Role of Human Cripto-1 in Tumor Angiogenesis

Article (PDF Available)inJournal of the National Cancer Institute 97(2):132-41 · February 2005with51 Reads
DOI: 10.1093/jnci/dji011 · Source: PubMed
Abstract
Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). CR-1 appears to have an important role in the multistep process of angiogenesis.
Role of Human Cripto-1 in Tumor Angiogenesis
Caterina Bianco, Luigi Strizzi, Andreas Ebert, Cindy Chang, Aasia Rehman,
Nicola Normanno, Liliana Guedez, Rita Salloum, Erika Ginsburg, Youping Sun,
Nadia Khan, Morihisa Hirota, Brenda Wallace-Jones, Christian Wechselberger,
Barbara K. Vonderhaar, Giovanna Tosato, William G. Stetler-Stevenson,
Michele Sanicola, David S. Salomon
Background: Human cripto-1 (CR-1) promotes cell trans-
formation and increases migration and invasion of various
mouse and human epithelial cell lines. We investigated
whether CR-1 also stimulates angiogenesis. Methods: We
used human umbilical vein endothelial cells (HUVECs) to
measure in vitro migration with fibronectin-coated Boy-
den chambers, invasion with Matrigel-coated Boyden
chambers, proliferation with a tetrazolium salt, and dif-
ferentiation with an in vitro Matrigel assay. We investi-
gated new blood vessel formation in vivo by use of
Matrigel-filled silicone cylinders implanted under the skin
of nude mice and by use of a breast cancer xenograft
model with CR-1-transfected or control Neo-transfected
MCF-7 human breast cancer cells. We also used a block-
ing anti-CR-1 monoclonal antibody to investigate the role
of CR-1 in angiogenesis in vivo and in vitro. All statistical
tests were two-sided. Results: CR-1 stimulated HUVEC
proliferation, migration, and invasion and induced
HUVEC differentiation into vascular-like structures on
Matrigel. In vivo, recombinant CR-1 protein induced mi-
crovessel formation in Matrigel-filled silicone cylinders,
and microvessel formation was statistically significantly
inhibited with a blocking anti-CR-1 monoclonal antibody
(CR-1 and antibody 127% of microvessel formation
compared with that in untreated control cylinders and
CR-1 alone 259%; difference 132%, 95% confidence
interval [CI] 123% to 140%; P<.001). Tumors formed
by CR-1-transfected MCF-7 cells in the cleared mammary
fat pad of nude mice had higher microvessel density than
tumors formed by control Neo-transfected MCF-7 cells
(CR-1-transfected cells 4.66 vessels per field and Neo-
transfected cells 2.33 vessels per field; difference 2.33
vessels per field, 95% CI 1.2 to 2.8; P .004). Conclu-
sion: CR-1 appears to have an important role in the
multistep process of angiogenesis. [J Natl Cancer Inst
2005;97:132– 41]
Human cripto-1 (CR-1) is the founding member of the epi-
dermal growth factor (EGF)-CFC (named for the founding
members of the family: cripto in humans, FRL1 in Xenopus, and
cryptic in mice) family of proteins (1). CR-1 is overexpressed in
50%– 80% of different types of primary human carcinomas, such
as breast, cervix, colon, stomach, pancreas, lung, ovary, and
testis carcinomas (1). Proteins in the EGF-CFC family contain a
modified EGF-like domain, a cysteine-rich CFC domain, and a
short hydrophobic carboxyl terminus that is essential for mem-
brane attachment by a glycosylphosphatidylinositol moiety (1).
CR-1 can apparently function as an oncogene by inducing in
vitro cell transformation, migration, invasion, epithelial-to-
mesenchimal transition, and branching morphogenesis in mouse
and human mammary epithelial cells and in human breast and
cervical carcinoma cells (2–5). CR-1 overexpression can induce
tumor formation, as shown by the development of mammary
hyperplasias and papillary adenocarcinomas in transgenic mice
Affiliations of authors: Tumor Growth Factor Section, Mammary Biology and
Tumorigenesis Laboratory (CB, LS, CC, YS, NK, MH, BW-J), Molecular and
Cellular Endocrinology Section, Mammary Biology and Tumorigenesis Labo-
ratory (EG, BKV), Extracellular Matrix Section, Laboratory of Pathology (LG,
RS, WGS-S), Experimental Transplantation and Immunology Branch (GT),
Center for Cancer Research, National Cancer Institute, National Institutes of
Health, Bethesda, MD; Department of Gynecology, Charite Campus Benjamin
Franklin, Berlin, Germany (AE); Department of Molecular and Cellular Biology,
University of Michigan, Ann Arbor, MI (AR); Division of Haematological
Oncology and Department of Experimental Oncology, ITN-Fondazione Pascale,
Naples, Italy (NN); Upper Austrian Research GmbH Zentrum, Linz, Austria
(CW); Biogen-Idec Inc., Cambridge, MA (MS).
Correspondence to: David S. Salomon, PhD, Tumor Growth Factor Section,
Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bldg. 10, Rm. 5B39,
Bethesda, MD 20892 (e-mail: salomond@mail.nih.gov).
See “Notes” following “References.”
DOI: 10.1093/jnci/dji011
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Press 2005, all rights reserved.
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that stably overexpress a human CR-1 transgene in the mam-
mary gland (5).
Besides performing a crucial role in cellular transformation,
members of the EGF-CFC family have been shown to be essen-
tial for early embryonic development (6). During embryogene-
sis, CR-1 functions as an essential coreceptor for nodal, a
member of the transforming growth factor (TGF-) family (7).
CR-1 apparently recruits nodal to the serine-threonine kinase
activin type I (ALK4)/activin type II receptor complex by inter-
acting with nodal through its EGF-like domain and with ALK4
through its CFC domain and allows nodal to induce Smad-2
phosphorylation and activation (8,9).
Despite the clear association between CR-1 overexpression
and human tumors, the mechanism used by CR-1 to promote
cellular transformation is not clear. CR-1 can activate two major
signaling pathways—a nodal/ALK4/Smad-2 signaling pathway
and a nodal- and ALK4-independent signaling pathway that
leads to activation of ras/raf/mitogen-activated protein kinase
(MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT sig-
naling pathways (7,9 –15). Activation of MAPK and PI3-K/AKT
pathways requires CR-1 to bind a cell-surface heparan sulfate
proteoglycan, glypican-1, and then to activate the cytoplasmic
tyrosine kinase c-Src (13).
