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VASCULAR BIOLOGY
Netrin-4 induces lymphangiogenesis in vivo
Frederic Larrieu-Lahargue,1 Alana L. Welm,2 Kirk R. Thomas,1,3 and Dean Y. Li1,2,4
1Program in Molecular Medicine, 2Department of Oncological Sciences, Huntsman Cancer Institute, 3Division of Hematology, Department of Internal Medicine,
and 4Department of Medicine, University of Utah, Salt Lake City
Netrin-4, a laminin-related secreted protein is an axon guidance cue recently
shown essential outside of the nervous
system, regulating mammary and lung
morphogenesis as well as blood vascular
development. Here, we show that
Netrin-4, at physiologic doses, induces
proliferation, migration, adhesion, tube
formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2
(FGF-2), hepatocyte growth factor (HGF),
vascular endothelial growth factor-A
(VEGF-A), and vascular endothelial
growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and
their specific inhibition antagonizes Netrin-4–induced proliferation. Although Netrin receptors Unc5B and neogenin, are
expressed by human lymphatic endothelial cells, suppression of either or both
does not suppress Netrin-4–promoted in
vitro effects. In vivo, Netrin-4 induces
growth of lymphatic and blood vessels in
the skin of transgenic mice and in breast
tumors. Its overexpression in human and
mouse mammary carcinoma cancer cells
leads to enhanced metastasis. Finally,
Netrin-4 stimulates in vitro and in vivo
lymphatic permeability by activating small
GTPases and Src family kinases/FAK, and
down-regulating tight junction proteins.
Together, these data provide evidence
that Netrin-4 is a lymphangiogenic factor
contributing to tumor dissemination and
represents a potential target to inhibit
metastasis formation. (Blood. 2010;115(26):
5418-5426)
Introduction
The lymphatic and blood vascular systems share many structural
similarities, but with distinct functions. The lymphatic system is an
open-ended network of endothelial cell-lined vessels working to
maintain fluid homeostasis by unidirectionally transporting tissue
fluid, extravasated plasma proteins, lipids, and cells from the
interstitial space to the circulatory system via the thoracic duct. The
lymphatic system has also been demonstrated to be a route for
tumor metastasis.1
A vast number of lymphangiogenic factors, some previously
identified as regulators of blood vascular endothelium,2 have been
shown to induce physiologic and/or tumor lymphangiogenesis and
tumor spreading.1 Netrins are laminin-like secreted proteins, initially identified as axonal guidance molecules.3 In mammals, the
netrin family includes 5 ligands that act through 6 putative
receptors, including deleted in colorectal cancer (DCC), neogenin,
and the members of the Unc5 subfamily.3 Like Netrin-1, Netrin-4
promotes neurite outgrowth4 and regulates blood endothelial cell
biology both positively and negatively.5-7 Nothing is known
currently about the roles of Netrins in the lymphatic vasculature.
Here, we provide evidence that Netrin-4 functions as a
pro-lymphangiogenic factor. We show that Netrin-4 induces
proliferation, migration and survival of lymphatic endothelial
cells through activation of p42/p44 MAPkinase, Akt/PI3kinase
and mTor signaling pathways. We demonstrate that neogenin
and Unc5b are expressed by lymphatic endothelial cells, yet
their silencing does not suppress Netrin-4–induced biologic
effects. Moreover, overexpression of Netrin-4 in mouse skin or
in human breast tumors increases the density of lymphatics.
Finally, we show that mice bearing Netrin-4–overexpressing
tumors develop more metastases by an increased lymphatic
permeability. Taken together, the data demonstrate that Netrin-4
functions as a pro-lymphangiogenic factor.
Methods
Refer to supplemental Methods for details (available on the Blood Web site;
see the Supplemental Materials link at the top of the online article).
Animals and in vivo experiments
Animal experiments were conducted with approval from the University of
Utah Institutional Animal Care and Use Committee. In vivo experiments
were performed as described8,9 and in supplemental Methods.
Cell culture and in vitro assays
Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys),
dermal human microvascular endothelial cells (HMVEC-ds), and human
umbilical vein endothelial cells (HUVECs) were obtained from Lonza and
cultured in EBM-2 supplemented with the EGM-2MV kit according to
manufacturer’s instructions (Lonza) for 7 passages maximum. Human
MCF7 breast cancer cells were a kind gift of Dr Alex Swarbrick (Garvan
Institute, Australia) and were grown according to the ATCC recommendations. Mouse 66C14 mammary carcinoma line was provided by Dr Gary
Sahagian (Tufts University). In vitro assays were performed as described7,10
and in supplemental Methods.
Submitted November 2, 2009; accepted April 11, 2010. Prepublished online as
Blood First Edition paper, April 20, 2010; DOI 10.1182/blood-2009-11-252338.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
5418
BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
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BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
NETRIN-4 AND LYMPHANGIOGENESIS
5419
Figure 1. Netrin-4 induces HMVEC-dLy proliferation,
migration, tube formation and survival. (A) Mitogenic
potential of different doses of Netrin-4 on lymphatic
dermal human microvascular endothelial cells (HMVECdLys) compared with several known lymphangiogenic
growth factors (FGF-2 or bFGF, HGF, VEGF-A (VA),
VEGF-C (VC), and complete (CM) or basal cell (BM)
culture media. Cell number was assessed using dojindo
reagent 72 hours after treatment and expressed as fold
increase versus control. (B) HMVEC-dLy proliferation
under a single dose of Netrin-4 (500 ng/mL), FGF-2
(40 ng/mL), VEGF-A (50 ng/mL), or VEGF-C (300 ng/mL)
assessed every 24 hours for 3 days. (C) Chemotactic
effects of different doses of Netrin-4 and VEGF-C on
HMVEC-dLys in a Boyden chamber assay. (D) HMVECdLy adhesion on various matrixes: Fibronectin (FN),
Netrin-4, Collagen I (Col. I), and Poly-L-Lysin (PLL) at
10 ␮g/mL. (E) In vitro tube formation by HMVEC-dLys
under different doses of Netrin-4, FGF-2 (bF), HGF,
VEGF-A (VA), VEGF-C (VC), or complete media (CM).
