adhesions on laminin as assessed by FAK-P staining,
in marked contrast to the NRP2lowpopulation (Fig. 5C).
PKC mediates NRP2-dependent a6b1 activation and focal
To investigate the mechanism by which NRP2 promotes a6b1
integrin activation and focal adhesion formation, we focused on a
previous finding from our lab that PKC stimulates a6b1 integrin
activation and its association with the cytoskeleton (Shaw et al.,
1990). On the basis of this observation, we hypothesized that
NRP2 contributes to PKC activation and that PKC enhances
a6b1 integrin interactions with laminin and focal adhesion
formation. Indeed, loss of NRP2 expression reduced PKC
activation substantially as assayed using a phosphorylated-pan-
PKC (Ser660) antibody (Fig. 6A). This effect of NRP2 on PKC
activation is not dependent on laminin adhesion (data not shown),
excluding the possibility that a6 signaling mediates this
activation. We assessed whether NRP2 contributes to PKCa
activation by immunoprecipitating extracts with a PKCa-specific
antibody. As shown in Fig. 6A, loss of NRP2 expression
reduced the amount of phosphorylated-pan-PKC captured by
the PKCa-specific antibody specifically. This result provides
evidence that NRP2 contributes to PKCa activation. Additional
evidence to support a role for PKC in regulating a6b1 integrin
function was obtained using a PKC inhibitor (G06983). This
inhibitor prevented the localization of a6b1 integrin to focal
adhesions at the leading edge, although it did not have much
effect on cell adhesion (Fig. 6B). We also assessed whether
the effects of NRP2 depletion on laminin adhesion and
focal adhesion formation could be ‘rescued’ by expressing a
constitutively active form of PKC (myr-PKC). Expression of this
construct in NRP2-depleted cells increased adhesion to laminin
specifically (Fig. 6C) and it increased focal adhesion formation
significantly (P50.01; Fig. 6D).
Focal adhesion signaling on laminin is dependent on NRP2
Our data on the importance of NRP2 in regulating the
localization and function of a6b1 integrin suggest that it
Fig. 3. Neuropilin-2 associates with the a6b1 integrin.
(A) Left panel: MDA-MB-435 cells were extracted in a
Triton X-100 buffer and immunoprecipitated using either a
NRP2 antibody (C9) or mouse IgG. Immunoprecipitated
proteins were separated by SDS-PAGE and immunoblotted
using antibodies to either a6 integrin, a3 integrin or NRP2.
Middle panel: extracts of MDA-MB-435 cells were
immunoprecipitated using either an a6 antibody (J8H) or
mouse IgG. Immunoprecipitated proteins were separated on
SDS-PAGE and immunoblotted using antibodies to either a6
integrin or NRP2. Right panel: SUM-1315 cells were
extracted in a Triton X-100 buffer and immunoprecipitated
using either a NRP2 antibody (C9) or mouse IgG.
Immunoprecipitated proteins were separated by SDS-PAGE
and immunoblotted using antibodies to either a6 integrin, a3
integrin or NRP2. TCL, total cell lysate. (B) MDA-MB-435
cells were plated on laminin (5 mg/ml) and
immunofluorescence was examined using antibodies to
NRP2 or a6 integrin. Samples were imaged by confocal
microscopy (606 objective). Arrows indicate an area of
colocalization. (C) MDA-MB-435 transfectants (shGFP,
shNRP2-1, shNRP2-2 and shNRP-3) were analyzed by flow
cytometry using an a6 antibody (Ab; GoH3) or rat IgG.
Journal of Cell Science 125 (2) 500
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contributes to a6b1-integrin-mediated signaling. To test this
possibility, we assessed FAK activation in control and NRP2-
depleted cells by immunoblotting with a FAK-P antibody. As
shown in Fig. 7A, NRP2-depleted MDA-MB-435 cells were
unable to activate FAK upon adhesion to laminin, unlike the
control cells, but they were able to activate FAK on collagen. To
investigate the role of VEGF in FAK activation, we measured
FAK activation upon VEGF depletion and detected a substantial
reduction in FAK activation on laminin compared with collagen
(Fig. 7B). We extended our analysis of FAK activation to the
NRP2highand NRP2lowpopulations of cells isolated from tumors
(Fig. 1). Indeed, the NRP2highpopulation expressed much higher
levels of FAK-P than did the NRP2lowpopulation, as assessed by
immunofluorescence (Fig. 5C). This result was substantiated
by immunoblotting using the FAK-P antibody (Fig. 7C).