Several approaches have been developed to inhibit CR-1
expression in tumor cells, including the use of monoclonal
antibodies and antisense technology. We have previously gen-
erated a panel of mouse anti-CR-1 monoclonal antibodies
(mAbs) that bind to different functional epitopes in the EGF-like
and CFC domains of CR-1 protein. Anti-CR-1 mAb A8.G3.5,
directed against the CFC domain, blocks the binding of CR-1 to
ALK4 and to activin B and inhibits up to 70% of the in vivo
growth of human testicular and colon cancer xenografts
in nude mice (16). CR-1 antisense oligonucleotides inhibit the in
vitro and in vivo growth of human breast, colon, ovarian, and
testicular carcinoma cells (17,18). In fact, treatment of nude
mice bearing human GEO colon carcinoma xenografts with
CR-1 antisense oligonucleotides reduces the microvessel density
in GEO tumors, suggesting that CR-1 might participate in neo-
vascularization during tumor formation (18).
We investigated whether CR-1 is involved in tumor angio-
genesis by evaluating the effect of CR-1 at different stages of
neovascularization of human vascular endothelial cells, includ-
ing proliferation, migration, invasion, and differentiation into
vascular-like structures. We also investigated whether the neu-
tralizing anti-CR-1 mAb A8.G3.5 can block angiogenesis in
vitro and in vivo.
MATERIALS AND METHODS
Cell Culture, Growth Factors, Inhibitors, and Antibodies
Human umbilical vein endothelial cells (HUVECs, purchased
from Clonetics Cambrex, Rockland, ME) were grown in the
endothelial cell growth medium system EBM-2 (Clonetics Cam-
brex) supplemented with endothelial growth supplements
(EGM-2 single quotes; Clonetics Cambrex) and 2% fetal bovine
serum (Invitrogen, Carlsbad, CA). For all the experiments,
HUVECs were used at passage 4 or less. Human MCF-7 breast
cancer cells were cultured and transfected with a human CR-1
expression vector or with an empty control Neo vector, as
previously described (10). A human glycosylphosphatidylinositol-
truncated recombinant CR-1 protein containing an Fc tag was
expressed in Chinese hamster ovary cells and purified, as pre-
viously described (19). Recombinant vascular endothelial
growth factor (VEGF) and basic fibroblast growth factor (bFGF)
proteins were purchased from R&D Systems (Minneapolis,
MN). The MAPK inhibitor PD98059, the PI3-K inhibitor
LY294002, the c-Src inhibitor PP2, and the VEGF receptor
tyrosine kinase inhibitor (VEGFRI) 4,4-chloro-2-fluorophe-
nylamino-6,7-dimethoxyquinazoline were purchased from Cal-
biochem (San Diego, CA). The ALK4 inhibitor SB-431542 was
kindly provided by GlaxoSmithKline (20). The blocking anti-
CR-1 mAb A8.G3.5, specific for the CFC domain, and the
nonblocking anti-CR-1 mAb B3.F6.17, specific for the amino-
terminal domain, were gifts from Biogen-Idec (16).
Reverse Transcription-Polymerase Chain Reaction (PCR)
Analysis for ALK4 and Nodal Expression in HUVECs
Total RNA (2 g) was prepared from HUVECs and reverse
transcribed to cDNA with Superscript II (Invitrogen) and with
random primers in a reaction volume of 20 L; 2 L of this
reaction mixture was used for PCR amplification with Platinum
PCR Supermix (Invitrogen). For nodal, PCR was performed for
30 cycles for 30 seconds at 94 °C, 45 seconds at 62 °C, and 45
seconds at 72 °C. For ALK4, PCR was performed for 30 cycles
as follows: 1 minute at 94 °C, 1 minute at 55 °C, and 1 minute
at 72 °C. Primers for amplification of human ALK4 and nodal
have been previously described (10,16).
Cell Proliferation Assay
HUVECs were cultured at 3 10
4
cells per well on 96-well
microtiter plates in EBM-2 medium containing 2% fetal bovine
serum and endothelial growth supplements (Clonetics Cam-
brex). After 24 hours, the cells were washed twice with
phosphate-buffered saline (PBS), and then EBM-2 medium
(Clonetics Cambrex) without supplements and serum but con-
taining recombinant CR-1 (1, 10, 50, or 100 ng/mL), VEGF (10
ng/mL), or bFGF (10 ng/mL) proteins was added. These con-
centrations of CR-1 and bFGF or VEGF have been previously
shown to stimulate proliferation of epithelial or endothelial cells
(9 –15,21). To measure cell proliferation, we used a colorimetric
assay that is based on the cleavage of the tetrazolium salt
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate (WST-1; Roche, Indianapolis, IN) to
formazan by mitochondrial dehydrogenase in viable cells. Cul-
tures were incubated for 48 hours at 37 °C, WST-1 (10 L per
well, dilution 1:10) was added, and cultures were incubated for
4 hours at 37 °C. The reaction product was quantified by
measuring the optical density of the formazan product at 450
nm. The experiment was repeated three times, and the samples
were tested in triplicate.
Migration and Invasion Assays
Cell migration and invasion assays, respectively, were per-
formed in fibronectin-coated Boyden chambers and Matrigel-
coated Boyden chambers (QCM-FN Quantitative Cell Migration
Assay and Cell Invasion Assay Kit, Chemicon, Temecula, CA).
For both migration and invasion assays, Dulbecco’s modified
Eagle medium (DMEM) containing 5% fetal bovine serum was
used in the lower Boyden chamber as the chemoattractant.
HUVECs were cultured in EBM-2 medium (Clonetics Cambrex)
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without serum and supplements for 24 hours, harvested by
trypsinization, and resuspended in DMEM containing 5% bo-
vine serum albumin at 4 10
5
cells per milliliter. We placed 0.5
mL of this cell suspension in the upper chamber with the
components indicated at the following concentrations: recombi-
nant VEGF at 10 ng/mL, recombinant CR-1 protein at 200
ng/mL, anti-CR-1 mAb A8.G3.5 at 25 g/mL, anti-CR-1 mAb
B3.F6.17 at 25 g/mL, VEGFRI at 1 or 10 mM, the MAPK
inhibitor PD98059 at 10 M, the PI3-K inhibitor LY294002 at
10 M, the c-Src inhibitor PP2 at 10 M, and the ALK4
inhibitor SB-431542 at 10 M. The Boyden chambers were
incubated overnight at 37 °C. Cells on the top side of the filter
were removed, and cells that had migrated and invaded the
Matrigel through the filter and attached to the bottom of the
membrane were stained with crystal violet stain solution
(Chemicon). The crystal violet stain solution was eluted with
10% acetic acid extraction buffer (Chemicon) and transferred to
wells of a 96-multiwell plate, and the absorbance was read at
595 nm in each well. Experiments were repeated three times
with duplicate samples.