(F) Inhibition of serum deprivation–induced HMVECdLys apoptosis under different doses of Netrin-4, FGF-2,
VEGF-A (VA), VEGF-C (VC), and complete media (CM).
*P ⬍ .05.
Statistical analysis
Data are shown as mean plus or minus SEM of 6 to 9 samples from 2 to
3 independent experiments. All statistical analysis was carried out using
Statview (SAS Institute) and a standard Student 2-tailed t test or a Fisher
exact test (for the determination of the lymph node metastasis score). A
P value less than .05 was defined as statistically significant.
Results
Netrin-4 is a lymphatic endothelial mitogen and
chemoattractant
Netrin-4 regulates angiogenesis in vitro and in vivo.5,7 Because the
blood and lymphatic systems share common structural and functional features, we asked if the lymphatic system also responded to
Netrin-4. We initially studied Netrin-4 effects on the proliferation,
migration, adhesion, tube formation, and survival of HMVECdLys. The mitogenic effects of Netrin-4 were compared with
known lymphangiogenic factors FGF-2, HGF, VEGF-A (VA) and
VEGF-C (VC) and complete media (CM) or basal media (BM),
and quantified using the colorimetric Cell Counting kit-8 Dojindo.
Results were expressed as fold increase compared with control. As
shown in Figure 1A, Netrin-4 led to a dose-dependent increase in
cell proliferation with a maximum of activity at 500 ng/mL (6nM)
when HMVEC-dLys were treated for 72 hours and also induced a
time-dependent cell proliferation (Figure 1A-B). Lymphatic endothelial cell proliferation was also induced by a higher concentration
of Netrin-4 (10 ␮g/mL, supplemental Figure 1A). Lymphangiogenic activity of certain growth factors is suppressed by inhibiting
VEGF-C/VEGFR-3 pathway.11 To rule out a similarly indirect role
of Netrin-4, HMVEC-dLys were treated for 24 hours with 1 ␮g/mL
of VEGFR-3 extracellular domain/human Fc construct in presence
of Netrin-4, VEGF-C, HGF (supplemental Figure 1B) and their
proliferation determined as described in the previous paragraph. As
expected, VEGF-C–induced proliferation was blocked by the
VEGFR-3/Fc construct while no effect was detected with Netrin-4
or HGF treatments.
Netrin-4 also exhibited a concentration-dependent chemotactic
activity on HMVEC-dLys in a modified Boyden chamber (Figure
1C). Maximum migration was obtained at a dose of Netrin-4 as low
as 50 ng/mL (0.6nM). As Netrin-4 shares homology to the
N-terminal parts of the laminin short arms,4 we wondered if
Netrin-4 could promote HMVEC-dLys adhesion. Cell culture wells
were coated with bovine serum albumin (BSA), Fibronectin (FN),
Collagen I (Col.I), Poly-L-Lysine (PLL), or Netrin-4. Cells were added
for 30 minutes, washed, and the number of attached cells quantified
using the colorimetric dojindo. As seen in Figure 1D, HMVEC-dLys
adhered most to collagen I, followed by fibronectin and poly-L-Lysine.
Netrin-4 also induced cell adhesion but at a lower level (Figure 1D).
To assess the capacity of Netrin-4 to induce in vitro tube
formation, HMVEC-dLys were seeded on the top of growth
factor-reduced Matrigel and treated with various doses of Netrin-4
for 12 hours. Formation of vascular tubes was assessed by counting
lengths per photograph using ImageJ (National Institutes of Health)
and represented as fold increase in tube length per surface area
compared with control. Netrin-4 promoted a dose-dependent tube
formation as VEGF-A (Figure 1E). Finally, Netrin-1 has been
demonstrated to suppress endothelial cell death.12 To determine
whether Netrin-4 could induce HMVEC-dLys survival, cells were
incubated for 24 hours in serum-free media supplemented with
different doses of Netrin-4. Cell death was quantified by the Cell
Death Detection Elisa kit (Roche) and expressed as fold decrease in
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5420
LARRIEU-LAHARGUE et al
apoptosis versus control. Netrin-4 strongly reduced serumdeprivation–induced cell death (Figure 1F).
Taken together, these data showed that Netrin-4 directly induces
lymphangiogenesis in vitro and at a level at least comparable to
already-identified lymphangiogenic factors.
