Post on 12-Jan-2023
IGF-II regulates metastatic properties of choriocarcinomacells through the activation of the insulin receptor
L.E. Diaz1, Y-C. Chuan2, M. Lewitt2, L. Fernandez-Perez3, S. Carrasco-Rodrıguez1,M. Sanchez-Gomez1,4 and A. Flores-Morales2
1Hormone Laboratory, Department of Chemistry, Universidad Nacional de Colombia, Bogota, Colombia; 2Department of Molecular
Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 3Department of Clinical Sciences, Molecular Endocrinology Group,
University of Las Palmas de G.C., Canary Institute for Cancer Research, RTICCC, Spain
4Correspondence address. Tel: þ57-1-316-5000 ext. 14466; Fax: þ57-1-316-5220; E-mail: mysanchezd@unal.edu.co
Choriocarcinoma is a highly malignant tumor that can arise from trophoblasts of any type of gestational event but most often from
complete hydatidiform mole. IGF-II plays a fundamental role in placental development and may play a role in gestational
trophoblastic diseases. Several studies have shown that IGF-II is expressed at high levels in hydatidiform moles and choriocarci-
noma tissues; however, conflicting data exist on how IGF-II regulates the behaviour of choriocarcinoma cells. The purpose of this
study was to determine the contribution of the receptors for IGF-I and insulin to the actions of IGF-II on the regulation of
choriocarcinoma cells metastasis. An Immuno Radio Metric Assay was used to analyse the circulating and tissue levels of
IGF-I and IGF-II in 24 cases of hydatidiform mole, two cases of choriocarcinoma and eight cases of spontaneous abortion at
the same gestational age. The JEG-3 choriocarcinoma cell line was used to investigate the role of IGF-II in the regulation of
cell invasion. We found that mole and choriocarcinoma tissue express high levels of IGF-II compared to first trimester placenta.
Both IGF-I and IGF-II regulate choriocarcinoma cell invasion in a dose dependent manner but through a different mechanism.
IGF-II effects involve the activation of the InsR while IGF-I uses the IGF-IR. The positive effects of IGF-II on invasion are the
result of enhanced cell adhesion and chemotaxis (specifically towards collagen IV). The actions of IGF-II but not those of
IGF-I were sensitive to inhibition by the insulin receptor inhibitor HNMPA(AM)3. Our results demonstrate that the insulin
receptor regulates choriocarcinoma cell invasion.
Keywords: choriocarcinoma; hydatidiform mole; metastasis; IGF-II; insulin receptor
Introduction
Gestational trophoblastic neoplasias comprise a group of
pregnancy-related disorders that include pre-malignant complete and
partial hydatidiform mole as well as malignant lesions such as
invasive mole, choriocarcinoma, and placental site trophoblastic
tumour (PSTT) (Altieri et al., 2003). Complete moles are diploid
and nearly always androgenetic in origin. By contrast, partial moles
are triploid, consisting of one maternal and two paternal sets of
chromosomes. After uterine evacuation, 10–20% of complete and
0–5% of partial moles undergo malignant change. For complete
and, to a lesser extent, partial moles this malignant change includes
invasive mole, choriocarcinoma and PSTT (Soper, 2006).
Choriocarcinoma is a highly malignant tumor that can arise from
trophoblasts of any type of gestational event but most often from com-
plete hydatidiform mole (Seckl et al., 2000). The pathogenesis of these
tumors is poorly understood. Due to the complete absence of maternal
genome in complete hydatidiform mole, the function of paternally
expressed genes has been studied in relation to choriocarcinoma
development (Lustig-Yariv et al., 1997; He et al., 1998; Kim et al.,
2003). IGF-II is expressed in many tissues from the paternally
derived allele. Several studies have shown that IGF-II is expressed
in hydatidiform moles and choriocarcinoma tissues although this
expression is not related to paternal imprinting (Kim et al., 2003).
Therefore, IGF-II appears to play an important role in the pathogenesis
of choriocarcinoma in addition to its well known function in the
regulation of normal placenta development (Kim et al., 2003).
IGF-I, IGF-II, the receptors for IGF-I, IGF-II and insulin as well as
several IGF binding proteins are produced by trophoblastic cells,
establishing a complex network of autocrine and paracrine actions
that is yet poorly understood (Han and Carter, 2000; Constancia
et al., 2002). Specifically, conflicting reports exist on how IGF-II
regulates the behaviour of choriocarcinoma cells (Korner, 1995;
Mckinnon et al., 2001). Initial reports implicated the Mannose 6-P/
IGF-II receptor in proliferative actions of IGF-II on choriocarcinoma
cell lines (Mckinnon et al., 2001) but later reports have clearly demon-
strated that this receptor has mainly inhibitory effects on IGF-II
actions (Li and Sahagian, 2004). Here, we have used the JEG-3 chor-
iocarcinoma cell line to study the contribution of the receptors for
IGF-I and insulin to the actions of IGF-II, specifically on cell
adhesion, invasion and proliferation. Our findings indicate that
IGF-I and IGF-II use different receptors to regulate the invasive
properties of choriocarcinoma cells although they share downstream
mechanisms involving ERK and PI3- kinase activation.
Materials and Methods
Subjects and clinical samples
Twenty-four patients (mean age 21.9+7.8 years) with complete hydatidiform
mole were diagnosed in gestational weeks eight through 20 with clinical,
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For
Permissions, please email: journals.permissions@oxfordjournals.org 567
Molecular Human Reproduction Vol.13, No.8 pp. 567–576, 2007
Advance Access publication June 6, 2007 doi:10.1093/molehr/gam039
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
histopathology, microsatellite and gonadotropin (hCG) analysis; and two
patients diagnosed with choriocarcinoma were included in the study. Placenta
from eight patients (mean age 27.4+7.5 years) with non-molar abortion
during weeks eight through 18 and confirmed negative for choriocarcinoma
or hydatidiform mole were used in the study. All patients were selected from
hospitals in Bogota, Colombia and written consent was obtained. All
procedures were approved by the local ethics committee. Blood samples
were obtained and serum was separated by centrifugation. Tissues were
obtained by curettage and maintained at 2708C until further analysis.