Protein Phosphorylation and Western Blot Analysis
HUVECs were cultured in 60-mm-diameter plates (1.5 10
6
cells per plate) and were serum starved in complete serum-free
medium (Cellgro, Mediatech, Herndon, VA) for 24 hours. We
have previously shown that CR-1-mediated phosphorylation of
MAPK is detected within 5 minutes and that the CR-1-mediated
phosphorylation of c-Src, AKT, and Smad-2 is detected within
15–30 minutes (9,13). Cells were stimulated with CR-1 protein
at either 200 or 400 ng/mL for 5 minutes to assess MAPK
phosphorylation or for 30 minutes to assess Smad-2, AKT, and
c-Src phosphorylation. HUVECs were also pretreated with the
inhibitors 10 M PD98059 (for MAPK), 10 M LY294002 (for
PI3-K), 10 M PP2 (for c-Src), or 10 M SB-431542 (for
ALK4) for 30 minutes at 37 °C and then stimulated with CR-1
at 400 ng/mL as a control for the specificity of the various
inhibitors (13,20). Levels of phosphorylated and total MAPK,
AKT, Smad-2, and c-Src were measured by western blot anal-
ysis as previously described (9). Densitometric analysis of the
bands on the western blots was performed with the NIH Image
program (http://rsb.info.nih.gov/nih-image/). Density of the
bands was normalized to total protein expression of the non-
phosphorylated forms and expressed as relative band intensity
units (U).
In Vitro Capillary Tube Formation on Matrigel
HUVECs that had been cultured in EBM-2 medium contain-
ing serum and endothelial cell supplements were washed twice
with PBS and trypsinized, and then 5 10
4
HUVECs per well
were cultured in 96-well microtiter plates coated with Matrigel
(In Vitro Angiogenesis Assay Kit, Chemicon) in the presence of
the following components, as indicated: recombinant VEGF at
10 ng/mL, the anti-CR-1 mAb A8.G3.5 at 25 g/mL, the anti-
CR-1 mAb B3.F6.17 at 25 g/mL, recombinant CR-1 protein at
200 ng/mL, VEGFRI at 1 or 10 mM, 10 M PD98059, 10 M
LY294002, 10 M PP2, and 10 M SB-431542. After 18 hours,
tube formation was assessed under an inverted light microscope,
and cultures were photographed. The experiment was repeated
three times, with duplicate samples.
VEGF and bFGF Immunoassays
To assess secreted VEGF and bFGF levels in cell culture
medium, we used a quantitative sandwich enzyme immunoassay
(human bFGF and human VEGF Quantikine, R&D Systems). In
this assay, we compared Neo-transfected MCF-7 cells, which
have low levels of endogenous CR-1 expression, with CR-1-
transfected MCF-7 cells (10). Cell culture supernatant, from
80% confluent control Neo-transfected MCF-7 cells or CR-1-
transfected MCF-7 cells cultured in DMEM containing 10%
fetal bovine serum, was added to 96-well microtiter plates (200
L per well) that were precoated with a monoclonal antibody
specific for either VEGF or bFGF (R&D Systems) and incubated
for 2 hours at room temperature. After several washings with
PBS to remove any unbound protein, a horseradish peroxidase-
conjugated polyclonal antibody against human VEGF or bFGF
(R&D Systems) was added to the wells and the reaction mixture
was incubated for 2 hours at room temperature. The reaction was
developed by adding substrate solution containing hydrogen
peroxide and the chromogen tetramethylbenzidine at 200 L per
well (R&D Systems). After color development, the reaction was
stopped by adding 50 L of 2 N sulfuric acid to each well. The
intensity of the color in each well was read at 450 nm. VEGF
and bFGF standard curves ranging from 15.6 to 1000 pg/mL
were used to determine VEGF and bFGF concentrations in the
culture medium.
Directed In Vivo Angiogenic Assay (DIVAA)
Surgical-grade silicone tubes (1 cm long, with an internal
diameter of 0.15 cm; New Age Industries, Southampton, PA)
were closed at one end with metal plugs. For each angioreactor
implant, the lumen of each tube was filled with 18 Lof
Matrigel (Collaborative Research, Becton Dickinson) containing
the following components as indicated: recombinant VEGF at
50 ng/mL, recombinant CR-1 at 100 ng/mL, recombinant bFGF
at 50 ng/mL, anti-CR-1 mAb A8.G3.5 at 25 g/mL, and 1 10
4
Neo-transfected MCF-7 cells or 1 10
4
CR-1-transfected
MCF-7 cells (22). The tubes were held at 37 °C to allow the
Matrigel to solidify. Athymic nude mice (females, 6 8 weeks of
age; National Cancer Institute, Frederick, MD) were anesthe-
tized by intraperitoneal injection with 0.015 mL of 2.5% Avertin
(Aldrich, St. Louis, MO; 0.02 mg/g of body weight). Animal
care was in accordance with institutional guidelines, and all
experiments were performed under an approved protocol by the
National Institutes of Health. Two tubes were inserted into a skin
pocket in the flank of each anesthetized nude mouse, and the
pocket was sealed with surgical staples. After 9 days, the mice
were injected intravenously with fluorescein isothiocyanate
(FITC)-dextran, to quantify the vascular volume within the
angioreactors (25 mg/mL; 100 L per mouse; Sigma, St. Louis,
MO). After 20 minutes, the tubes were removed from the skin
pockets and photographed with an inverted light microscope.
Matrigel was removed from angioreactors and digested in 200
L of dispase solution (Collaborative Research). The FITC
fluorescence that was trapped in the implant was measured with
an HP Spectrophotometer (Perkin-Elmer, Foster City, CA) re-
flects the volume of blood circulating through the newly formed
capillary vessels (i.e., the vascular volume). The experiment was
repeated three times, with at least three mice per treatment
condition.
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MCF-7 Cell Xenograft Tumors in Nude Mice
The mammary fat pad of female nude mice (4 6 weeks of
age) was cleared of epithelium by the method of DeOme (23).