Netrin-4 activates intracellular signaling pathways in lymphatic
endothelial cells
Stimulation of HMVEC-dLys in vitro by Netrin-4 suggested that
this factor could activate intracellular signaling pathways as has
been reported for VEGF-C, IGFs and PDGF-BB.1 We thus
investigated phosphorylation of MAP Kinase Erk1 and 2, Akt and
Ribosomal protein S6, a target of mTor. HMVEC-dLys were
treated with Netrin-4 at variable doses (Figure 2A-C) or for
variable times (Figure 2D-F). Both Netrin-4 and VEGF-C induced
phosphorylation of Erk1/2, Akt, and S6 in concentration and
time-dependent manners (Figure 2A-F). To rule out the possibility
of a nonproteinaceous contamination of our Netrin-4 solution, we
incubated Netrin-4 with native or heat-inactivated proteinase K,
and alternatively, filtered the Netrin-4 solution through a 30-kDa
exclusion column. Netrin-4 exposed to heat inactivated, but not
native, proteinase K could activate Erk1/2 (supplemental Figure 2).
BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
Furthermore, Erk1/2 phosphorylation is only observed with the
more than 30-kd fraction.
To determine whether these intracellular signaling pathways
had functional relevance to Netrin-4’s biologic activity, we treated
HMVEC-dLys with Netrin-4 (500 ng/mL) in the absence or
presence of U0126, LY294001, Akt inhibitor IV, and rapamycin,
which are MEK, PI3K, Akt, and mTor inhibitors, respectively.
Degrees of phosphorylation inhibition were then examined by
Western blotting (supplemental Figure 3A,C,E,G), densitometry
(supplemental Figure 3B,D,F,H), and inhibitors effect examined by
a proliferation assay (supplemental Figures 3I-K, 4A-B). Although
Netrin-4–stimulated Erk1/2 phosphorylation was completely inhibited by 10 ␮m U0126 (supplemental Figure 3A-B), this concentration only partially suppressed Netrin-4–induced cell proliferation
(supplemental Figure 3I). Interestingly, both PI3K and Akt inhibitors strongly inhibited Netrin-4–promoted cell proliferation (supplemental Figure 3J and S4A) and Akt activation (supplemental
Figure 3C-D and E-F). Although Rapamycin completely inhibited
Netrin-4–induced phospho-S6 (supplemental Figure 3G-H), its
effect on Netrin-4 proliferation was only partial (supplemental
Figure 3K). Finally, simultaneous blockade of Erk, Akt and
S6 pathways strongly inhibited Netrin-4–stimulated proliferation
(supplemental Figure 4B). These data show that Netrin-4 induces
phosphorylation of intracellular signaling pathways essential for
Netrin-4–mediated lymphangiogenesis.
Netrin-4–mediated in vitro effects are not mediated by Unc5B
or neogenin
Figure 2. Netrin-4 activates intracellular signaling pathways of HMVEC-dLys.
Determination of the phosphorylation of Akt (Ser 473; Serine 473, panels A and D,
respectively), p42/p44 (Thr 202/Tyr 204; Threonin 202/Tyrosine 204, panels B and E,
respectively), and Ribosomal protein S6 (Ser 235/236; Serine 235/236, panels C and
F) by Western blotting after Netrin-4 treatment of HMVEC-dLy. Experiments were
performed in triplicate.
We next investigated whether the effects of Netrin-4 in lymphatic
endothelium were mediated by the canonical neuronal Netrin-1
receptors, DCC, or Unc5 family members.3 We first determined
which Netrin-1 receptors were expressed in prox-1-positive
HMVEC-dLys (supplemental Figure 5A). Quantitative RT-PCR
detected only Unc5b and at a much lower level, neogenin mRNAs
(supplemental Figure 5B). Inactivation of Netrin-1 receptors was
conducted using a small interfering RNA (siRNA) strategy and
examined by qRTPCR (supplemental Figure 5C,G,K). Control or
Netrin-1 receptor siRNA transfected cells underwent migration
(supplemental Figure 5D,H,L), proliferation (supplemental Figure
5E,I,M) or Erk1/2 activation (supplemental Figure 5F,J,N) assay as
indicated previously. Unc5B mRNA level was reduced by 80% in
the Unc5b versus control siRNA or untransfected conditions
(supplemental Figure 5C), a knockdown score largely sufficient to
detect its potential actvity as reported by Castets et al.12 Nevertheless, neither Netrin-4–induced migration (supplemental Figure
5D), proliferation (supplemental Figure 5E) nor Erk1/2 phosphorylation (supplemental Figure 5F) was reduced or abolished in the
Unc5B siRNA transfected cells. Similar results were obtained
using a second set of Unc5B siRNA (Dharmacon; data not shown).
Complete depletion of neogenin receptor (supplemental Figure 5G)
resulted in increased apoptosis as reported previously by Srinivasan et al13 (data not shown). However, Netrin-4–mediated effects
were still preserved (supplemental Figure 5 H-J). Finally, the dual
knockdown had also no effect (supplemental Figure 5K-N).
Adenosine receptor A2b has been reported as another potential
Netrin-1 receptor and is expressed by HMVEC-dLys (supplemental
Figure 6A). DPSPX or Enprofylline, both inhibitors of A2b, failed
to suppress Netrin-4–stimulated cell proliferation (supplemental
Figure 6B) or Erk1/2 activation (supplemental Figure 6C). These
data provide evidence that none of the canonical Netrin-1 receptors
mediate Netrin-4 function in lymphatic endothelium.