Materials
Recombinant human IGF-I was obtained from Genentech (San Francisco, CA),
recombinant human IGF-II from Upstate Biotechnology, Inc. (Lake Placid,
NY), recombinant human insulin from Sigma Chemicals (St Louis, MI) and
recombinant IGFBP-1 was purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). The monoclonal mouse antihuman IGF-I receptor antibody
(aIR3) and polyclonals against subunit b-IGF-IR, subunit b-insulin receptor,
Akt 1/2, phospho-Akt (Ser 473), Erk 1/2 were purchased from Santa Cruz Bio-
technology (Santa Cruz, CA). A polyclonal goat antihuman a5b1 antibody
(MAB1969) (Conforti et al., 1989) was purchased from Chemicon Inter-
national Inc. (Mississauga, ON). U0126, a specific inhibitor of MEK kinase,
was purchased from Promega (Madison, WI). LY294002, a PI3 kinase inhibitor
was obtained from Cell Signalling (Beverly, MA). HNMPA(AM)3
[Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester], an
inhibitor of insulin receptor (InsR) tyrosine kinase activity (Saperstein et al.,
1989), was purchased from Alexis, Biochemicals (Alexis, San Diego, CA).
Mouse monoclonal antibody (Clon 4G10) against phosphotyrosine and
IGF-II neutralizing antibody S1F2 were purchased from Upstate Biotechnol-
ogy, Inc. (Lake Placid, NY). A polyclonal rabbit antihuman anti-p-Erk 1/2
(Thr202/Tyr204) was obtained from Cell Signalling Technology (Beverly,
MA). Goat antimouse IgG (H þ L) was obtained from Southern Biotech
(USA).
Measurement of IGFs
Total IGF-I, IGF-II and IGFBP-1 concentrations were determined by immunor-
adiometric assay (IRMA) using a commercial kit DSL (Webster, TX; IGF-I
DSL-10-5600, IGF-II DSL-10-2600 and IGFBP-1 DSL-10-7800). Standards
and samples were tested in duplicate. One part frozen tissue was weighed,
thawed and homogenized in two parts 0.06 M Tris-hydrochloride (pH 7.2) in
a Polytron homogenizer (Kinematica Gmbh, Luzern, Switzerland) at 48C.
Total IGF-I was extracted from crude tissue homogenate with buffer, according
to the manufacturer’s instructions. Total protein in the homogenates was deter-
mined by the Bradford method.
Cell culture and treatments
The choriocarcinoma cell line JEG-3 was maintained as previously described
(Zhang and Shiverick, 1997). For the experiments, cells were sub-cultured in
60 mm culture for 24–48 h before the experiments were performed. Stimu-
lation with 10 nM IGF-I, 10 nM IGF-II, 10 nM insulin or 10 mg/ml IGFBP-1
was performed on cells kept in serum-free media for 12 h. LY294002
(40 mM), U0126 (20 mM) and HNMPA(AM)3 (10, 50 or 100 mM) were
added to the cells 2 h prior to stimulation.
Western blotting
JEG-3 cells were cultivated and treated as described above. Cells were har-
vested using RIPA buffer (Tris 50 mM, pH 7.4, NaCl 150 mM, Triton X-100
1%) supplemented with protease inhibitors and protein concentration was
determined with BCA Protein Assay Reagent (Pierce, USA). Equal amounts
of protein extracts were denatured and separated on a 12% SDS-PAGE gel
(Invitrogen Life Technologies, UK) and blotted onto a PVDF membrane
(Amersham, Life Science, UK). Membranes were then blocked in a
Tris-buffered saline solution with 5% BSA or 5% non-fat dry milk and
probed with primary antibody. The membranes were further probed with
HRP-conjugated goat anti-mouse/anti-rabbit to visualize the specific band.
Immunoprecipitation
Immunoprecipitations of cell extracts were performed by incubating lysates
with the indicated antibodies. The reaction mixture was gently rocked at 48Covernight. Protein G-agarose (Amersham, Life Science, UK) was used to
absorb immunocomplexes that were washed extensively with ice-cold RIPA
buffer containing 0.1% Triton X-100. The pellet was resuspended in loading
buffer, boiled for 5 min, and centrifuged at 14 000 rpm (5 000 g) for 5 min.
The supernatant was collected and subjected to western blotting.
Matrigel invasive assay
Matrigel invasion assays were performed as essentially described before
(Chuan et al., 2006). Matrigel was purchased from Beckton Dickinson (San
Jose, CA) and stored at 2208. 0.2 ml aliquot containing 200 000 cells was
seeded on the upper chamber of the Matrigel coated transwell filter. Serum-free
DMEM containing IGF-I, IGF-II or IGFBP-1 was added to the lower chamber
and incubated for 24 h at 378C in a humidified atmosphere of 5% CO2. Each
assay was carried out in triplicate on at least three different occasions.
Matrigel adhesion assay
Matrigel adhesion assay was performed as described (Zhang and Yeh, 2004),
with some modifications. JEG-3 cells were suspended in serum-free media.
The cell suspension (2.5 � 105 cells in 0.5 ml medium/well) was added to
24 wells pre-coated with Matrigel and allowed to attach by incubation at
378C for 60 min. Unattached cells were removed by repeated washing with
PBS. Attached cells were stained with 0.5% Crystal Violet for 10 min. After
washing with water, the stained cells were extracted with 0.25 ml of 10%
acetic acid and the absorbance of the dye extract was measured at 570 nm.
Cell proliferation assays
Cell proliferation was assessed using the MTT assay (Mosmann, 1983).
Measurements were done at the time of seeding (t ¼ 0) and 24 h after treat-
ment. Each plate was scanned and measured in a Spectra Shell microplate
reader (SLT, Austria) at 570 nm.