After the mice were anesthetized by intraperitoneal injection
with 0.015 mL of 2.5% Avertin (Aldrich; 0.02 mg/g of body
weight), we made a single incision laterally across the abdomen,
revealing the fourth mammary gland. We removed the nipple-
associated epithelial portion of the fourth mammary gland with
an electrical cauterizing scalpel, leaving a mammary fat pad
cleared of its epithelium. Neo-transfected or CR-1-transfected
MCF-7 cells were grown until they were 80% confluent,
trypsinized, and resuspended in DMEM to 5 10
6
cells per 100
L; all 100 L was injected into the cleared mammary fat pad
(five animals per group). At the same time, the mice also were
implanted subcutaneously in the intrascapular region with a
10-mg cholesterol-based pellet containing 0.72 mg of 17-
estradiol (Innovative Research of America, Sarasota, FL) to
allow the growth of estrogen-dependent MCF-7 cells (24). Tu-
mor volumes were measured with a caliber twice a week. After
3 weeks, mice were killed and tumors were excised. Results
shown are representative of two independent experiments with
similar results.
Immunohistochemistry and Assessment of Vessel Density
To evaluate tumor vessel density, MCF-7 tumor xenografts
were surgically removed from the mice after 3 weeks, fixed in
PBS-buffered formalin, and embedded in paraffin. Five-
micrometer-thick sections of the paraffin-embedded tissue were
deparaffinized in xylene, rehydrated in a series of graded eth-
anols, and predigested with a ready-to-use pepsin solution
(Digest-All3, Zymed, San Francisico, CA) for 10 minutes at
37 °C. Endogenous peroxidase activity was blocked by a
5-minute incubation in 3% H
2
O
2
. To identify intratumoral blood
vessels, the sections were then incubated for 1 hour at room
temperature with rabbit anti-CD31 polyclonal antibody (sc-
8306; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100.
This antibody is used to measure CD31, which is expressed on
endothelial cells and, therefore, identifies blood vessels. In neg-
ative controls, the primary antibody was replaced with an irrel-
evant control isotype IgG. Immunostaining was carried out with
the Vectastain ABC kit (Vector, Burlingame, CA) by the man-
ufacturer’s instruction. Color was developed with AEC peroxi-
dase substrate (Vector) by following the manufacturer’s instruc-
tions, and sections were counterstained with hematoxylin.
Average tumor vessel counts were obtained as described (25).
Briefly, areas with relatively high vascular density, commonly
referred to as hot spots, were identified microscopically at a
magnification of 100. CD31-positive vessels in these areas
were counted in three separate fields at a magnification of 400.
The mean value was calculated, and tissue vessel density was
expressed as the number of vessels per field.
Statistical Analysis
Student’s t test was used to assess the statistical significance
of the differences between various groups in the western blot
analysis. For all other experiments, the statistical significance of
differences between groups was evaluated by using the nonpara-
metric Wilcoxon rank sum test. Results were expressed as the
mean values and 95% confidence intervals (CIs). Statistical
calculations were performed with the use of Statistical Package
for Social Sciences software package, version 11.0 (SPSS, Chi-
cago, IL). All statistical tests were two-sided, and data were
considered statistically significant at P.05.
RESULTS
CR-1 and the Nodal/ALK4/Smad-2 and Glypican-1/c-Src/
MAPK/AKT Signaling Pathways in HUVECs
HUVECs express the signaling pathway components ALK1,
ALK5, glypican 1, c-Src, MAPK, and AKT (26 –29). Because
CR-1 can signal through the activation of the glypican-1/c-Src/
MAPK/AKT or nodal/ALK4/Smad-2 signaling pathway, we in-
vestigated whether HUVECs also express nodal and ALK4. By
use of reverse transcription-PCR and primers specific for human
nodal and ALK4, we detected the expression of both nodal and
ALK4 in HUVECs (Fig.1, A). We next determined whether
treatment of HUVECs with recombinant CR-1 enhances the
activation (i.e., phosphorylation) of intracellular signaling mol-
ecules that function downstream of glypican-1 or nodal and
ALK4. The level of MAPK phosphorylation was threefold
higher in CR-1-treated HUVECs than in untreated HUVECs
(Fig. 1, B). The level of AKT phosphorylation was twofold
higher and the level of c-Src phosphorylation was threefold
higher in CR-1-treated HUVECs than in untreated HUVECs
(Fig. 1, C and D). In addition, CR-1 treatment activated a
nodal/ALK4/Smad-2 pathway in HUVECs, as shown by a two-
fold increase in the level of Smad-2 phosphorylation (Fig. 1, E).
Pretreatment of HUVECs with the specific inhibitors of MAPK
(PD98059), AKT (LY294002), c-Src (PP2), or ALK4 (SB-
431542) statistically significantly interfered with the CR-1-
induced phosphorylation of the corresponding signaling compo-
nent in HUVECs (e.g., for phosphorylated AKT, CR-1 at 400
ng/mL and LY294002 55 U versus CR-1 159 U; difference
104 U, 95% CI 93 to 114 U; P .005; and, for phosphor-
ylated c-Src, CR-1 at 400 ng/mL and PP2 65.5 U versus CR-1
169 U; difference 103.5 U, 95% CI 94 to 108 U; P
.003) (Fig. 1, B–E). Thus, all components of both nodal/ALK4/
Smad-2 and glypican-1/c-Src/MAPK/AKT signaling pathways
are expressed in HUVECs, appear to be activated by CR-1, and
may contribute to the function of CR-1.
CR-1 and Proliferation of Human Endothelial Cells
Proliferation of endothelial cells (HUVECs) is an important
step during early angiogenic events. We evaluated the ability of
purified recombinant CR-1, VEGF, and bFGF proteins to stim-
ulate HUVEC proliferation. All three proteins stimulated
HUVEC proliferation under serum-free conditions (Fig. 2).
VEGF treatment increased proliferation of HUVECs by 53%
(95% CI 42% to 63%; P .004), and bFGF treatment
increased proliferation of HUVECs by 218% (95% CI 204%
to 231%; P .004), both compared with the proliferation of
untreated HUVECs. CR-1 also induced a statistically significant
dose-dependent increase in the proliferation of HUVECs. The
activity of CR-1 at 10 ng/mL was comparable to that of VEGF
(57% increase, 95% CI 47% to 67%; P .039), and the
activity of CR-1 at 100 ng/mL was comparable to that of bFGF
(212% increase, 95% CI 200% to 223%; P .004). Thus,
CR-1 could induce the proliferation of HUVECs as well as the
potent angiogenic molecules VEGF and bFGF.