Interestingly, VEGF expression was much higher in the
NRP2highcells than in the NRP2lowcells (Fig. 7C). Therefore,
we depleted endogenous VEGF and observed a substantial
reduction in FAK activation. Importantly, stimulation of these
VEGF-depleted cells with exogenous VEGF restored FAK
activation (Fig. 7C).
We also evaluated Src activation as a function of NRP2
expression because Src is the kinase involved in FAK tyrosine
phosphorylation (Zhao and Guan, 2009). Similar to FAK
activation, NRP2 is necessary for Src activation induced by
laminin but not collagen attachment (Fig. 7D). However, NRP2
Fig. 4. Neuropilin-2 regulates the
localization of a6b1 integrin in focal
adhesions and its interaction with the
cytoskeleton. (A) MDA-MB-435
transfectants (shGFP, shNRP2-1 and
shNRP2-2) were plated on laminin or
collagen and stained with a FAK-P (Y397)
antibody. The percentages of cells with
focal adhesions were quantified as shown
in the bar graphs. Original magnification:
606. (B) MDA-MB-435 transfectants
(shGFP, shNRP2-1 or shNRP2-2) were
used to identify the location of a6b1
integrin by immunofluorescence
microscopy. Original magnification: 606.
The photomicrographs on the right are
higher magnification images of the left
panel to provide better resolution of focal
adhesions at the leading edge. The number
of cells with discrete localization of a6b1
integrin in focal adhesions was counted
and plotted as a percentage of total cells.
This experiment was repeated three times
with consistent results. *P,0.05.
(C) MDA-MB-435 transfectants (shGFP,
shNRP2-1 and shNRP2-2) were extracted
in either Triton X-100 or RIPA lysis buffer
and immunoblotted using antibodies to a6
(AA6A) and actin.
Neuropilin-2 regulates a6b1 integrin501
Journal of Cell Science
downregulation did not cause any change in ERK activation
(Fig. 7D, right panel). We also assessed whether the effects of
NRP2 depletion on cell invasion could be ‘rescued’ by expressing
a constitutively active form of Src (CA-Src). Expression of this
construct in NRP2-depleted cells increased cell invasion on
Matrigel (Fig. 7E).
Given that the NRP2lowpopulation adheres much less avidly
to laminin than does the NRP2highpopulation (Fig. 1), we
hypothesized that FAK activation is crucial for strong adhesion
on laminin. To test this hypothesis we expressed constitutively
active FAK K38A in NRP2lowcells and observed a significant
(P50.001) increase in adhesion to laminin but not to collagen
and this adhesion was dependent on a6b1 integrin (Fig. 7F).
These data suggest a positive feedback mechanism in which CA-
FAK enhances the activity of a6b1 integrin.
The data reported here reveal a previously unknown mechanism
for activation of the a6b1 integrin by NRP2. Specifically, we
conclude that VEGF–NRP2 signaling activates PKC and that
PKC contributes to the functional activation of a6b1 integrin
enabling it to interact more avidly with laminin, form focal
adhesions and signal FAK activation. Unexpectedly, we
discovered that NRP2 itself is located in focal adhesions on
laminin and that it is essential for the formation of these
structures. The relevance of these findings is validated by our
observation that the NRP2highpopulation of tumor cells harvested
from freshly resected, invasive breast carcinomas expressed high
levels of a6b1 integrin and active FAK, adhered avidly to
laminin and formed focal adhesions in contrast to the NRP2low
Our study supports the hypothesis that the activation state of
the a6b1 integrin is tightly regulated by autocrine and paracrine
factors present in the tumor microenvironment, highlighting the
reported functional importance of this integrin in cancer (e.g.
Lathia et al., 2010; Sroka et al., 2010; Wewer et al., 1997).