Cell motility assay
Cell migration assays were performed using modified Boyden chambers with a
6.5-mm diameter, 10-mm thickness, porous (8.0 mm) polycarbonate membrane
separating the two chambers (Transwellw; Costar, Cambridge, MA). Briefly,
the lower surface of the membrane was coated with fibronectin, collagen IV,
laminin or BSA (20 mg/ml in phosphate-buffered saline, pH 7.4 for 2 h at
378C). Excess ligand was removed, and the lower chambers were loaded
with medium DMEM with IGF-I (10 nM), IGF-II (10 nM), insulin (10 nM)
or IGFBP-1 (10 ng/ml). 100 000 serum-starved JEG-3 cells were plated on
the upper wells of transwell chambers and incubated at 378C for 24 h. Cells
on the upper surface of membranes were completely removed; the cells that
migrated on the lower surface of the filter were fixed in 4% formaldehyde,
stained with hematoxylin and counted under a microscope. Each assay was
carried out in triplicate on at least three different occasions. To verify
whether IGF-I, IGF-II, insulin or IGFBP-1-stimulated migration of the JEG-3
cells were mediated through IGF-IR, IR or a5b1 integrin activation, the
same procedures were performed in JEG-3 cells that were preincubated with
aIR3 (1 mg/ml), HNMPA(AM)3 (50 mM) or MAB1969 (5 mg/ml) for 60 min.
RNA extraction, cDNA synthesis and RT–PCR
RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (5 mg) from
different samples were first treated with DNase I (Promega) and then reverse
transcribed using Superscript II (Invitrogen) in a reaction volume of 20 ml.
The synthesized cDNA was used to measure the InsR expression by
PCR. The reactions were performed in 20 ml with 1 ml of the respective
cDNA sample and 0.4 mM primers, Taq DNA polymerase, buffer and
nucleotides (Invitrogen). The primers used were: for GAPDH,
50GTGAAGGTCGGAGTCAACG30 and 50GGTGGAGACGCCAGTGGAC30
and for the InsR, 50AACCAGAGTGAGTATGAGGAT30 and
50CCGTTCCAGAGCGAAGTGCTT30.
Diaz et al.
568
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
Results
IGF-II is highly expressed in gestational trophoblasticdisease tissue
Trophoblastic cells are known to express IGF-II. Although the steady
state mRNA levels of IGF-II have been measured in different
trophoblastic diseases (Kim et al., 2003), no information is yet
available regarding the protein levels of this growth factor in these
tissues. Because we intend to study IGF-II effects on trophoblastic
cells, it was important to estimate IGF-II protein concentration
within the tissue. We used IRMA to analyse IGF-II and IGF-I
levels in choriocarcinoma, complete hydatidiform mole as well as
first trimester placenta tissue samples. As shown in Fig. 1, placenta
contains an average concentration of 1.38+0.21 nM (0.84+0.23 ng/mg protein) of IGF-I and 2.66+0.63 nM (0.43+0.18 ng/mg protein) of IGF-II. Hydatidiform moles, express signifi-
cantly (P , 0.05) higher levels of IGF-II in comparison with placenta
with levels of 7.33+0.58 nM (1.12+0.12 ng/mg protein). In
contrast, a lower amount of IGF-I (P , 0.05) was observed in moles
as compared to placenta with measured levels of 0.92+0.16 nM
(0.29+0.11 ng/mg protein). During this investigation, two choriocar-
cinoma samples were available for study. The individual levels of
IGF-II were 7.95 nM (1.23 ng/mg protein) and 9.37 nM (1.28 ng/mg
protein), which place them in the upper range of concentration
values measured in the samples. In addition, we observed high
IGF-II circulating levels in sera of patients with hydatidiform mole
compared to those with spontaneous abortions at the same gestational
age (Fig. 1B). In summary, these results indicate that mole and
choriocarcinoma tissue contain high levels of IGF-II, sufficient to
saturate all known high affinity receptors (Denley et al., 2005).
IGF-II regulates choriocarcinoma cell invasion
We next studied the role of IGF-II in the regulation of choriocarci-
noma cell invasion. JEG-3 choriocarcinoma cells were used since
they are known to express all known receptors for IGF-I and
IGF-II (Ritvos et al., 1988; O’Gorman et al., 1999). In preliminary
experiments measuring ERK1/2 phosphorylation, we determined
that maximal receptor activation is achieved with 5 nM of both
IGF-I and IGF-II, which is in line with the known Kd for these
receptors (Denley et al., 2005). The invasive properties of JEG-3
choriocarcinoma cells were measured using a matrigel cell invasion
assay. As shown in Fig. 2A, both IGF-I and IGF-II treatment induced
JEG-3 matrigel invasion in a dose dependent manner. Maximal
effects were obtained with concentrations of 10 nM for both
ligands. Similar treatment has little effect on cell proliferation
within the time frame analysed (Fig. 2B). Interestingly, despite the
fact that receptor saturating concentrations of IGF-I (Kd ¼ 0.2 nM)
are used (Denley et al., 2005), concomitant addition of IGF-II
further increases cell invasion suggesting that IGF-II uses receptors
other than IGF-IR to promote cell invasion (Fig. 2C). We also tested
the effects of IGFBP-1 alone or in combination with the type I and
type II IGFs. IGFBP-1 is known to be expressed in placenta and is
thought to negatively regulate IGF-I activity (Crossey et al., 2002)
but also to have IGF-I independent actions through direct
interactions with integrin a5b1 receptor (Jones et al., 1993).
Treatment with IGFBP-1 alone induced JEG-3 invasion and has an
additive effect to the actions of IGF-II. In contrast, no additive
effect was observed on the actions of IGF-I which is indicative of
a negative interaction between the two growth factors (Crossey
et al., 2002).
In order to better understand the nature of IGF-I, IGF-II and
IGFBP-1 actions on choriocarcinoma cells, the effects on cell
invasion of IGF-IR blocking antibody aIR3 and anti-a5b1 integrin
receptor antibody were measured. As shown in Fig. 3A, treatment
with aIR3 Ab completely inhibits the effects of IGF-I on cell
invasion and has a small although significant effect in the actions
of IGF-II. The actions of IGFBP-1 on cell invasion were not affected
by treatment with aIR3 Ab. On the other hand, treatment with
anti-a5b1 integrin reduces the actions of IGFBP-1 but was inactive
towards IGF-I or IGF-II (Fig. 3B). An isotype Ab (IgG) did not
influence basal or IGF-I/ IGF-II induced cell invasion (Fig. 3C).