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CR-1 and HUVEC Migration and Invasion
To form new blood vessels in a tumor, endothelial cells must
invade the tumor’s extracellular matrix and migrate through its
basement membrane into the remodeled perivascular space. To
determine whether CR-1 affects endothelial cell migration and
invasion, HUVECs were incubated in Boyden chambers with
recombinant CR-1 protein or recombinant VEGF protein alone
or in combination for 18 hours. For the migration assay, we used
membranes coated with the extracellular matrix protein fi-
bronectin, which promotes cell adhesion and allows cell migra-
tion. For the invasion assay, we used membranes coated with
Matrigel, which cells must degrade to reach the underside of the
membrane. Treatment with CR-1 or VEGF individually in-
creased the number of migrating and invading cells approxi-
mately twofold (for the migration assay, CR-1 265% versus
control 100%, difference 165%, 95% CI 158% to 172%,
P .031; for the invasion assay, CR-1 195% versus control
100%, difference 95%, 95% CI 93% to 97%, P .031)
(Fig. 3). The combination of VEGF and CR-1 proteins did not
enhance HUVEC migration or invasion further (Fig. 3). In
addition, treatment of HUVECs with a specific VEGF receptor
tyrosine kinase inhibitor (VEGFRI) induced a dose-dependent
inhibition of VEGF-enhanced cell migration or invasion but did
not affect the level of CR-1-induced migration or invasion,
suggesting that CR-1 is not acting through the autocrine produc-
tion of VEGF.
To elucidate the type of signaling pathways involved in
CR-1-induced HUVEC migration and invasion, we used specific
inhibitors of signaling molecules and the neutralizing anti-CR-1
mAb A8.G3.5, which has been shown to inhibit tumor growth in
vivo (16), and the control nonblocking anti-CR-1 mAb B3.F6.17
(also of the IgG1 isotype) (16). CR-1-induced enhanced migra-
tion and invasion of HUVECs was statistically significantly
inhibited by adding the blocking mAb A8.G3.5 with CR-1 (55%
and 42% inhibition in migration and invasion assays, respec-
tively, P .031 for both sets of results), the c-Src inhibitor PP2
(approximately 50% inhibition in both assays), or the PI3-K
inhibitor LY2940002 (83% and 48% inhibition in migration and
invasion assays, respectively) (Fig. 3). In contrast, no statisti-
cally significant decrease in the endothelial cell migration and
invasion in response to CR-1 was observed in the presence of the
MAPK inhibitor PD98059 or the control nonblocking anti-CR-1
Fig. 1. Expression of signaling pathway components in human umbilical vein
endothelial cells (HUVECs). A) Reverse transcription–polymerase chain reac-
tion using specific primers for ALK4 (435 base pairs [bp]) and nodal (251 bp)
in HUVECs. In the lane to the left is shown HaeIII fragments of X174
replicative form DNA, shown as DNA markers. BE) Mitogen-activated protein
kinase (MAPK), AKT, c-Src, and Smad-2 activation by cripto-1 (CR-1) in
HUVECs. Serum-starved HUVECs were stimulated with recombinant CR-1
protein at 200 ng/mL or 400 ng/mL for 5 minutes for MAPK phosphorylation or
for 30 minutes for AKT, c-Src, and Smad-2 phosphorylation, as indicated.
Phosphorylation was analyzed by western blot analysis with antibodies against
phosphorylated and nonphosphorylated forms of MAPK (B), AKT (C), c-Src
(D), and Smad-2 (E). Cells were also pretreated with the MAPK inhibitor
PD98059 at 10 M(B), the phosphatidylinositol 3-kinase (PI3-K) inhibitor
LY294002 at 10 M(C), the c-Src inhibitor PP2 at 10 M(D), or the ALK4
inhibitor SB-431542 at 10 M(E) and then stimulated with recombinant CR-1
protein (400 ng/mL).
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mAb B3.F6.17. The ALK4 inhibitor SB-431542 induced a non-
statistically significant reduction in endothelial cell migration,
whereas it had no effect on CR-1-induced endothelial cell inva-
sion (Fig. 3). Finally, the blocking anti-CR-1 mAb A8.G3.5 had
no effect on VEGF-enhanced migration and invasion in
HUVECs. Thus, c-Src and PI3-K/AKT appear to be directly
involved in mediating CR-1 chemotactic function in HUVECs,
and the neutralizing anti-CR-1 mAb A8.G3.5, which recognizes
an epitope in the CFC domain of CR-1, appears to block the in
vitro proangiogenic function of CR-1 in HUVECs by inhibiting
CR-1-induced endothelial cell migration and invasion.
CR-1 and In Vitro Morphologic Differentiation of
HUVECs
To explore the ability of CR-1 to induce angiogenesis, we
evaluated the formation of vascular tube-like structures by CR-
1-treated cells in an in vitro Matrigel assay. In the absence of
growth factors, HUVECs did not form tube-like structures and
remained round and isolated on the Matrigel bed (Fig. 4).
Addition of recombinant CR-1 protein induced formation of
distinct rings and cords of cells (Fig. 4) that were visible by 18
hours. VEGF induced similar structures. Coadministration of
CR-1 and VEGF did not further increase the formation of
vascular structures over that observed with CR-1 or VEGF
alone. VEGFRI strongly blocked the formation of VEGF-
induced vascular structures by HUVECs but did not affect
CR-1-induced capillary-like tube formation. The combination of
CR-1 and anti-CR-1 mAb A8.G3.5, PP2, or LY294002 mark-
edly decreased formation of vascular structures, but the combi-
nation of CR-1 and MAPK inhibitor PD98059, the ALK4 in-
hibitor SB-431542, or the nonblocking mAb B3.F6.17 did not
affect the CR-1-induced formation of vascular structures. Fi-
nally, the blocking anti-CR-1 mAb A8.G3.5 did not interfere
with VEGF-induced formation of vascular structures. Thus,
CR-1 appears to stimulate differentiation of endothelial cells
through a c-Src/PI3-K/AKT signaling pathway.
CR-1 and Neovascularization In Vivo
We had shown that CR-1 stimulates endothelial cell prolif-
eration, migration, invasion, and differentiation in vitro. How-
ever, agents that affect angiogenesis in vitro do not always affect
angiogenesis the same way in vivo. Therefore, we used a re-
cently described quantitative in vivo angiogenesis assay—the
directed in vivo angiogenesis assay—to determine the angio-
genic potential of CR-1-overexpressing MCF-7 human breast
cancer cells or of purified recombinant CR-1 protein (22). We
have previously shown (10) that CR-1 overexpression in MCF-7
Fig. 2. Cripto-1 (CR-1) and the proliferation of human umbilical vein endothelial
cells (HUVECs). Serum-starved HUVECs were stimulated with vascular endo-
thelial growth factor (VEGF, 10 ng/mL), basic fibroblast growth factor (bFGF,
10 ng/mL), or CR-1 (1, 10, 50, or 100 ng/mL). After 48 hours, 10 L per well
of the reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate (WST-1) was added, and the assay was quantified by
measuring the optical density (OD) of the dye solution at 450 nm. Data are
expressed as percentage of increase over control. Error bars upper 95%
confidence interval. *P .004, compared with control. All statistical tests were
two-sided.