Moreover, the fact that both NRP2 (Yasuoka et al., 2009) and
a6b1 integrin (Friedrichs et al., 1995; Wewer et al., 1997) have
been associated with aggressive breast cancer supports this
pathophysiological mechanism. This mode of a6b1 integrin
regulation was foreshadowed in earlier work from our lab
demonstrating that activation of a6b1 integrin in macrophages is
regulated by inflammatory stimuli such as IFN-c and LPS (Shaw
and Mercurio, 1989). A common theme in these studies is that
PKC contributes to the functional activation of a6b1 integrin and
its association with the cytoskeleton (Shaw et al., 1990). The
possibility that PKC activation is dependent on laminin adhesion
and not NRP2 signaling in our studies is discounted by our
finding that NRP2 contributes to PKC activation on all substrata
tested. The mechanism by which VEGF–NRP2 signaling
activates PKC is worth considering in light of recent findings.
Specifically, we note that VEGF–NRP2 can activate TORC2 and
that TORC2 can phosphorylate and stabilize conventional PKCs
(Muders et al., 2009; Sarbassov et al., 2004). This potential
Fig. 5. Neuropilin-2 localizes to focal
adhesions and contributes to focal adhesion
formation on laminin. (A) MDA-MB-435
cells were plated on laminin and
immunofluorescence staining was performed
using a NRP2 antibody and phalloidin.
Original magnification: 606. (B) MDA-MB-
435 cells were plated on laminin and
immunofluorescence staining was performed
using antibodies to FAK-P (Y397) and NRP2.
Samples were imaged using TIRF
microscopy. Analysis of the TIRF images was
performed as described in Materials and
Methods. NRP2 and FAK colocalization was
calculated as number of pixels where
NRP2.threshold and FAK.threshold
divided by the number of pixel where
NRP2.threshold; and FAK colocalization
with NRP-2 was calculated as the number of
pixels where NRP2.threshold and
FAK.threshold divided by the number of
pixel where FAK.threshold. Averaging all
experiments, the colocalization of NRP2 with
FAK was 57.8±4.6% and FAK with NRP-2
was 54.6±6.8% (means ± s.e.m., n510).
(C) NRP2highand NRP2lowpopulations
(Fig. 1) were plated on laminin and stained
with a FAK-P (Y397) antibody. Original
Journal of Cell Science 125 (2) 502
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mechanism is relevant because TORC2 has been implicated
in regulating the actin cytoskeleton by modulating PKC
phosphorylation (Sarbassov et al., 2004).
We are particularly intrigued by our observation that VEGF–
NRP2 isneededfora6b1 integrintoactivateFAK,because FAKis
emerging as a central player in the biology of mammary gland
development and breast cancer (Nagy et al., 2007; Provenzano
et al., 2008; van Miltenburg et al., 2009). In fact, we reported
contributes to branching morphogenesis in the developing
mammary gland (Goel et al., 2011). Interestingly, FAK
activation is dependent on a6b1-integrin-mediated adhesion
to laminin, which excludes the possibility that VEGF–NRP2
activatesFAK directly. The most compelling data we obtained that
highlightthe importance of FAK is that the NRP2highpopulation of
tumors isolated from breast carcinomas expresses considerably
higher levels of FAK-P than the NRP2lowpopulation and that the
ability of NRP2lowcells to adhere to laminin and form focal
adhesions could be rescued by expression of constitutively active
FAK. This rescue experiment suggests that CA-FAK can enhance
FAK stimulation of VEGF expression.
The potential relationship of our FAK data to the biology of
breast tumor stem cells merits consideration. FAK has been
implicated in the function of such cells (Luo et al., 2009), which
are characterized by high expression of a6 integrin (CD49f)
Fig. 6. PKC mediates NRP2-dependent a6b1 integrin
activation and focal adhesion formation. (A) Extracts
from MDA-MB-435 transfectants (shGFP and shNRP2)
were immunoblotted using antibodies to pan-
phosphorylated-PKC (Ser660) or PKCa. These extracts
were also immunoprecipitated using the PKCa and
immunoblotted using antibodies to pan-phosphorylated
PKC (Ser660) and PKCa. (B) MDA-MB-435 cells were
treated with either DMSO or a PKC inhibitor (G06983;
10 nM) for 30 minutes and a6b1 integrin location was
analyzed by immunofluorescence microscopy. Original
magnification: 606. (C,D) MDA-MB-435 transfectants
(shGFP and shNRP2) were transfected with either vector
alone or a FLAG-tagged, myristylated PKC construct
(myr-PKC). Expression of this construct was verified by
immunoblotting for PKCa and FLAG (C, right panel).