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BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
Netrin-4 is expressed in the lymphatic vasculature in mouse
embryos and adults
Netrin-4 expression has been identified in basement membranes
of kidney, ovaries, and blood vasculature.4 We asked whether
Netrin-4 expression was also associated with mouse developmental
and adult lymphangiogenesis. Netrin-4 was detected by immunostaining in the developing dorsal aorta (DA) and the anterior
cardinal vein (ACV) in embryonic days (E) 10.5 mouse embryos
(supplemental Figure 7A-C). Moreover, many of the prox1positive lymphatic progenitor cells budding from the ACV also
expressed Netrin-4 (supplemental Figure 7C). By E14.5, Netrin-4
is still detected in differentiated prox1-positive lymphatic endothelial cells lining the lymph sac (LV) and the blood vascular system;
jugular vein (JV) and carotid artery (CA; supplemental Figure
7D-F). LYVE-1-positive lymphatic vessels costained with Netrin-4
in adult mouse intestinal villi and crypts, lymph nodes and skin
(supplemental Figure 7G-I, J-L, and M-O, respectively). Interestingly, Netrin-4 was also detected in human breast tumor lymphatic
and blood vessels (supplemental Figure 7P-R). Together, these data
demonstrate that Netrin-4 is expressed in association with embryonic and adult lymphatic vessels and might play an important role
in lymphatic development and maintenance of its integrity.
Netrin-4 overexpression in mouse skin induces in vivo
lymphangiogenesis
To define a biologic function of Netrin-4 in vivo, we generated a
transgenic mouse in which Netrin-4 was ectopically expressed in
skin under control of the keratin 14 promoter (Figure 3A). The
allele, Rosa26 Netrin-4, contains the Netrin-4 cDNA inserted downstream of the murine Rosa26 promoter. Between the promoter and
the cDNA is a strong transcriptional stop signal flanked by loxP
sites. Expression of Cre recombinase excises the stop signal and
allows expression of Netrin-4. Rosa26 Netrin-4/⫹ mice were bred with
Keratin 14 Cre mice, with the expectation that progeny carrying
both genes would express Netrin-4 in basal cells of stratified
squamous epithelia and hair follicles (Figure 3B). Tg (K14 cre/⫹);
Figure 3. Netrin-4 overexpression in mouse skin
results in an increased lymphatic density. (A) Schematic of the Netrin-4 overexpression system and genotyping of the various mice. Netrin-4 cDNA was inserted into
the constitutively active Rosa26 locus downstream of a
LoxP-flanked transcriptional stop signal. In presence of a
cre recombinase driven by the keratin14 promoter (indicated as Prom.), the stop signal is excised allowing
expression of Netrin-4. (B) Expression of the LacZ gene
reporter (Rosa26 LacZ/⫹) in mouse skin keratinocytes in
presence of the Keratin14 cre recombinase (K14 cre/⫹).
Scale bars: 200 and 50 ␮m. (C) Quantification of Netrin-4
mRNA by qRT-PCR in the skin of Netrin-4–overexpressing mice versus littermate controls. (D) Three-week-old
skin-overexpressing Netrin-4 mice are viable but hairless,
smaller, and redder than controls (scale bar: 1 cm).
Pictures were taken on an Olympus IX71 microscope, at
100⫻ and 400⫻ magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus
software. (E) Skin sections of 3-week-old Netrin-4–
overexpressing and littermates control mice costained
with an anti–LYVE-1 and an antipodoplanin (indicated as
podo.) antibody (dotted box represents the enlarged area
shown; scale bars: 200 and 20 ␮m). (F) Quantification of
the LYVE1⫹/podoplanin⫹ staining density using ImageJ
and normalized to the control group. Pictures in panel E
are representative of the quantification reported in panel F.
NETRIN-4 AND LYMPHANGIOGENESIS
5421
Rosa26 Netrin-4/⫹ mice showed a 4-fold increase of Netrin 4 mRNA
in skin compared with littermate controls (Figure 3C) leading to a
protein expression level equal to 1 ng/␮g protein of total skin lysate
(supplemental Figure 8A). These mice were also smaller, redder
and had poorly developed fur, but were fully viable (Figure 3D).
Interestingly, a similar phenotype has been reported for mice
overexpressing VEGF-C in skin. Immunostaining of skin sections
of 3-week-old mice for the lymphatic specific markers, LYVE1 and
podoplanin, or for blood vessel marker, PECAM-1, showed an
increase in lymphatic and blood vessel density in Netrin-4–
overexpressing versus control mice (Figure 3E and supplemental
Figure 8B). Hair follicles, which strongly express K14 Cre, were
surrounded by lymphatic or blood vessels in Netrin-4 but not
control mice (Figure 3E and supplemental Figure 8B). Quantification of LYVE-1/Podoplanin or PECAM-1-positive structures confirmed that lymphatic and blood vessel density was significantly
higher in Netrin-4 expressing mice than littermate controls (Figure
3F and supplemental Figure 8B). Macrophages have been shown to
promote lymphangiogenesis and angiogenesis by secreting various
growth factors.14 Immunostaining on skin sections using the
macrophage marker F4/80, did not show any difference in macrophage density in Netrin-4–overexpressing versus control mice
(supplemental Figure 8C).
