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ETV6-NTRK3 transformation requires insulin-like growth factor 1
receptor signaling and is associated with constitutive IRS-1 tyrosine
phosphorylation
Kevin B Morrison
1,2
, Cristina E Tognon
1,2
, Mathew J Garnett
1,2
, Cheri Deal
3
and
Poul HB Sorensen*
,1,2
1
Department of Pathology, BC Research Institute for Children’s and Women’s Health, and the University of British Columbia,
Vancouver, BC V5Z 4H4, Canada;
2
Department of Pediatrics, BC Research Institute for Children’s and Women’s Health, and the
University of British Columbia, Vancouver, BC V5Z 4H4, Canada;
3
Department of Pediatrics, Ste-Justine Hospital Research
Center, Montreal, Quebec H3T 1C5, Canada
Congenital fibrosarcoma (CFS) and cellular mesoblastic
nephroma (CMN) are pediatric spindle cell malignancies
that share two specific cytogenetic abnormalities: trisomy
of chromosome 11 and a t(12 ;15)(p13;q25) translocation.
The t(12;15) rearrangement creates a transcriptionally
active fusion gene that encodes a chimeric oncopro tein,
ETV6-NTRK3 (EN). EN transforms NIH3T3 fibro-
blasts through constitutive activation of both the Ras-
mitogen-activated protein kinase (MAPK) pathway and
the phosphatidylinositol- 3 ’kinase (PI3K)-Akt pathway.
However, the role of trisomy 11 in CFS and CMN
remains unknown. In this study we demonstrate elevated
expression of the chromosome 11p15.5 insulin-like
growth factor 2 gene (IGF2) in CFS and CMN tumors.
Moreover, we present evidence that an intact IGF
signaling axis is essential for in vitro EN-mediated
transformation. EN only very weakly transformed so-
called R-murine fibroblasts derived from mice with a
targeted disruption of the IGF1 receptor gene (IGFR I),
but transformation activity was fully restored in R7
cells engineered to re-express IGFRI (R+ cells). We
also observed that the major IGFRI substrate, insulin-
receptor substrate-1 (IRS-1), was constitutively tyrosine
phosphorylated and could be co-immunoprecipitated with
EN in either R7 or R+ cells expressing the EN
oncoprotein. IRS-1 association with Grb2 and PI3K p85,
which link IGFRI to the Ras-MAPK and PI3K-Akt
pathways, respectively, was enhanced in both cell types
in the presence of EN. However, activation of the Ras-
MAPK and PI3K-Akt pathways was markedly attenu-
ated in EN-expressing R7 cells compared to EN-
transformed R+ cells. This suggests that IRS-1 may
be functioning as an adaptor in EN signal transduction,
but that a link to EN transformation pathways requires
the presence of IGFRI. Our findings indicate that an
intact IGF signaling axis is essential for EN transforma-
tion, and are consistent with a role for trisomy 11 in
augmenting this pathway in EN expressing tumors.
Oncogene (2002) 21, 5684 – 5695. doi:10.1038/sj.onc.
1205669
Keywords: ETV6-NTRK3; IGF1 receptor; IRS-1
Introduction
Congenital fibrosarcoma (CFS) and cellular mesoblastic
nephroma (CMN) are spindle cell malignancies of
infancy and young childhood that are closely related
both clinically and histopathologically (Fisher, 1996;
O’Malley et al., 1996). These tumors are genetically
related as well, sharing common genetic abnormalities
including expression of the t(12;15)(p13;q25)-associated
ETV6-NTRK3 gene fusion (Knezevich et al., 1998b) and
trisomy of chromosome 11 (Knezevich et al., 1998a;
Rubin et al., 1998). The ETV6-NTRK3 fusion product
(EN) contains the sterile-alpha domain (SAM; also
called the pointed or helix – loop – helix domain) of the
ETS transcription factor, ETV6, fused to the cytoplas-
mic portion containing the protein tyrosine kinase
domain (PTK) of the neurotrophin-3 receptor, NTRK3.
The SAM domain enables EN to undergo homo-
dimerization and ligand-independent activation of the
PTK domain, which is required for its potent transfor-
mation activity in NIH3T3 cells (Wai et al., 2000). We
have recently shown that the transforming properties of
EN are associated with constitutive activation of
NTRK3 signaling pathways, namely the Ras-MAPK
and phosphatidylinositol-3’kinase (PI3K)-Akt cascades
(Tognon et al., 2001). However, the adaptor proteins
that link EN to activation of these pathways have yet to
be identified, as we failed to show a direct association
between EN and known NTRK3-associating molecules
including Shc, Grb2, PI3K p85, ABL, SH2Bb, Src, or
Ship2 (Tognon et al., 2001; Wai et al., 2000).