Cells were detached after 48 hours and cell adhesion
assays were performed using BSA, laminin (2.5 mg/ml) or
collagen (2.5 mg/ml; C, left panel). These cells were also
plated on laminin and FAK-P (Y397) and the location was
analyzed by immunofluorescence microscopy (D).
Original magnification: 606. The percentage of cells with
focal adhesions was determined from three independent
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Fig. 7. Focal adhesion signaling on laminin is dependent on NRP2. (A) MDA-MB-435 and transfectants (shGFP, shNRP2-1 and shNRP2-2) were serum-
deprived and plated on laminin or collagen. Cell extracts were immunoblotted using antibodies to FAK-P (Y397) or total FAK. (B) MDA-MB-435 cells were
transfected with either control siRNA or VEGF siRNA. Cells were either plated on laminin or collagen and cell extracts were analyzed by immunoblotting to
assess the expression of FAK-P and total FAK. Densitometric analysis was performed to quantify the immunoblotting results. (C) Left panel: NRP2lowand
NRP2highpopulations (Fig. 1) plated on laminin were treated with VEGF (100 ng/ml for 30 minutes) and cell extracts were analyzed by immunoblotting to assess
expression of FAK-P (Y397) and total FAK. Middle panel: NRP2lowand NRP2highpopulations were plated on laminin and cell extracts were analyzed by
immunoblotting to assess expression of VEGF and actin. Right panel: NRP2highpopulation were infected with either shGFP- or shVEGF-expressing lentivirus and
plated on laminin. Cells were treated with VEGF and cell extracts were analyzed by immunoblotting to assess expression of FAK-P (Y397) and total FAK.
(D) Left panel: MDA-MB-435 transfectants (shGFP or shNRP2) were plated on laminin or collagen for 30 minutes, and Src-P (Y418) and total Src levels were
assessed by immunoblotting. Right panel: MDA-MB-435 transfectants (shGFP, shNRP2-1, shNRP2-2 and shNRP-3) were plated on tissue culture plates, and
ERK-P and total ERK levels were assessed by immunoblotting. (E) MDA-MB-435 transfectants (shGFP, shNRP2-1 and shNRP2-2) were transfected with either
vector alone or a constitutively active Src construct (CA-Src). Expression of this construct was verified by immunoblotting with a Src-P (Y418). Invasion assays
were performed as described in the legend to Fig. 2. (F) Left and middle panels: the NRP2lowpopulation of tumor cells (Fig. 1) was transfected with either vector
or CA-FAK K38A. Cells were plated on laminin or collagen for 30 minutes and number of attached cells was counted in 20 fields; the results are shown as fold
change relative to vector (middle). *P,0.01. Immunoblotting verified expression of CA-FAK in cells (left). Right panel: the NRP2lowpopulation was transfected
with either vector or CA-FAK K38A. Cells were incubated with either IgG or a6 antibody (GoH3) for 1 hour at 4˚C. Cells were plated on laminin for 30 minutes
and the number of attached cells was counted in 20 fields and is shown as fold change relative to vector-transfected cells.
Journal of Cell Science 125 (2)504
Journal of Cell Science
(Lathia et al., 2010). Given our findings in this study including
the observation that the NRP2highpopulation isolated from
tumors expresses high levels of a6 integrin, it is reasonable to
postulate that VEGF–NRP2 signaling regulates a6b1-integrin-
mediated activation of FAK in breast tumor stem cells. This
hypothesis is consistent with other reports that have implicated
both VEGF signaling (Bao et al., 2006; Lichtenberger et al.,
2010) and the a6 integrin (Lathia et al., 2010) in the function of
tumor stem cells.