Netrin-4 promotes tumor lymphangiogenesis and tumor
metastasis
Metastasis is the principal cause of cancer mortality. In the past
decade, converging data have illuminated the importance of tumor
lymphatic vasculature in breast cancer dissemination. We investigated the capacity of Netrin-4 to promote tumor lymphangiogenesis and subsequent dissemination in 2 Netrin-4–overexpressing
breast cancer models: a subcutaneous xenograft of poorly invasive
human breast carcinoma MCF-7 cells; and an orthotopic transplant
of metastatic murine mammary carcinoma 66c14 cells. Netrin-4
was only detected in MCF-7s stably transfected with a vector
encoding mouse Netrin-4 and was secreted into the cell supernatant
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5422
LARRIEU-LAHARGUE et al
(SUP; like VEGF-C) or bound to the plasma membrane (Mb;
Figure 4A). Interestingly, 2 bands were seen in the supernatant
fraction compared with the membrane-bound fraction, suggesting
partial cleavage of Netrin-4 during its release from the plasma
membrane.15 MCF7 morphology and proliferation were not altered
either by Netrin-4 or VEGF-C overexpression (Figure 4B-C).
Green fluorescent protein (GFP) and firefly luciferase activity,
provided by the expression vector or a second vector, were
expressed at a similar level by all 3 lines (Figure 4B). To determine
whether Netrin-4 could induce tumor lymphangiogenesis, MCF7
tumors were grown subcutaneously on the backs of NOD/SCID
mice for 12 weeks, sectioned and stained for lymphatic or
blood-specific markers, LYVE-1/VEGFR-3 or CD31, respectively.
Netrin-4 tumors showed a higher density of intratumoral LYVE-1⫹/
VEGFR-3⫹, CD31 stained lymphatic and blood vessels, which
were only detected at tumor margins in the control group (Figure
4D and supplemental Figure 9A). More intratumoral lymphatic
vessels were also found in VEGF-C tumors, where they formed
dense continuous networks compared with the dilated and individualized vessels found in Netrin-4 groups. Quantification of LYVE1⫹/VEGFR-3⫹ or CD31-positive structures confirmed that vessel
density was significantly higher in Netrin-4 and VEGF-C expressing tumors than controls (Figure 4E and supplemental Figure 9A).
F4/80 immunostaining of tumor sections revealed that density of
BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
tumor-associated F4/80⫹ macrophages was not changed in
Netrin-4 versus control group (supplemental Figure 9B). Interestingly, GFP-positive tumor cells were noticed inside or tightly
surrounding the enlarged dilated lymphatic vessels in Netrin-4
tumors (Figure 4F). This sign of tumor cell invasion was confirmed
by intraperitoneal injection of luciferin substrate into tumorbearing mice and subsequent bioluminescence detection. Indeed,
axillary lymph nodes contained viable luciferase-expressing tumor
metastases in 8 of 22 Netrin-4 tumor-bearing mice (36%) and 6 of
15 VEGF-C tumor bearing mice (40%). No lymphatic metastases
were observed in the control tumor cohort (0 of 15, 0%; Figure 4G).
Microscopic analysis of luciferase-positive lymph nodes confirmed
the presence of GFP-positive metastases in mice bearing Netrin-4–
or VEGF-C–expressing tumors, but none in control groups (Figure
4H). Moreover, dense layers of lymphatic and blood vessels
surrounded cancer cell–invaded lymph nodes compared with
controls (Figure 4H and supplemental Figure 9C).
To validate our finding that Netrin-4 promoted metastasis via
tumor lymphatic vasculature in a second model; we used the mouse
mammary cancer cell line, 66c14, known to metastasize via the
lymphatic system.16 Netrin-4 overexpression did not modify in
vitro cell morphology, adhesion or proliferation rates compared
with empty vector–transfected or untransfected cells (data not
shown). Neither was a significant difference observed for the
Figure 4. Netrin-4–overexpressing MCF7 tumors have more
lymphatic vessels and are more metastatic. (A) Human breast
cancer MCF7 cells (WT) stably transduced with a Netrin-4 (Net4),
VEGF-C or empty vector (control). Protein expression was determined in the cell supernatant (SUP), the fraction secreted and
bound to the cytoplasmic membranes (Mb) or in the total cell
lysate (CL) by Western blot using an anti–Netrin-4 or an anti–
VEGF-C antibody. (rNet4; Recombinant Netrin-4). All control,
Netrin-4, and VEGF-C MCF7 tumor cells also express at similar
levels both GFP and luciferase (B) and proliferate in vitro at an
identical rate (C; Ctrl; control empty vector, Net4; Netrin-4; scale
bars in panel B: 100 ␮m and 1 cm). (D) Control, Netrin-4, and
VEGF-C–overexpressing MCF7 cells injected subcutaneously
into the midline of the back of NOS/SCID mice. Tumors were
removed after 12 weeks, sectioned and costained with an anti–
VEGFR-3/Flt-4 and anti–LYVE-1 antibody (scale bar: 100 ␮m).
(E) VEGFR-3⫹/LYVE-1⫹ costaining density was quantified using
ImageJ and represented as fold increase over the control-empty
vector tumor condition. (F) GFP-positive tumor cells in the lumen
of tumor lymphatic vessels stained with LYVE-1 antibody in the
Netrin-4–overexpressing tumors (scale bars: 100 and 20 ␮m).
(G) Luciferase-positive tumor cells metastasized into the auxillary
lymph nodes of the Netrin-4 and VEGF-C tumor-bearing mice.