Trisomy 11 has been reported in the majority of
cases of CFS and CMN (Bernstein et al., 1994; Dal Cin
et al., 1998; Kaneko et al., 1991; Knezevich et al.,
Received 18 February 2002; revised 9 May 2002; accepted 14 May
2002
*Correspondenc e: PHB Sorensen, BC Research Institute for
Children’s and Women’s Health, Room 3082-950 West 28th Ave.,
Vancouver, BC Canada V5Z 4H4; E-mail: psor@interchange.ubc.ca
Oncogene (2002) 21, 5684 – 5695
ª
2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00
www.nature.com/onc
1998a,b; Mascarello et al., 1994; Rubin et al., 1998;
Schofield et al., 1993, 1994). As well, high level
expression of the peptide hormone insulin-like grow th
factor 2 (IGF2) have been reported in CMN tissue
(Sharifah et al., 1995). Given that the IGF2 gene is
localized to the 11p15.5 region of chromosome 11, we
speculated that the extra copy of chromosome 11
might lead to over-expression of IGF2 in CFS and
CMN, and therefore that activation of the IGF
pathway is important for EN-mediated tumorigenesis.
IGF1 and IGF2 both bind to IGFRI and stimulate
multiple signaling cascades, includi ng mitotic, anti-
apoptotic, and transformation-associated pathways
(reviewed in Baserga, 2000). Ligand binding results in
receptor PTK autophosphorylation and activation,
followed by tyrosine phosphorylation of key substrates
including insulin receptor substrate-1 (IRS-1), Shc, and
PI3 kinase (PI3K) p85 subunit (reviewed in LeRoith et
al., 1995). Two major downstream signaling pathways
are associated with IGFRI activation: IRS-1 and Shc
binding lead to activation of the Ras-Ra f1-MEK-
ERK1/2 MAP kinase mitogenic cascade (Downward,
1996; Joneson and Bar-Sagi, 1997), and binding of the
PI3K p85 subunit leads to activation of the PI3K- AKT
cell survival pathway (Dudek et al., 1997; Franke et al .,
1997a,b). A large amount of literature points to a
critical role for this signaling system in cellular
transformation. IGFRI activation or ligand over-
expression has been described in a number of adult
malignancies including breast and prostate cancer as
well as in pediatric malignancies such as rhabdomyo-
sarcoma, Wilms’ tumor , neuroblastoma, Ewing family
tumors, and acute leukemias (reviewed in Baserga,
1995, 1999; Toretsky and Helman, 1996; Werner,
1998). The functional significance of the IGF signaling
axis in oncogenesis has been demonstrated experimen-
tally by the anti-tumorigenic effects of IGFRI antisense
oligonucleotides (Neuenschwander et al., 1995; Pass et
al., 1996; Resnicoff et al., 1994a,b) and dominant
negative IGFRI constructs (Burgaud et al., 1995;
D’Ambrosio et al., 1996; Dunn et al., 1998; Prager et
al., 1994; Reiss et al., 1998). Moreover, a number of
dominantly-acting oncogenes such as simian virus 40 T
antigen (Sell et al., 1993), Ha-Ras (Sell et al., 1994),
and c-Src (Valentinis et al., 1997), viruses such as
bovine (Morrione et al., 1995) and human papilloma
virus (Steller et al., 1996), and overexpressed growth
factor receptors such as PDGFR and EGFR (Coppola
et al., 1994), fail to transform mouse embryo
fibroblasts generated from IGFRI knockout mice
(known as R7 cells). Among oncoproteins involved
in childhood solid tumors, this requirement for intact
IGFRI signaling includes the EWS-FLI1 and PAX3-
FKHR fusion protein s of Ewing family tumors and
alveolar rhabdomyosarcoma, respectively (Toretsky et
al., 1997; Wang et al., 1998).
In this study we asked whether an intact IGFRI
signaling axis is required for the transformation
activity of the EN chimeric oncoprotein. We first
screened CFS and CMN primary tumors and found
high levels of IGF2 expression in all cases analysed. To
test whether the IGF pathway is important for EN-
mediated transformation, we co mpared the transform-
ing activity of EN in IGFR I null R7 cells to that
observed in R+ cells, which are R7 cells engineered
to re-express IGFRI using retroviral gene transfer. We
found that while the fusion protein was very weakly
transforming in R7 cells, transformation activity was
fully restored when EN was expressed in R+ cells.
Expression of the fusion protein correlated with
constitutive tyrosine phosphorylation of IRS-1 in both
cell types, and IRS-1 was able to bind and pull down
Grb2 and the p85 regulatory subunit of PI3K equally
well in each cell line. Moreover, co-immunoprecipita-
tion experiments suggested that EN and IRS-1 were
present in complexes in both R7 and R+ cells.
However, while activation of pathways essential for
EN transformation, the Ras-MAPK and PI3K-Akt
cascades, was markedly attenuated in EN-expressing
R7 cells, these pathways were enhanced in EN-
expressing R+ cells. Although these data are consis-
tent with IRS-1 serving as an adaptor protein for EN,
they indica te that it requires the presence of IGFRI in
order to efficiently activate downstream pathways
essential for EN transformation.