Fig. 3. Cripto-1 (CR-1) and migration and invasion of human umbilical vein
endothelial cells (HUVECs). HUVECs were seeded on fibronectin-coated filters
for the migration assay (A) and Matrigel-coated filters for the invasion assay (B).
Endothelial cells were incubated with vascular endothelial growth factor (VEGF,
10 ng/mL) alone or in combination with monoclonal antibody (mAb) A8.G3.5
(25 g/mL) or with VEGF receptor tyrosine kinase inhibitor (VEGFRI, 1 or 10
M), or CR-1 (200 ng/mL) alone or in combination with VEGF (10 ng/mL), the
mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10 M), phos-
phatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (10 M), c-Src inhibitor
PP2 (10 M), ALK4 inhibitor SB-431542 (10 M), mAb A8.G3.5 (25 g/mL),
mAb B3.F6.17 (25 g/mL), or VEGFRI (1 or 10 M). After an overnight
incubation, cells that migrated or invaded were stained with crystal violet stain
solution (Chemicon). Stain was eluted with 10% acetic acid extraction buffer
(Chemicon) and transferred to a 96-multiwell plate, and absorbance was read at
595 nm in each well. Error bars upper 95% confidence interval. *, P .031,
compared with control; **, P .031, compared with VEGF-treated cells; and
***, P .031, compared with CR-1-treated cells. All statistical tests were
two-sided.
Journal of the National Cancer Institute, Vol. 97, No. 2, January 19, 2005 ARTICLES 137
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cells is associated with enhanced levels of invasion and a more
aggressive phenotype in vitro. VEGF and bFGF are secreted in
the supernatant of Neo-transfected MCF-7 cells and CR-1-
transfected MCF-7 cells, but expression of CR-1 did not appear
to increase the levels of VEGF and bFGF, as determined by
immunoassay (data not shown). Neo-transfected MCF-7 cells or
CR-1-transfected MCF-7 cells and recombinant CR-1, bFGF, or
VEGF proteins were embedded in Matrigel inside semiclosed
silicone cylinders (i.e., angioreactors) and implanted subcutane-
ously into nude mice. We also combined CR-1 and VEGF
proteins to evaluate whether they cooperate to enhance new
vessel formation in vivo. After 9 days, the implants were re-
moved, and the degree of vascularization within these angio-
reactors was quantified after the intravenous injection of FITC-
dextran into the nude mice as an indirect measure of the volume
of blood circulating through the newly formed vessels within the
silicone cylinders (22).
Implants containing either CR-1 protein or CR-1-overexpressing
MCF-7 cells contained visible capillaries within the angioreac-
tors, whereas the cells transfected with the empty vector had
very few new vessels (Fig. 5, A–C). Implants containing either
purified recombinant CR-1 protein or CR-1-overexpressing
MCF-7 cells had a statistically significantly greater volume of blood
in newly formed vessels than implants containing Matrigel alone
or control Neo-transfected MCF-7 cells (recombinant CR-1 pro-
tein alone 259% versus control 100%, difference 159%,
95% CI 147% to 171%, P.001; and CR-1-transfected
MCF-7 cells 243% versus Neo-transfected MCF-7 cells
129%, difference 114%, 95% CI 109% to 118%, P.001)
(Fig. 5, D). Combined treatment with VEGF and CR-1 resulted
only in a slight increase of new vessel formation in vivo,
compared with CR-1 or VEGF alone. As expected, a statistically
significant angiogenic response was also detected in angioreac-
tors containing VEGF or bFGF (P.001); these responses were
similar to but less than that induced by CR-1. Finally, we
investigated the ability of anti-CR-1 mAb A8.G3.5 to inhibit
CR-1-induced angiogenesis in vivo. Addition of anti-CR-1 mAb
A8.G3.5 to CR-1 statistically significantly inhibited new vessel
formation in angioreactors (CR-1 and A8.G3.5 127% versus
CR-1 alone 259%, difference 132%, 95% CI 123% to
140%, P.001), suggesting that this neutralizing anti-CR-1 an-
tibody has substantial antiangiogenic activity in vivo (Fig. 5, D).
CR-1 and New Vessel Formation in CR-1-Expressing
MCF-7 Xenografts
We next determined whether the CR-1 expressed by CR-1-
transfected MCF-7 cells would enhance tumorigenesis of these
cells in nude mice and/or neovascularization of the tumors.
17-Estradiol-dependent Neo-transfected MCF-7 cells or CR-1-
transfected MCF-7 cells were injected into the cleared mammary
fat pad of nude mice implanted subcutaneously with 17-
estradiol pellets (five mice per group). If mice were not im-
planted subcutaneously with 17-estradiol pellets, MCF-7 cells
did not grow (10). After 10 days, mice that received CR-1-
transfected MCF-7 cells started to develop tumors, whereas no
tumors were detected in the control group, which received
Neo-transfected MCF-7 cells (CR-1-transfected MCF-7 tumors
0.11 mm
2
[mean] versus Neo-transfected MCF-7 tumors 0
mm
2
[mean]; difference 0.11 mm
2
, 95% CI 0.07 to 0.14
mm
2
; P .032; Fig. 6, A).
After 20 days, however, all mice in
both groups had tumors, with no statistically significant differ-
ence in tumor volume between them (Fig. 6, A). Finally, the
endothelial cell marker CD31 identified a statistically signifi-
cantly higher microvessel density in sections of CR-1-
transfected MCF-7 tumors than Neo-transfected MCF-7 tumors
(CR-1-transfected MCF-7 tumors 4.66 vessels per field versus
Neo-transfected MCF-7 2.33 vessels per field; difference
2.33 vessels per field, 95% CI 1.2 to 2.8 vessels per field;
P .004; Fig. 6, B and C).