(H) luciferase-positive lymph nodes contain GFP-positive tumor
cells and are surrounded by enlarged LYVE-1 stained vessels in
mice bearing Netrin-4 and VEGF-C–overexpressing tumors, but
not in control condition (scale bar: 200 ␮m). 66C14 murine
mammary carcinoma line metastasizing via the lymphatic system
to lungs were stably transfected with a Netrin-4 encoding or
empty-vector and injected into the exposed inguinal right mammary fat pad of Balb/C mice. Netrin-4–overexpressing 66C14
tumor bearing mice die faster than controls (I) and present more
metastatic nodules per lung (J; scale bar: 5 mm). Pictures were
taken on an Olympus IX71 microscope, at 100⫻, 200⫻, and 400⫻
magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software. The number of metastatic
nodules per lung was quantified by visual inspection (K). *P ⬍ .05.
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BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
growth rate between Netrin-4–overexpressing, control, and parental primary tumors (data not shown). However, Netrin-4 tumorbearing mice died more quickly than control and parental cohorts
(Figure 4I). Macroscopic analysis of lungs revealed a significant
increase in the number of metastatic nodules per lung from
Netrin-4–expressing tumors versus control tumors (Figure 4J), was
confirmed by quantification of metastatic nodules per lung (Figure
4K). Together, these data demonstrate that Netrin-4 induces tumor
lymphangiogenesis and increases metastasis formation via the
lymphatic system.
Netrin-4 stimulates in vitro and in vivo lymphatic permeability
Lymphatic vessel structure and integrity as well as increased
invasiveness or migration properties of cancer cells have been
identified as avenues for tumor dissemination via the lymphatic
vasculature.17 Specialized cell-cell junctions expressing VEcadherin and tight junction-associated proteins such as Zona
Occludens 1 (ZO-1) have been reported between lymphatic endothelial cells.18 Disruption of functional organization of such
structures might lead to vascular fragility and increase in permeability. We therefore examined whether Netrin-4 could change lymphatic permeability. VEGF-A–induced endothelial cell permeability can be regulated by both small GTPases and protein kinases.19
Similarly, we found that Netrin-4 increased active RhoA (GTP-
Figure 5. Netrin-4 induces in vitro lymphatic permeability.
(A) Induction of GTP-RhoA and Rac1 by Netrin-4, VEGF-A, and
VEGF-C treatment of HMVEC-dLys. (B) Stimulation of the phosphorylation of Tyrosine 416 of Src kinase family (SFK, Tyr416) and
the Tyrosin 861 but not the Tyrosine 391 of focal adhesion kinase
(FAK; Tyr861 and Tyr391) by Netrin-4 (500 ng/mL) and VEGF-C
(500 ng/mL). (C) Measurement of the electrical resistance of the
cell monolayer over 24 hours using the ECIS system or
(D) immunostained using an anti–VE-cadherin antibody to visualize cell junctions (scale bar: 50 ␮m) of HMVEC-dLys seeded
either on Fibronectin or Fibronectin plus Netrin-4. (E) Membrane
fraction proteins prepared from HMVEC-dLys seeded as previously mentioned in panels C and D analyzed for ZO-1, VEcadherin, and beta-catenin expression (equivalent loading assessed by coomassie blue staining). (F) Control, Netrin-4, VEGF-C
overexpressing MCF7 tumor sections stained for the cell junction
protein ZO-1 or the lymphatic marker LYVE1 (scale bar: 20 ␮m).
Data presented in panels A through E are from 1 experiment and
representative of 2 independent experiments. Pictures were taken
on an Olympus IX71 microscope, at 400⫻ magnification using a
DP30BW Olympus camera and the MicroSuite Basic Edition
Olympus software.
NETRIN-4 AND LYMPHANGIOGENESIS
5423
bound) and, to a lower extent, Rac1 in HMVEC-dLys, (Figure 5A).
Phosphorylation of Src family kinase (SFK, on Tyrosine 416) and
FAK (on Tyrosine 861 but not 397) was also induced by Netrin-4
stimulation of HMVEC-dLys in a time-dependent manner (Figure
5B). Netrin-4 also increased permeability, as measured by a
decrease in transendothelial resistance, in a HMVEC-dLys monolayer in vitro (Figure 5C). VE-cadherin and actin staining of
HMVEC-dLy revealed looser and more punctate cell junctions in
Netrin-4 versus control (Figure 5D). Expression of the tight
junction protein ZO-1 and VE-cadherin were down-regulated in the
membrane fraction of HMVEC-dLys after stimulation with
Netrin-4 compared with the control. However, beta-catenin or p120
catenin levels were unchanged (Figure 5E and data not shown).
These results prompted us to examine whether expression of cell
junction molecules were changed in lymphatic vessels in Netrin-4
versus control tumors. We found that ZO-1 immunostaining was
fainter, thinner and more punctate in LYVE1-positive structures in
both Netrin-4– and VEGF-C–overexpressing tumors compared
with control tumors (Figure 5F). To further investigate Netrin-4
effect on lymphatic leakage, Tg (K14 cre/⫹); Rosa26 Netrin-4/⫹ mice
(mice overexpressing Netrin-4 mice in the skin) and their littermate controls, Tg (K14 cre/⫹); Rosa26 ⫹/⫹ were tested in a
modified Miles assay.20 As shown in supplemental Figure 10,
higher lymphatic-leakage was detected in Netrin-4–overexpressing
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
5424
LARRIEU-LAHARGUE et al
versus control mice. These data provide evidence that Netrin-4
induces lymphatic permeability by disorganizing lymphatic cellcell junctions.