These results strongly suggest that receptors other than IGF-I
receptor or integrin a5b1 are used by IGF-II to promote choriocarci-
noma cell invasion.
Figure 1: IGF-I and IGF-II protein expression in hydatidiform mole (A) Boxplots showing the absolute levels of IGF-I and IGF-II in hydatidiform mole incomparison to placenta from spontaneous abortions. (B) Box plots showing thelevels of IGF-II in sera from patients with complete hydatidiform mole in com-parison to those of similar age with spontaneous non-molar abortions. Statisti-cal analysis was performed using analysis of variance (ANOVA). Differentletters indicate statistically significant differences (P , 0.05)
IGF-II regulation of trophoblast invasion
569
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
IGF-II induces choriocarcinoma cell invasion through theactivation of the insulin receptor
The InsR, specifically the isoform A, can bind IGF-II with high
affinity (Kd ¼ 0.9 nM), Therefore, we wanted to test the effect of
IGF-I and IGF-II on IGF-IR and InsR activation. We first analysed
the expression levels of InsR isoforms in placenta tissue. Fig. 4A
shows that both InsR-A and InsR-B are expressed in placenta
(Benecke et al., 1992), hydatidiform mole and in JEG-3 trophoblastic
cell lines. We next used JEG-3 cells to study InsR and IGF-IR
activation. As shown in Fig. 4B, IGF-I treatment induced the tyrosine
phosphorylation of the IGF-I receptor, an effect that was inhibited
by blocking antibody aIR3. No induction of InsR tyrosine
phosphorylation by IGF-I was observed. On the other hand, IGF-II
treatment induced the InsR tyrosine phosphorylation with no
evidence of IGF-I receptor activation. The IGF-IR blocking antibody
had no discernible effect on IGF-II induced InsR activation. On the
other hand, treatment with InsR inhibitor HNMPA(AM)3 caused a
dose dependent inhibition of IGF-II-induced InsR tyrosine
phosphorylation. This inhibition was observed from 10 to 100 nM.
Similar concentrations also inhibited insulin-mediated activation of
the InsR. In contrast, HNMPA(AM)3 was able to inhibit IGF-I
activation of the IGF-I receptor at only the higher concentration. In
summary, both the direct measurement of IGF-II effects on receptor
activation as well as the actions of HNMPA(AM)3 demonstrate that
Figure 2: Regulation of JEG-3 trophoblastic cell invasion by IGF-I, IGF-IIand IGFBP-1. (A) Matrigel invasion assays to measure the treatment effectof increasing concentrations of IGF-I and IGF-II on JEG-3 cell invasion.(B) Effects of IGF-I (10 nM), IGF-II (10 nM), foetal calf serum-FCS (10%)and BSA (2%) on cell proliferation measured by MTT assay after 24 h treat-ment. (C) Matrigel invasion assays to measure the effects of JEG-3 cells treat-ments with different combinations of IGF-I (10 nM or 20 nM as indicated),IGF-II (10 nM or 20 nM as indicated) or IGFBP1 (10 ng/ml). In each casethe results of at least three independent replicates are shown. Statistical analysiswas performed using ANOVA. Different letters indicate statistically significantdifferences (P , 0.05) between cells treated with a single ligand and thosetreated with a combination of two growth factors
Figure 3: Effects of IGF-I receptor blocking Ab, aIR-3 (1 mg/ml) (A) andintegrin a5b1 blocking Ab MAB1969 (25 mg/ml) (B) on IGF-I, IGF-II andIGFBP1 induced cell invasion; the effects of non-immune goat IgG(1 mg/ml) on IGF-I and IGF-II induced cell invasion is shown for comparison(C) In each case the results of at least three independent replicates are shown.Statistical analysis was performed using ANOVA. Different letters indicatestatistically significant differences (P , 0.05)
Diaz et al.
570
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
Figure 4: Activation of IGF-IR and InsR by IGF-I, IGF-II and insulin in JEG-3 cells (A) RT–PCR based detection of insulin receptor isoforms A and B expressionin placenta from non-molar spontaneous abortions (lines 1–4 and 11), in complete hydatidiform mole (lines 5–10 and 12) and in JEG-3 trophoblastic cell line (line13). (B) The upper panel shows the tyrosine phosphorylation of the IGF-IR and InsR upon treatment with IGF-I (10 nM) or IGF-II (10 nM) given alone or incombination with either different doses of IR inhibitor HNMPA(AM)3 or the IGF-IR blocking Ab aIR-3 (1 mg/ml). The lower panel shows the tyrosine phosphoryl-ation of the IGF-I receptor and InsR receptor upon treatment with IGF-I (10 nM) or insulin (10 nM) given alone or in combination with either different doses of IRinhibitor HNMPA(AM)3 or the IGFR blocking Ab aIR-3 (1 mg/ml). (C) Effects of IR inhibitor HNMPA(AM)3 on IGF-I (10 nM), IGF-II (10 nM) and insulin(10 nM) induced matrigel cell invasion. Statistical analysis was performed using ANOVA. Different letters indicate statistically significant differences (P , 0.05)
IGF-II regulation of trophoblast invasion
571
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
the InsR is the key mediator of IGF-II actions in choriocarcinoma
cells. The analysis of cell invasion (Fig. 4C) shows that
HNMPA(AM)3 treatment at concentrations (50 mM) that specifically
inhibit IR activation by IGF-II also blocked ligand induced cell
invasion. In contrast, IGF-I induced cell invasion was not affected
by similar concentrations of HNMPA(AM)3 although inhibitory
effects are evident at 100 mM, parallel to inhibition of IGF-IR
activation. As expected, IGF-I induced cell invasion was inhibited
by the IGF-IR (aIR3) blocking antibody.