DISCUSSION
We show, to our knowledge for the first time, that CR-1 has
a strong angiogenic activity in cultured endothelial cells (i.e.,
Fig. 4. Cripto-1 (CR-1) and human umbilical vein endothelial cell (HUVEC)
differentiation into vascular-like structures. HUVECs were cultured in Matrigel-
precoated wells in absence (control) or in presence of vascular endothelial
growth factor (VEGF), VEGF plus monoclonal antibody (mAb) A8.G3.5 (25
g/mL), VEGF plus VEGF receptor tyrosine kinase inhibitor (VEGFRI, 1 or 10
M), CR-1 plus VEGF, CR-1, CR-1 plus mAb A8.G3.5 (25 g/mL), CR-1 plus
mAb B3.F6.17 (25 g/mL), CR-1 plus PP2 (10 M), CR-1 plus LY294002 (10
M), CR-1 plus PD98059 (10 M), CR-1 plus SB-431542 (10 M), or CR-1
plus VEGFRI (1 or 10 M). VEGF and CR-1 proteins were used at 10 ng/mL
or 200 ng/mL, respectively.
138 ARTICLES Journal of the National Cancer Institute, Vol. 97, No. 2, January 19, 2005
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HUVECs). CR-1 enhanced the proliferation of HUVECs in a
dose-dependent manner, suggesting that it can function as a
mitogenic factor for endothelial cells, as do VEGF and bFGF
(30). CR-1 also induced migration and invasion of HUVECs, as
does VEGF. Moreover, CR-1-treated HUVECs aligned to form
vascular-like structures on Matrigel, indicating that CR-1 can
stimulate differentiation in cultured endothelial cells. Because
the specific inhibitor VEGFRI did not block CR-1-induced mi-
gration, invasion, and capillary-tube formation in HUVECs,
CR-1 does not appear to function through the autocrine produc-
tion of VEGF.
We also demonstrated that CR-1 can stimulate endothelial
cells by activation of a c-Src/MAPK/PI3-K/AKT or a nodal/
ALK4/Smad-2 signaling pathway. Specific inhibitors of c-Src or
PI3-K strongly inhibited CR-1-induced migration, invasion, and
differentiation of HUVECs. We have previously shown that
activation of c-Src and PI3-K/AKT signaling pathways in mam-
mary epithelial cells or in human cervical and breast cancer cell
Fig. 6. Overexpression of cripto-1 (CR-1) in MCF-7 cells and microvessel density.
A) Neo-transfected MCF-7 cells (MCF-7 Neo) or CR-1-transfected MCF-7 cells
(MCF-7 CR-1) were inoculated in the cleared fat pad of nude mice implanted with
17-estradiol pellets (five mice per group). Tumor size at 10 and 20 days after
injection of the cells was determined with a caliper. B) Immunohistochemistry for
the endothelial cell marker CD31 in CR-1-transfected MCF-7 cell tumor sections.
Brown color and arrowheads indicate endothelial cells and representative blood
vessels, respectively. C) Microvessel quantification. Tissue vessel density was
expressed as the number of vessels per high-power magnification field. Error bars
upper 95% confidence interval. *P .039. All statistical tests were two-sided.
Fig. 5. Cripto-1 (CR-1) and angiogenesis. Angio-
genesis was assessed in vivo by using the directed
in vivo giogenesis assay. Paired angioreactors re-
covered at 9 days after implantation with Neo-
transfected MCF-7 cells (A), CR-1-transfected
MCF-7 cells (B), or recombinant CR-1 protein
(100 ng/mL) (C). In the figure, angioreactors are
oriented with the open end to the left and with the
sealed end to the right. Arrows vascular struc-
tures within the lumen. D) Quantification of di-
rected in vivo angiogenesis assay by fluorescein
isothiocyanate (FITC)– dextran injection. Results
are expressed as relative fluorescence units (per-
centage of control) of FITC-dextran. Recombinant
proteins and monoclonal antibody (mAb) were
used at the following concentration: basic fibro-
blast growth factor (bFGF, 50 ng/mL), vascular
endothelial growth factor (VEGF, 50 ng/mL),
CR-1 (100 ng/mL) alone or in combination with
mAb A8.G3.5 (25 g/mL) or VEGF (50 ng/mL).
MCF-7 Neo Neo-transfected MCF-7 cells;
MCF-7 CR-1 CR-1-transfected MCF-7 cells.
Error bars upper 95% confidence interval. *,
P.001, compared with control; **, P.001, com-
pared with CR-1-containing angioreactors; ***,
P.001, compared with no-transfected MCF-7
cells. All statistical tests were two-sided.
Journal of the National Cancer Institute, Vol. 97, No. 2, January 19, 2005 ARTICLES 139
by guest on June 12, 2013http://jnci.oxfordjournals.org/Downloaded from
lines is required for CR-1-dependent cell migration, invasion,
and transformation (2,4,10,12,13). The PI3-K/AKT signaling
pathway has been identified as a major regulator of VEGF-
induced endothelial cell motility (27,28). However, the inability
of a specific MAPK inhibitor to block CR-1-induced HUVEC
migration, invasion, and morphologic differentiation in our
study demonstrates that these CR-1-induced responses do not
require activation of a MAPK signaling cascade. Similarly, the
MAPK inhibitor PD98059 had no effect on VEGF-induced
endothelial cell migration (27).
The inhibition of CR-1-induced migration, invasion, and
capillary-like tube formation induced by treatment with a neu-
tralizing anti-CR-1 mAb, but not by the nonblocking anti-CR-1
mAb B3.F6.17, strongly indicates that CR-1 directly modulates
angiogenesis in HUVECs. The mAb A8.G3.5 binds to an
epitope in the CFC domain of CR-1, thereby preventing CR-1
binding to ALK4 and perturbing nodal signaling (16). Surpris-
ingly, blockade of ALK4/Smad-2 signaling pathway with the
ALK4 inhibitor SB-431542 had a marginal effect on the CR-1-
induced proangiogenic phenotype in HUVECs in vitro. How-
ever, the CFC domain may also bind to other proteins that act
through an ALK4-independent signaling pathway. For example,
CR-1 can bind to glypican-1, Vg1/GDF1, Lefty, activin, and
tomoregulin 1 (13,16,31–34). Therefore, mAb A8.G3.5 may
interfere with the binding of CR-1 to one or more of these
signaling molecules in addition to blocking a nodal/ALK4 sig-
naling pathway. Alternatively, CR-1 may function as an activin
antagonist (16,34). Because activin has been recently shown to
inhibit proliferation and differentiation of vascular endothelial
cells in vitro, mAb A8.G3.5 might disrupt the binding of CR-1
to activin B and thereby restore activin B growth suppression in
endothelial cells (35). In agreement with our results, blocking
the function of activin with SB-431542 does not inhibit prolif-
eration and/or differentiation of endothelial cells (35). Interest-
ingly, treatment of HUVECs with the combination of CR-1 and
SB-431542 actually produced a higher level of differentiation
than treatment with CR-1 alone (Fig. 4), suggesting that CR-1
and SB-431542 cooperate in blocking activin signaling in endo-
thelial cells.