Discussion
Although blood and lymphatic systems perform different functions, they share a variety of common features. Netrins, initially
characterized as axon-guidance cues, have been recently described
as regulators of blood vasculature.5-7,12,21 In the present study, we
provide evidence that Netrin-4 acts in vitro and in vivo as a
lymphangiogenic factor comparable in activity to FGF-2, HGF,
VEGF-A and VEGF-C, but appears to act independent of the
canonical Netrin-1 receptors, neogenin and Unc5B.
First, we show that that Netrin 4 induces proliferation, migration, tube formation and survival of human adult HMVEC-dLys in
a time- and dose-dependent manner. This is consistent with a report
showing that Netrin-4 stimulated in vitro cell functions of
HMVECs at low concentrations, but was inhibitory at higher doses,
and the demonstration that HUVECs and HUAECs in vitro showed
a bi-phasic response to Netrin-1.6,7,12,21 Netrins, as axon guidance
cues, were originally described as bifunctional factors, promoting
attraction or repulsion, dependent on their concentration, cell
expression or dimerization status of its receptors.3 A combination
of receptor expression, receptor affinity, receptor dependence and
ligand concentration might also be invoked to explain the multiple
activities of Netrins in the endothelium.6,7,12,21
We observed that HMVEC-dLys express canonical neuronal
Netrin-1 receptors Unc5B and neogenin mRNA but their inhibition
failed to suppress Netrin-4–promoted in vitro effects. Interestingly,
we noticed significant cell death in neogenin-knocked down cells
compared with control cells, despite the extremely low amount of
siRNA (in nanomolar range), while no significant change of
morphology was observed with the Unc5B RNAi. A similar
phenotype was noticed in neogenin knockout mice in comparison
to littermate controls.13 These data might indicate that neogenin
signals continuously to induce lymphatic cell survival in presence
of its ligand, Netrin-1, which is also expressed by lymphatic cells
(data not shown), according to the ligand/receptor dependence
model.12 There is a single report that Neogenin and Unc5B
receptors mediate Netrin-4 inhibitory effects in HUAECs pretreated with VEGF-A5. The apparent discrepancy between our
findings and data published by Lejmi et al might be explained by
the different types and origins of the cells studied and/or by their
different proliferation status after VEGF-A or FGF-2 stimulation.
Interestingly, Nacht et al, who also showed a Netrin-4 inhibitory
effect on HMVECs, failed to detect any binding of Netrin-4 to
DCC, neogenin or Unc5B by both coimmunoprecipitation and
Biacore surface Plasmon resonance based technology6; nor has
Netrin-4 binding to canonical Netrin-1 receptors been seen in
neurons.3 Together, these data indicate that the function of Netrins
in endothelium is much more complicated than that of other
endothelial factors such as VEGFs. This is likely to be compounded
by the multiplicity of receptors and by ligand-receptor dependency
effects. Further, the existence of unidentified netrin receptors is
strongly probable; new noncanonical Netrin-1 receptors on nonendothelial cells have been reported in the past years.3,22 Also,
because Netrins are laminin like proteins,15 the ECM could either
regulate local concentrations of Netrins by serving as a growth
factor reservoir, or could act as a coreceptor.22
BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
We showed that Netrin-4 stimulated phosphorylation of protein
kinase ERK1/2 and Akt (PKB) in a time- and concentrationdependent fashion. Although intracellular signaling pathways controlling lymphatic endothelial cell migration, proliferation, and
survival have not been studied exhaustively, similar results were
reported for the lymphangiogenic factors PDGF-BB, IGF-1 and
2, VEGF-A and VEGF-C1. More interestingly, we have demonstrated that the ribosomal protein S6 kinase, target of the mTor
complex, was activated by both Netrin-4 and VEGF-C, as previously shown for the lymphangiogenic factor FGF-2.23 In addition,
the Netrin-4–induced proliferation of HMVEC-dLys was partially
inhibited in vitro by specific kinase inhibitors and Rapamycin, a
specific inhibitor of mTOR, which has also been shown to inhibit in
vivo lymphangiogenesis and metastasis formation.24
The kinetics of Netrin-4 action, similar to VEGF-C or PDGFBB, and the absence of effect using a VEGFR-3/Fc chimera argue
strongly that this factor stimulates in vitro lymphangiogenesis
directly, for example, not by changing the levels of expression of
other endothelial growth factors.
We show that Netrin-4 is expressed by the first differentiated
lymphatic endothelial cells and the adult lymphatic vasculature.
Interestingly, Netrin-4 is also detected, at a higher extent in blood
vessels particularly in the intestine. The relevance of this expression pattern is still unknown, and its resolution awaits the ability to
mutate Netrin-4 specifically in each vascular bed using a blood or
lymphatic specific cre recombinase and a Netrin-4 floxed allele. It
also remains undetermined if Netrin-4 acts in vivo on the vasculature as a growth factor and/or in an autocrine manner. Indirect in
vivo effects cannot be ruled out totally, as Netrin-1 has already
been shown to act directly on endothelial, smooth muscle, inflammatory or even cancer cells.12,25-27 In addition, functional redundancy between Netrin-1 and Netrin-4 could also be envisioned as
Netrin-1 expression has also detected in both mouse blood and
lymphatic vasculature (data not shown).