IGF-II regulates cell invasion through its activation ofAKT, ERK pathways and the induction of cell adhesionand motility
In order to understand the nature of IGF-II actions, we analysed signal-
ling pathways downstream of the IGFR and IR, which are potentially
important in the regulation of cell invasion. The activity of AKT was
measured by Western Blot using anti-phospho Ser 473; while active
ERK was measured using a specific Ab against Thr202/Tyr204. The
results are depicted in Fig. 5. We found that treatment with IGF-II,
as well as insulin, IGF-I and IGFBP-1 induced the activation of
ERK as well as AKT. Similar to previous findings regarding receptor
activation, IGF-IR blocking antibody, aIR3 inhibited IGF-I activation
of ERK and AKT but failed to inhibit IGF-II action on these kinases
(Fig. 5C). IGF-II stimulatory activity towards ERK and AKT was
instead sensitive to inhibition by low concentrations (10 mM) of the
IR inhibitor HNMPA(AM)3. The relevance of ERK and AKT
activation in cell invasion was next studied using matrigel invasion
assays. Treatment with PI3-kinase inhibitor LY294002 and MEK
inhibitor U0126, which reduce the activation of AKT and ERK
respectively, resulted in significant inhibition of IGFs-induced cell
invasion (Fig. 5D).
For cells to invade through a thick layer of matrigel a complex set of
biological processes have to take place. This includes the attachment of
cells to the matrigel, degradation of extracellular matrix and motility
towards specific matrix protein components. In order to understand
the nature of IGF-II actions on cell invasion, we measured both cell
adherence as well as motility towards three different extracellular
matrix proteins: collagen IV, fibrinogen and laminin (Fig. 6). We
found that IGF-II treatment induces the attachment of JEG-3 cells to
matrigel, an effect that was sensitive to inhibition by HNMPA(AM)3.
Similar effects could be observed when treating with insulin. IGF-I
has analogous effects through the activation of the IGF-I receptor.
The analysis of cell motility (Fig. 6B) showed that IGF-II treatment
resulted in a 3-fold increase in motility towards collagen IV. Similar
effects were observed after IGF-I treatment, but in addition, enhanced
motility towards fibrinogen and laminin was also observed. Similar to
what was observed when analysing cell invasion, IGF-II but not IGF-I
effects were sensitive to pharmacological inhibition with the InsR
kinase inhibitor HNMPA(AM)3 used at 50 mM. The above evidence
demonstrates qualitative differences in the way IGF-I and IGF-II
regulate cell motility.
Discussion
This study showed that the circulating and tissue levels of IGF-II are
elevated in gestational trophoblastic diseases, specifically complete
hydatidiform moles when compared to first trimester placenta from
spontaneous abortions. In addition, we have investigated the
mechanisms whereby IGF-II regulates the invasive properties of
trophoblastic cells. Our results show that IGF-II treatment, like
IGF-I, insulin and IGFBP-1, results in enhanced invasion of JEG-3
trophoblastic cells. IGF-II employs a distinct mechanism to initiate
its actions on trophoblasts, which differs from that used by
IGF-I. IGF-II effects are mainly initiated through the activation of
the InsR while IGF-I mostly uses the IGF-IR to initiate signalling.
On the other hand, both IGFs share common intracellular mechanisms
including the activation of ERK and AKT kinases, which are import-
ant for the regulation of cell invasion. The positive effects of IGF-II on
invasion are the result of enhanced cell adhesion and chemotaxis
(specifically towards collagen IV). In comparison to IGF-I, IGF-II
actions were more sensitive to inhibition by the InsR inhibitor
HNMPA(AM)3.
The role of IGF-II in normal placental development is well
established (Baker et al., 1993; Louvi et al., 1997). IGF-II is
abundantly expressed in human placenta, specifically in extravillous
cytotrophblasts invading into the endometrium (Han and Carter,
2000). Consequently, a role for IGF-II in the regulation of invasive
trophoblasts in normal placenta has been proposed. This study
showed that the protein expression levels of IGF-II are highly elevated
in gestational trophoblastic diseases compared to first trimester
placenta from spontaneous abortions. In the two cancer tissues we
obtained, high levels of IGF-II were also detected. Although, due to
the scarcity of tumour cases, no statistical analysis was performed,
the concentrations in choriocarcinoma are in the range observed for
complete hydatidiform mole. Increased IGF-II protein levels in
gestational trophoblastic disease are likely to be explained by
enhanced expression of this gene. Previous results have shown that
loss of imprinting of IGF-II gene locus occur in 43% of complete
hydatidiform moles concomitant to increased mRNA levels of the
gene (Kim et al., 2003). Therefore, enhanced local production of
IGF-II can potentially contribute to progression of gestational
trophoblastic diseases and choriocarcinoma pathology. In addition,
increased circulating levels of IGF-II in sera may contribute to the
invasive properties of choriocarcinoma cells in distant metastatic sites.
How IGF-II exerts its actions on trophoblastic cells is a matter of
great interest. At least two different receptors have been previously
proposed to initiate the actions of IGF-II on trophoblastic cells; the
IGF-II/Mannose-6 phosphate receptor (Mckinnon et al., 2001) and
the IGF-I receptor. Initial findings pointed to the IGF-II/Mannose-6
phosphate receptor based on the use of antibodies against the receptor
(Mckinnon et al., 2001), but numerous studies have demonstrated that
this receptor has mainly inhibitory actions on IGF-II (Ludwig et al.,
1996; O’Gorman et al., 1999, 2002). Other studies have postulated
that IGF-II actions are mediated by the IGF-I receptor (Pandini
et al., 2002). Here we demonstrate that IGF-II signalling in JEG-3
cells is initiated mainly through the activation of the InsR. Two
different lines of evidence support our findings. Firstly, IGF-II treat-
ment of JEG-3 cells mainly induced the tyrosine phosphorylation of
the InsR but little effect is observed in IGF-I receptor. In contrast,
IGF-I treatment induced the tyrosine phosphorylation of the IGF-I
receptor while no effect on InsR activation was observed. Secondly,
although both IGFs were able to activate ERK and AKT, IGF-II but
not IGF-I effects were inhibited by concentrations of
HNMPA(AM)3 that specifically inhibit the InsR (Fig. 5A). Pharmaco-
logical inhibition of the InsR with 50 mM HNMPA(AM)3 completely
blocked IGF-II induced cell invasion and motility towards collagen IV
but were ineffective against IGF-I. On the other hand a blocking
antibody against the IGF-I receptor has no effect on IGF-II actions
in the above described parameters but effectively inhibited all IGF-I
actions.