Our findings on the regulation of a6b1 integrin by NRP2
should be considered in the context of previous studies on the
regulation of integrin function by the NRPs. These studies have
focused entirely on NRP1 and no published data exist on integrin
regulation by NRP2. Interestingly, we found that the a6 integrins
do not associate with NRP1 (data not shown). NRP1 has been
reported to interact with the a5b1 integrin in endothelial cells and
promote FN adhesion independently of the known NRP1 ligands,
VEGF and semaphorins (Valdembri et al., 2009). The mechanism
involves NRP1-mediated trafficking and internalization of a6b1
integrin. Although this study is significant, we found no evidence
that NRP2 regulates the surface expression of a6b1 integrin. The
importance of integrin–NRP interactions in cancer was indicated
initially by the report that NRP1 interacts with b1 integrins in
pancreatic carcinoma cells and it modulates their growth, survival
and invasion (Fukasawa et al., 2007). Integrins might also
regulate NRP function. For example, the avb3 integrin was
shown to inhibit the contribution of NRP1 to angiogenesis by
sequestering it away from VEGFR2 (Robinson et al., 2009).
Together, the previously published data have established the
importance of NRP1-integrin interactions.
The distinguishing aspect of our results is not only that NRP2
interacts with a specific integrin and regulates its function
but also that NRP2 is located in the focal adhesion, which is
the nexus of integrin signaling (Dubash et al., 2009), and it
contributes to the formation of this structure through its ability to
activate a6b1 integrin and promote its association with the
cytoskeleton. Of note, a recent study concluded that NRP1 does
not localize to focal adhesions, establishing a key difference
between these two receptors (Evans et al., 2011). Importantly, we
also implicate FAK activation as the prime consequence of
VEGF–NRP2 regulation of a6b1 integrin and demonstrate the
significance of this mechanism in tumor cells isolated from
invasive breast carcinomas. The implication of these findings for
the pathogenesis of breast cancer, especially the function of
tumor stem cells, is significant.
Materials and Methods
Reagents and antibodies
Matrigel and collagen I were purchased from BD Biosciences (San Jose, CA);
laminin-1 (LN-1) from Invitrogen (Carlsbad, CA), fibronectin from Sigma (St
Louis, MO); VEGF-165 from Peprotech (Rocky Hill, NJ) and G06983 from
Calbiochem (Darmstadt, Germany). Antibodies against the following proteins
were used: a3 integrin (Millipore, Billerica, MA) used for immunoblotting, or
P1B5 (Gibco, Invitrogen, used for immunofluorescence); a6 integrin (AA6A,
provided by Anne Cress, University of Arizona Cancer Center, Tucson, AZ, USA;
J8H, provided by Arnoud Sonnenberg, The Netherlands Cancer Center,
Amsterdam, The Netherlands; and GoH3, purchased from Millipore); b1
integrin (AIIB2, Developmental Studies Hybridoma Bank, Iowa); b4 integrin
(439-9b, provided by Rita Falcioni (Regina Elena Cancer Institute, Rome, Italy);
NRP2 (goat IgG, R&D, Minneapolis, MN; C9 and H300, Santa Cruz
Biotechnology, Santa Cruz, CA); vinculin (Sigma); actin (Sigma); FAK-P
(Y397) [mouse IgG (BD Bioscience) used for immunoblotting; rabbit IgG
(AbCaM, Cambridge, MA, USA) used for immunofluorescence]; FLAG (Sigma);
anti-rabbit-FITC; anti-goat FITC; anti-goat TRITC; rat IgG; mouse IgG (Jackson,
West Grove, PA, USA); pan-phosphorylated-PKC S660; Src, phosphorylated-Src
Y418; ERK, phosphorylated ERK (Cell Signaling, Beverly, MA, USA); PKCa
(H7) and FAK (Santa Cruz Biotechnology); EpCAM (AbCaM) and VEGF
(Calbiochem). Rhodamine-conjugated phalloidin was purchased from Sigma.