The strong CR-1-induced angiogenic effects in endothelial
cells observed in vitro were then extended by in vivo experi-
ments. CR-1, either as a purified recombinant protein or when
overexpressed in MCF-7 breast cancer cells, stimulated various
endothelial cell responses that are associated with microvessel
formation in vivo. Anti-CR-1 mAb A8.G3.5 inhibited 50% of
the CR-1-induced neovessel formation, suggesting that this mAb
has potent antiangiogenic activity and that the strong antitumor
activity exerted by mAb A8.G3.5 in testicular and colon cancer
xenografts in nude mice may be mediated by its direct anti-
angiogenic activity and by its inhibition of tumor cell prolifer-
ation (16).
Interestingly, coadministration of CR-1 and VEGF did not
produce a greater effect than that observed with administration
of CR-1 or VEGF alone, suggesting that these two angiogenic
molecules may act through the same signaling pathways. In this
regard, PI3-K and AKT have been strongly implicated in medi-
ating different VEGF functions and, therefore, may be common
effector molecules in the process of angiogenesis induced by
VEGF and/or CR-1 (28).
In accord with the involvement of CR-1 in regulating new
blood vessel formation, overexpression of CR-1 in MCF-7
breast cancer cells enhanced tumor neovascularization in a xeno-
graft model. Because CR-1 overexpression in MCF-7 cells did
not increase the secretion of VEGF or bFGF, CR-1 may not be
acting indirectly in MCF-7 cells by inducing VEGF and/or
bFGF expression. Although CR-1-transfected MCF-7 cells pro-
duced tumors more quickly than control Neo-transfected MCF-7
cells, after 20 days the size of both types of MCF-7 mammary
tumor xenografts were comparable. We have previously shown
(10) that CR-1 overexpression fails to induce an estrogen-
independent phenotype in estrogen-responsive MCF-7 cells.
However, in serum-free conditions, CR-1 overexpression in
MCF-7 cells could enhance proliferation, survival, and invasion
(10). Therefore, in our xenograft model, estrogen supplementa-
tion, which is normally required for the proper growth of
estrogen-dependent MCF-7 cells in vivo, may account for some
of the reduction in CR-1-induced tumor growth observed.
In conclusion, we have demonstrated a critical role for CR-1
signaling in angiogenesis and have shown that a blocking anti-
CR-1 mAb inhibits microvessel formation in vivo. We believe
that further experiments are warranted to determine whether the
blocking anti-CR-1 mAb is a good therapeutic candidate to
inhibit angiogenesis.
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11.
NOTES
This work was supported in part by a grant from the Associazione Italiana per
la Ricerca sul Cancro (AIRC) to Nicola Normanno.
Manuscript received April 30, 2004; revised October 25, 2004; accepted
November 22, 2004.
Journal of the National Cancer Institute, Vol. 97, No. 2, January 19, 2005 ARTICLES 141
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    • "It has been suggested that CR-1 functions in conjunction with Nodal in cancer stem cell populations to promote tumorigenesis in melanoma and testicular tumors [17, 18]. Under some circumstances, CR-1 can promote cell proliferation, migration, invasion, or stimulate angiogenesis [19, 20]. On the other hand, it can promote apoptosis [21], or inhibit cell proliferation [22]. "
    [Show abstract] [Hide abstract] ABSTRACT: Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors.
    Full-text · Article · May 2015
    • "We believe that our findings could underscore the specific roles of trophoblast cells at the maternal-fetal interface. CRIPTO-1 signaling within tumor cells has previously been demonstrated to modulate cellular growth, survival, and invasion in several human cancers [30, 32, 33], and this could be especially relevant to the biology of trophoblast cells. In particular, extravillous cytotrophoblast cells are nonproliferative and exhibit a low apoptotic index during the late stages of gestation, which suggests the hypothesis that CRIPTO-1 contributes to trophoblast invasiveness or cell survival mechanisms [34]. "
    [Show abstract] [Hide abstract] ABSTRACT: CRIPTO-(CR)1 is a protein associated with tumorigenesis and metastasis. Here we demonstrate that CR-1 expression in normal and creta placentas is associated with various degrees of uterine invasion. Cytokeratin (CK) and CR-1 protein expression was visualized by immunohistochemical staining of formalin-fixed, paraffin-embedded placental specimens (control placentas, n = 9; accreta, n = 6; increta, n = 10; percreta, n = 15). The pattern of extravillous trophoblast (EVT) cell morphology was distinctive in creta placentas: densely-compacted cell columns and large star-shaped cells with a typically migratory phenotype, not common among third trimester control placentas. Quantification revealed higher CR-1 immunoreactivities in accreta (P = 0.001), increta (P = 0.0002), and percreta placentas (P = 0.001) than in controls. In contrast to controls, there was a significant positive relationship between CR-1 and CK reactivity in all creta placentas (accreta, P = 0.02; increta, P = 0.0001, and percreta, P = 0.025). This study demonstrated CR-1 expression in the placental bed, its increased expression in creta placentas, and EVT cells as the main CR-1-producing cell type. Morphological examination revealed an immature and invasive trophoblast profile in creta placentas, suggesting impairment of the trophoblast differentiation pathway. These findings provide important new insights into the pathophysiology of abnormal creta placentation and its gestational consequences.
    Full-text · Article · Aug 2014
    • "We performed endothelial cell tubule formation assays (2D assays) in accordance with the previous report 16. To incorporate a monolayer of tumor cells, we modified the 2D assays in the following manner. "
    [Show abstract] [Hide abstract] ABSTRACT: We have developed novel phenotypic fluorescent three-dimensional co-culture platforms that efficiently and economically screen anti-angiogenic/anti-metastatic drugs on a high-throughput scale. Individual cell populations can be identified and isolated for protein/gene expression profiling studies and cellular movement/interactions can be tracked by time-lapse cinematography. More importantly, these platforms closely parallel the in vivo angiogenic and metastatic outcomes of a given tumor xenograft in the nude mouse model but, unlike in vivo models, our co-culture platforms produce comparable results in five to nine days. Potentially, by incorporating cancer patient biopsies, the co-culture platforms should greatly improve the effectiveness and efficiency of personalized chemotherapy.
    Full-text · Article · Jun 2013
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