Mouse gene knockout studies first identified the important role of
the InsR in mediating the growth promoting effects of IGF-II in
early development (Louvi et al., 1997). The InsR exists in two
isoforms that originate by alternative splicing of exon 11 in the InsR
gene. The two isoforms are identical except 12 amino acids inserted
Diaz et al.
572
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
upstream of the third last residue of the extracellular alpha subunits of
the InsR-B isoform (Moller et al., 1989). The InsR-A isoform can bind
insulin and IGF-II with high affinity but binds IGF-I poorly (Frasca
et al., 1999; Denley et al., 2004). Therefore, it is likely that the
InsR-A is the receptor mediating IGF-II actions on trophoblast cells.
Indeed, previous reports show that InsR-A is highly expressed in
placenta as compared with normal adult tissues such as liver and
muscle, which mainly express InsR-B (Benecke et al., 1992).
Figure 5: Effect of IGF-I, IGF-II or insulin treatment on ERK and AKT activation in JEG-3 cells Phosphorylation of ERK and AKT kinases upon treatment withIGF-I (10 nM) (A and B), IGF-II (10 nM) (A and C), insulin (10 nM) (B) or IGFBP-1 (C) given alone or in combination with either different doses of (A) the InsRinhibitor, HNMPA(AM)3, (A and C) the IGF-IR blocking Ab, aIR-3 (1 mg/ml), (C) the MEK inhibitor UO126, (C) the PI3 kinase inhibitor LY94002 and (C) anIGF-II neutralizing antibody S1F2 (10 mg/ml). (D) Effects of MEK inhibitor UO126 and PI3 kinase inhibitor LY294002 on IGF-I, IGF-II induced cell invasion.Statistical analysis was performed using ANOVA. Different letters indicate statistically significant differences (P , 0.05)
IGF-II regulation of trophoblast invasion
573
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
An intriguing observation was that despite using similar
concentrations of IGF-II and insulin and the latter having slightly
higher affinity for InsR-A, IGF-II is significantly more potent than
insulin in the promotion of cell invasion and motility. Interestingly,
similar findings have been described in SKUT-1 cells in relation to
cell invasion (Sciacca et al., 2002). This suggests that different
signalling pathways are triggered by IGF-II and insulin despite
sharing a common receptor. It is important to notice that moderate
(10 nM) concentrations of IGF-II and insulin were used in the
experiments, which in the case of IGF-II will preferentially activate
Figure 6: IGF-I, IGF-II and IGFBP-1 regulation of JEG-3 trophobalstic cell adhesion and motility. (A) Effect of IGF-I (10 nM), IGF-II (10 nM) IGFBP-1 (10 ng/ml) and insulin (10 nM) treatment on JEG-3 cells adhesion to matrigel. The effects of combined treatment with IGF-I receptor blocking Ab, aIR-3 (1 mg/ml), theintegrin a5b1 blocking Ab, MAB1969 (25 mg/ml) and the InsR kinase inhibitor HNMPA(AM)3. (B) Results from assays measuring the effects of IGF-I (10 nM),IGF-II (10 nM) or insulin (10 nM) and IGFBP-1 (10 ng/ml) on JEG-3 cells motility towards specific extracellular matrix proteins: laminin, collagen IV and fibro-nectin. BSA was used as control. The effects of combined treatment with IGF-I receptor blocking Ab, aIR-3 (1 mg/ml), the integrin a5b1 blocking Ab, MAB1969(25 mg/ml) and the InsR kinase inhibitor, HNMPA(AM)3 are also shown. In each case the results of at least three independent replicates are shown. Statistical analy-sis was performed using ANOVA. Different letters indicate statistical significant differences (P , 0.05)
Diaz et al.
574
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
the InsR-A isoform while insulin will activate both InsR-A and B. It
has been long known that the different isoforms of the InsR have
distinct signalling properties despite having identical intracellular
domains (Sciacca et al., 2003; Pandini et al., 2004). The reasons for
this are not fully understood, although recent findings indicate that
both receptors localized to discrete regions in the cell membrane
and are differentially internalized in response to insulin (Uhles
et al., 2003); thereby suggesting the involvement of additional
membrane proteins in signalling. One possibility to explain the
enhanced invasive activity of IGF-II is through the activation of
hybrid insulin/IGF-I receptors that bind IGF-II but not insulin with
high affinity. We do not favour this hypothesis because: (i) we were
not able to detect IGF-I receptor activation upon IGF-II treatment
and (ii) IGF-II effects on cell invasion were inhibited by
HNMPA(AM)3 used at concentrations that specifically target the
InsR. To note is that, IGF-I can also bind hybrid receptors with high
affinity, yet we cannot detect increased InsR tyrosine phosphorylation
upon IGF-I treatment, again suggesting a minor role for hybrid recep-
tors in signalling. Clearly, the issue of how receptor hybrids contribute
to signalling requires further exploration, specially if we keep in mind
that the relative effect of HNMPA(AM)3 on hybrids is not known.
Another possibility to explain the different potency of IGF-II and
insulin in cell invasion assays is that InsR-A triggers different
signals depending on whether it is activated by IGF-II or insulin.