We have demonstrated that Netrin-4 induces in vivo lymphangiogenesis in several different, standard lymphangiogenic models:
overexpression in mouse skin; and 2 tumor models. Overexpression of Netrin-4 in the mouse skin phenocopied the reduction in fur
development and increased lymphangiogenesis induced by overexpression of VEGF-C, although it did not lead to a hyperplasia of
lymphatic vasculature induced by VEGF-C.28 The more robust
phenotype with VEGF-C might be explained by the transgenesis
strategy used by Jeltsch et al, leading to multiple insertions of the
construct in the mouse genome and a subsequent higher expression.28
Netrin-4 overexpression also induced tumor lymphangiogenesis. Lymphatic vessels with wider lumens containing cancer cells
were observed in Netrin-4 tumors and were correlated with
increased tumor metastasis to axillary lymph nodes. A previous
study in which subcutaneous MCF7 tumors overexpressed VEGF-C
cells led to similar results,9 emphasizing the relevance of lymphatic
vessels in metastatic disease. Several mechanisms have been
shown to support metastatic dissemination including increased
tumor blood and/or lymphatic density, or cancer cell migration and
invasiveness.17 Stimulation of tumor lymphangiogenesis is thought
to increase the surface area of tumor cells in contact with lymphatic
endothelial cells and then to facilitate tumor dissemination because
of their structures.29
Control of lymphatic permeability might also be a crucial
element contributing to metastasis. Several reports showed that
RhoA and Rac1, as well as focal adhesion kinase (FAK) and Src
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 1 JULY 2010 䡠 VOLUME 115, NUMBER 26
NETRIN-4 AND LYMPHANGIOGENESIS
family kinases (SFK) are responsible of VEGF-A–mediated vascular permeability.19 Here we showed that Netrin-4 activates Rac1,
RhoA and SFK, FAK phosphorylation and increased permeability
of lymphatic cells similar to known lymphatic factors.30 Phosphorylation of FAK and SFK have been shown to correlate with
enhanced cancer cell migration and invasion, while RhoA and Rac1
levels are up-regulated in numerous metastatic cancers.31-33 Vascular permeability in the lymphatic system is also regulated by
cell-cell junctions composed of VE-cadherin and tight junctionassociated proteins such as ZO-1.18 Expression of VEGF-A/
VEGFR-2 has been shown to promote vascular permeability
through the endocytosis of VE-cadherin.19 Disruption of tight
junctions using a VE-cadherin inhibitory antibody increased lung
metastasis.33 Similarly, expression of ZO-1 in endothelial tight
junctions is down-regulated in metastatic breast and liver tumors.34,35 We demonstrated that both VE-cadherin and ZO-1 are
reduced at the surface of HMVEC-dLy treated in vitro with
Netrin-4, and vascular structures in Netrin-4– and VEGF-C–
overexpressing breast tumors show a decrease in ZO-1 expression,
leading to open gaps between lymphatic endothelial cells. Given
our results, the contribution and mechanisms regulating such
specialized lymphatic cell-cell junctions18 might be clearly investigated during tumor metastasis.
Our findings clearly show that Netrin-4 is a lymphangiogenic
factor that induces metastasis via 2 probable mechanisms1; by
increasing lymphatic density and consequently augmenting surface
contact with cancer cells and2 by increasing lymphatic permeability
and facilitating intravasation and/or extravasation of tumor cells.
Our data suggest that Netrin-4 may contribute to lymphatic
metastasis, and support the strategy of targeting not only tumorderived functions, but also targeting functions of the host that
support the tumor. These data also warrant future studies to fully
identify the functional receptors for Netrins in endothelium and
therefore clarify their contribution in vascular biology and oncology.
5425
Acknowledgments
We gratefully thank Dr Charles L. Murtaugh for help with microscopy
and Dr Lisa Urness for a critical reading of the manuscript. Podoplanin
antibody (clone 8.1.1) was obtained from the Developmental Studies
Hybridoma bank, University of Iowa. 66c14 cells were provided by
Pr G. Gary Sahagian (Tufts University).
This work was supported by grants from the Fondation pour la
Recherche Médicale (F.L.-L.), the American Heart Association
(F.L.-L. and D.Y.L.), the Huntsman Cancer Institute Foundation
(A.L.W.), the US Department of Defense Breast Cancer Research
Program (A.L.W. and D.Y.L.), the US National Institutes of Health,
the H.A. and Edna Benning Foundation, the Juvenile Diabetes
Research Foundation, the Burroughs Wellcome Fund, and the
Flight Attendants Medical Research Institute (D.Y.L.).
Authorship
Contribution: F.L.-L., A.L.W., and K.R.T. contributed new
reagents, F.L.-L. and A.L.W. performed experiments, and F.L.-L.
and D.Y.L. designed the research, analyzed results, and wrote
the paper.
Conflict-of-interest disclosure: The authors are or were previously employed by the University of Utah, which has filed
intellectual property surrounding the therapeutic uses of vascular
guidance cues and with the intent to license this body of intellectual
property for commercialization.
Correspondence: Dean Y. Li, University of Utah, Eccles
Institute of Human Genetics, Bldg 533, Rm 4450, 15 North 2030
East, Salt Lake City, UT 84112; e-mail: [email protected].
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2010 115: 5418-5426
doi:10.1182/blood-2009-11-252338 originally published
online April 20, 2010
Netrin-4 induces lymphangiogenesis in vivo
Frederic Larrieu-Lahargue, Alana L. Welm, Kirk R. Thomas and Dean Y. Li
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