Isolation of epithelial cells from breast tumors
All human breast tissue was obtained in compliance with the laws and institutional
guidelines, as approved by the Institutional Review Board committee of the
University of Massachusetts Medical School. Epithelial cells were isolated from
discarded but freshly resected, invasive breast tumors as described previously
(Fillmore et al., 2010). Briefly, the tissue was minced and digested overnight with
a mixture of collagenase (Roche, Indianapolis, IN, USA) and hyaluronidase (MP
Biomedicals, Solon, OH, USA). The digested cells were plated briefly in serum
(1–2 hours) to deplete mammary fibroblasts. The organoids were dissociated into a
single cell suspension by trypsinization and filtered (40-mm filter; BD Biosciences)
to remove residual clustered cells. Immediately after dissociation, cells were sorted
on the basis of EpCAM and NRP2 expression and subsequently analyzed by flow
cytometry using GoH3 (a6), NRP2 (R&D), b4 (439-9B) or control IgG to quantify
expression of these receptors. In some experiments, cells were infected with
lentiviruses expressing VEGF shRNA (Open Biosystems, Huntsville, AL, USA;
clone ID TRCN0000003343 or TRCN0000003344).
Cell lines and transfectants
SUM-1315 cells were provided by Steve Ethier (Wayne State University School
of Medicine). MDA-MB-435 cells were obtained from the Lombardi Cancer
Center Breast Cancer Repository. Lentiviruses containing NRP2 shRNAs
(Open Biosystems;cloneID TRCN0000063308,
TRCN0000063312) or a GFP control (Open Biosystems; RHS4459) were
generated, titrated according to the manufacturer’s instructions and used to
infect cells following standard protocols. Stable cell transfectants were generated
by puromycin selection (2 mg/ml). In some experiments, cells were transfected
with VEGF siRNA (Smartpool, Dharmacon, Lafayette, CO, USA) or scrambled
To assay cell adhesion, 96-well plates were coated with varying concentrations of
either laminin or collagen overnight at 4˚C, blocked with BSA and washed with
PBS. Cells were detached, washed with PBS and plated (105cells per well). Cells
were incubated at 37˚C for 30 minutes in serum-free medium, washed, and
adherent cells were stained with Crystal Violet to quantify adhesion using a
colorimetric assay. In some experiments, cells were incubated with function
blocking antibodies on ice for 1 hour. Invasion assays were performed as described
previously (Shaw et al., 1997).
Cells were extracted in either a Triton X-100 buffer (1% Triton X-100, 150 mM
NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM PMSF and protease inhibitors) or RIPA
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate,
1 mM PMSF and protease inhibitors). The extracts were pre-cleared by
centrifugation and proteins were immunoprecipitated by incubating with primary
antibody and protein-A–Sepharose. Immunocomplexes were dissociated and
proteins were separated by SDS-PAGE and immunoblotted using antibodies as
specified in the figure legends. In some experiments (Fig. 4C), cell extracts were
prepared using a modified Triton X-100 buffer that removes most of the soluble
protein and phospholipid but leaves the cytoskeleton intact, to assess the interaction
of a6b1 integrin with the cytoskeleton (Rabinovitz and Mercurio, 1997).
Immunofluorescence and TIRF microscopy
immunofluorescence microscopy. Cells were fixed, permeabilized and blocked
using BSA. Cells were incubated with primary antibody overnight at 4˚C, washed
and incubated with fluorochrome-conjugated secondary antibodies. Images were
captured using fluorescence, confocal or TIRF microscopy. For TIRF, there were
10 data sets (i.e. n510). For each color image (either NRP-2 or FAK) of each data
set, we first estimated the background using morphological filter, a gray level
‘opening’ operation, and subtracted it from the image. Subsequently, we selected a
global intensity threshold (same for all pixels in that given image) for that
background-subtracted image and eliminated all pixels having intensity less than
this threshold. Colocalization was calculated on a pixel basis as follows. NRP-2
with FAK: number of pixels where NRP2.threshold and FAK.threshold divided
by the number of pixel where NRP2.threshold; FAK with NRP2: number of
pixels where NRP2.threshold and FAK.threshold divided by the number of
pixel where FAK.threshold.
ontoECM-coated coverslipsandprocessed for
We thank Isaac Rabinovitz and Chris Turner for helpful discussions.
We also thank Arnoud Sonnenberg and Anne Cress for providing a6
Neuropilin-2 regulates a6b1 integrin505
Journal of Cell Science
antibodies, Jun-Lin Guan for providing K38A FAK, Alex Toker for
providing myr-PKC and Leslie Shaw for CA-Src.
This work was supported by the National Institutes of Health [grant
number R01CA80789 to A.M.M.] Deposited in PMC for release
after 12 months.
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