Indeed, support for this hypothesis comes from the study of transcrip-
tional actions of IGF-II and insulin in fibroblasts that lack IGF-I
receptor and overexpress InsR-A (Pandini et al., 2003). This study
showed that although most transcriptional actions of insulin and
IGF-II are shared, some ligand specific effects could be detected
such as the induction of integrin aVb3 and adhesion molecule
ICAM. Interestingly, IGF-II and IGF-I but not insulin are known to
bind vitronectin, a ligand for integrin aVb3 to promote cell migration
(Kricker et al., 2003). Therefore, integrin aVb3 activation by IGF-II/
vitronectin complex can bear direct relationship to the enhanced
effects of IGF-II on JEG-3 cell migration as compared to insulin.
Clearly, a better understanding of how the InsR isoforms contribute
to IGF-II signalling is required before the different potency of
IGF-II and insulin in cell motility assays can be explained.
Despite the differences in signalling, insulin, IGF-I, IGF-II as well
as IGFBP-1 have the capacity to promote cell invasion using a core set
of signalling intermediaries. This is evident by the fact that PI3 kinase
and MEK inhibitors reduce cell invasion induced by the three ligands.
Consequently, in the context of the tissue, trophoblast invasion may be
primarily determined by factors that influence the availability of IGFs
and its binding proteins. Because we have shown that IGF-II but not
IGF-I is overexpressed in placental tissue from complete hydatidiform
moles and in corresponding sera, IGF-II is likely to be more influential
than IGF-I in the regulation of the invasive properties of trophoblasts
from complete moles. Supporting this hypothesis are previous findings
showing that extravillous cytotrophoblasts, which have a very high
invasive capacity, express the highest amounts of IGF-II in normal
placenta while syncitiotrophoblasts express little IGF-II (Han and
Carter, 2000). In order to promote cell invasion, IGF-II has to regulate
complex cellular mechanisms that control adhesion, migration and
extracellular matrix proteolysis. We have shown that IGF-II controls
invasion by at least regulating cell adhesion and motility.
Interestingly, IGF-II but also IGF-I and insulin promote motility
specifically towards collagen IV but not laminin or fibronectin
suggesting a specific regulation of integrin subunits that act as
collagen receptors (Denley et al., 2006). In contrast, IGFBP-1 the
main secretory product of the decidua also promotes invasion
towards fibronectin, in line with its known function as a ligand of
the main fibronectin receptor: the a5/b1 integrin complex (Jones
et al., 1993; Gleeson et al., 2001). The specific activation of fibronec-
tin receptor by IGFBP-1 but not IGF-II also provides an explanation
for its enhancing effects of IGF-II induced invasion. Future studies
are needed to clarify which specific integrin receptors are influenced
by IGF-II as well as the role of matrix proteolysis in IGF-II-induced
trophoblast invasion.
In conclusion, we have shown that IGF-II induces trophoblast cell
invasion through the activation of the InsR. Clearly, additional
studies are needed to better characterize the role of the different
InsR isoforms in the regulation of IGF-II actions. The development
of a specific inhibitor of the IGF-II/InsR-A receptor interaction may
have important applications. It could potentially be used for the
pharmacological treatment of choriocarcinomas and the several
types of tumours known to overexpress IGF-II, while preserving the
metabolic actions of insulin.
Acknowledgements
This work has been supported by grants to M.S.-G. from COLCIENCIAS andto A.F.M. from the Swedish Research Council and Wallenberg Foundation.Y.-C.C. was supported by Cancerfonden. L.F.-P. was supported by grantsfrom Pfizer Spain CN-78/02-05045 and MEC SAF2003-02117.
References
Altieri A, Franceschi S, Ferlay J, Smith J, La Vecchia C. Epidemiology andaetiology of gestational trophoblastic diseases. Lancet Oncol2003;4:670–678.
Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growthfactors in embryonic and postnatal growth. Cell 1993;75:73–82.
Benecke H, Flier JS, Moller DE. Alternatively spliced variants of the insulinreceptor protein. Expression in normal and diabetic human tissues. J ClinInvest 1992;89:2066–2070.
Chuan YC, Pang ST, Cedazo-Minguez A, Norstedt G, Pousette A,Flores-Morales A. Androgen induction of prostate cancer cell invasion ismediated by ezrin. J Biol Chem 2006;281:29938–29948.
Conforti G, Zanetti A, Colella S, Abbadini M, Marchisio PC, Pytela R,Giancotti F, Tarone G, Languino LR, Dejana E. Interaction of fibronectinwith cultured human endothelial cells: characterization of the specificreceptor. Blood 1989;73:1576–1585.
Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A,Fundele R, Stewart F, Kelsey G, Fowden A, Sibley C et al.Placental-specific IGF-II is a major modulator of placental and fetalgrowth. Nature 2002;417:945–948.
Crossey PA, Pillai CC, Miell JP. Altered placental development andintrauterine growth restriction in IGF binding protein-1 transgenic mice.J Clin Invest 2002;110:411–418.
Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW,Wallace JC. Structural determinants for high-affinity binding ofinsulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minusisoform of the IR. Mol Endocrinol 2004;18:2502–2512.
Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecularinteractions of the IGF system. Cytokine Growth Factor Rev2005;16:421–439.
Denley A, Brierley GV, Carroll JM, Lindenberg A, Booker GW, Cosgrove LJ,Wallace JC, Forbes BE, Roberts CT, Jr. Differential activation of insulinreceptor isoforms by insulin-like growth factors is determined by the Cdomain. Endocrinology 2006;147:1029–1036.
Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, Goldfine ID,Belfiore A, Vigneri R. Insulin receptor isoform A, a newly recognized,high-affinity insulin-like growth factor II receptor in fetal and cancer cells.Mol Cell Biol 1999;19:3278–3288.
Gleeson LM, Chakraborty C, McKinnon T, Lala PK. Insulin-like growthfactor-binding protein 1 stimulates human trophoblast migration bysignaling through alpha 5 beta 1 integrin via mitogen-activated proteinKinase pathway. J Clin Endocrinol Metab 2001;86:2484–2493.
Han VK, Carter AM. Spatial and temporal patterns of expression of messengerRNA for insulin-like growth factors and their binding proteins in the placentaof man and laboratory animals. Placenta 2000;21:289–305.
IGF-II regulation of trophoblast invasion
575
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from
He L, Cui H, Walsh C, Mattsson R, Lin W, Anneren G, Pfeifer-Ohlsson S,Ohlsson R. Hypervariable allelic expression patterns of the imprintedIGF2 gene in tumor cells. Oncogene 1998;16:113–119.
Jones JI, Gockerman A, Busby WH, Jr, Wright G, Clemmons DR. Insulin-likegrowth factor binding protein 1 stimulates cell migration and binds to thealpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc NatlAcad Sci U S A 1993;90:10553–10557.
Kim SJ, Park SE, Lee C, Lee SY, Kim IH, An HJ, Oh YK. Altered imprinting,promoter usage, and expression of insulin-like growth factor-II gene ingestational trophoblastic diseases. Gynecol Oncol 2003;88:411–418.
Korner C, Nurnberg B, Uhde M, Braulke T. Mannose 6-phosphate/insulin-likegrowth factor II receptor fails to interact with G-proteins analysis of mutantcytoplasmic receptor domains. J Biol Chem 1995;270:287–295.
Kricker JA, Towne CL, Firth SM, Herington AC, Upton Z. Structural andfunctional evidence for the interaction of insulin-like growth factors(IGFs) and IGF binding proteins with vitronectin. Endocrinology2003;144:2807–2815.
Li J, Sahagian GG. Demonstration of tumor suppression by mannose6-phosphate/insulin-like growth factor 2 receptor. Oncogene 2004;23:9359–9368
Louvi A, Accili D, Efstratiadis A. Growth-promoting interaction of IGF-II withthe insulin receptor during mouse embryonic development. Dev Biol1997;189:33–48.
Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, EfstratiadisA. Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued fromperinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol1996;177:517–535.
Lustig-Yariv O, Schulze E, Komitowski D, Erdmann V, Schneider T, de GrootN, Hochberg A. The expression of the imprinted genes H19 and IGF-2 inchoriocarcinoma cell lines. Is H19 a tumor suppressor gene? Oncogene1997;15:169–177.
McKinnon T, Chakraborty C, Gleeson LM, Chidiac P, Lala PK. Stimulation ofhuman extravillous trophoblast migration by IGF-II is mediated by IGF type2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK.J Clin Endocrinol Metab 2001;86:3665–3674.
Moller DE, Yokota A, Caro JF, Flier JS. Tissue-specific expression of twoalternatively spliced insulin receptor mRNAs in man. Mol Endocrinol1989;3:1263–1269.
Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J Immunol Methods1983;65:55–63.
O’Gorman DB, Costello M, Weiss J, Firth SM, Scott CD. Decreasedinsulin-like growth factor-II/mannose 6-phosphate receptor expressionenhances tumorigenicity in JEG-3 cells. Cancer Res 1999;59:5692–5694.
O’Gorman DB, Weiss J, Hettiaratchi A, Firth SM, Scott CD. Insulin-likegrowth factor-II/mannose 6-phosphate receptor overexpression reduces
growth of choriocarcinoma cells in vitro and in vivo. Endocrinology2002;143:4287–4294.
Pandini G, Frasca F, Mineo R, Sciacca L, Vigneri R, Belfiore A. Insulin/insulin-like growth factor I hybrid receptors have different biologicalcharacteristics depending on the insulin receptor isoform involved. J BiolChem 2002;277:39684–39695.
Pandini G, Medico E, Conte E, Sciacca L, Vigneri R, Belfiore A. Differentialgene expression induced by insulin and insulin-like growth factor-IIthrough the insulin receptor isoform A. J Biol Chem 2003;278:42178–42189.
Pandini G, Conte E, Medico E, Sciacca L, Vigneri R, Belfiore A. IGF-IIbinding to insulin receptor isoform A induces a partially different geneexpression profile from insulin binding. Ann N Y Acad Sci 2004;1028:450–456.
Ritvos O, Rutanen EM, Pekonen F, Jalkanen J, Suikkari AM, Ranta T.Characterization of functional type I insulin-like growth factor receptorsfrom human choriocarcinoma cells. Endocrinology 1988;122:395–401.
Saperstein R, Vicario PP, Strout HV, Brady E, Slater EE, Greenlee WJ,Ondeyka DL, Patchett AA, Hangauer DG. Design of a selective insulinreceptor tyrosine kinase inhibitor and its effect on glucose uptake andmetabolism in intact cells. Biochemistry 1989;28:5694–5701.
Sciacca L, Mineo R, Pandini G, Murabito A, Vigneri R, Belfiore A. In IGF-Ireceptor-deficient leiomyosarcoma cells autocrine IGF-II induces cellinvasion and protection from apoptosis via the insulin receptor isoform A.Oncogene 2002;21:8240–8250.
Sciacca L, Prisco M, Wu A, Belfiore A, Vigneri R, Baserga R. Signalingdifferences from the A and B isoforms of the insulin receptor (IR) in 32Dcells in the presence or absence of IR substrate-1. Endocrinology2003;144:2650–2658.
Seckl MJ, Fisher RA, Salerno G, Rees H, Paradinas FJ, Foskett M, NewlandsES. Choriocarcinoma and partial hydatidiform moles. Lancet2000;356:36–39.
Soper JT. Gestational trophoblastic disease. Obstet Gynecol 2006;108:176–187.
Uhles S, Moede T, Leibiger B, Berggren PO, Leibiger IB. Isoform-specificinsulin receptor signaling involves different plasma membrane domains.J Cell Biol 2003;163:1327–1337.
Zhang L, Shiverick KT. Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, alters cell proliferation and c-myc and growth factor expressionin human placental choriocarcinoma JEG-3 cells. Biochem Biophys ResCommun 1997;231:117–120.
Zhang M AS, Yeh S. RRR-alpha-tocopheryl succinate inhibits human prostatecancer cell invasiveness. Oncogene 2004;23:3080–3088.
Submitted on March 12, 2007; resubmitted on April 19, 2007; accepted onApril 23, 2007
Diaz et al.
576
by guest on February 3, 2014http://m
olehr.oxfordjournals.org/D
ownloaded from