Results
IGF2 is overexpressed in CFS and CMN primary tumors
Given the strong association between the expression of
the ETV6-NTRK3 gene fusion and the presence of
trisomy 11 in CFS and CMN (Knezevich et al., 1998a;
Rubin et al., 1998), we postulated that the extra copy
of chromosome 11 might lead to increased expression
of a gene critical for EN transformation. Since IGF2
over-expression in CMN has already been described
(Sharifah et al., 1995), we assessed mRNA levels of this
gene in EN-expressing tumors. Northern blotting was
used to analyse IGF2 transcripts in primary tumor
sample specimens of fusion positive CFS and CMN
with trisomy 11. We also tested cases of infantile
fibromatosis and the classical form of mesoblastic
nephroma, which are benign counterparts of CFS and
CMN, respectively, that lack this gene fusion as well as
trisomy 11 (Bourgeois et al., 2000; Knezevich et al.,
1998a). A typical result is shown in Figure 1,
demonstrating high IGF2 expression in CFS and
CMN, but low to negligible levels in infantile
fibromatosis and classical mesoblastic nephroma. This
was confirmed by quantita tive RT – PCR of five cases
of fusion positive CFS and CMN with trisomy 11,
which demonstrated 10 – 30-fold higher levels of IGF2
expression as compared to normal fibroblast cultures
(data not shown). These findings indicate that over-
expression of the IGF2 gene is associated with trisomy
11 in CFS and CMN.
Expression of EN in R7 and R+ murine fibroblasts
Given the high IGF2 mRNA levels in EN-expressing
tumors, we hypothesized that the IGF signaling axis
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5685
Oncogene
may serve an important function in EN-mediated
transformation. NIH3T3 cells are known to express
moderately high levels of IGFRI protein which can be
difficult to block experimentally (Baserga, 1999).
Therefore to directly test whether IGFRI signaling is
required for EN transformation, we chose to assess the
in vitro transforming properties of EN in R7 cells,
which are murine 3T3 fibroblast cells derived from
IGFRI knockout mice (Baker et al., 1993; Liu et al.,
1993). We used retroviral gene transfer to generate R7
cell lines containing vector alone or stably expressing
EN or an activated form of Ha-Ras (R7,R7EN and
R7Ha-Ras, respectively). As co ntrols, we stably re-
expressed IGFRI in R7 cells, creating so-called R+
cells positive for IGFRI. Expr ession of IGFRI in these
cells was confirmed by immunoprecipitation with the a-
IR3 IGFRI monoclonal antibody followed by Western
blotting with an a-IGFRI b-subunit antibody, demon-
strating the 97 kDa IGFRI b-subunit (see Figure 2a).
Retroviral gene transfer was similarly used to generate
R+ cell lines containing vector alone or stably
expressing EN or activated Ha-Ras (R+, R+EN
and R+Ha-Ras, respectively). Immunoblotting
demonstrated equivalent expression of the character-
istic 68 and 73 kDa doublet of EN (Wai et al., 2000) in
R7EN and R+EN cells, and confirmed that levels
were similar to those in EN-transformed NIH3T3 cells
(see Figure 2B).
EN fails to transform R7 cells
To assess whether IGFRI signaling is required for
EN-mediated oncogenesis, we compared the ability of
EN to transform R7 versus R+ cells using
morphologic criteria and anchorage-independent soft
agar colony growth as parameters of transformation
(Ponten, 1971). Vector alone and activated Ha-Ras
constructs were used as controls. R7 and R+ cells
showed identical non-transformed phenotypes (shown
for R7 cells in Figure 3), while R7EN (Figure 3)
and R7Ras cells demonstrated slight increases in
spindle morphology and nuclear-to-cytoplasmic ratios.
This suggests that EN and Ha-Ras are only weakly
transforming in R7 cells. In contrast, both R+EN
(Figure 3) and R+Ras cells were morphologically
transformed with spindling, elaboration of cellular
processes, increased nuclear-to-cytoplasmic ratios and
refractility, and focus formation with loss of contact
inhibition. Of note, cell viability was similar in each
cell line (data not shown). To confirm these results,
cells were plated in soft agar to determine their
Figure 1 IGF2 transcripts are over-expressed in CFS and cellular
mesoblastic nephroma. Total RNA was extracted from primary
tumor samples and subjected to Northern blot analysis using an
IGF2 exon 9-specific probe. Arrows show the 6.0 and 4.8 kb
IGF2 transcripts in congenital fibrosarcoma (CFS) and the cellu-
lar form of mesoblastic nephroma (MN). This is not observed in
the classical form of MN nor in infantile fibromatosis (IFB),
which are benign lesions lacking the ETV6-NTRK3 gene fusion
or trisomy 11. b-actin loading controls are shown in the lower
panel
Figure 2 Generation of ETV6-NTRK3 (EN) and IGFRI expres-
sing R+ cells. (a) Retroviral gene transfer was used to re-express
IGFRI in R7 fibroblasts derived from mice with a targeted dis-
ruption of the IGFRI gene, creating so-called R+ cells. Expres-
sion of IGFRI in R+ cells was compared to parental R7 cells
and IGFRI-expressing P6 control cells by immunoprecipitation
from whole cell lysates using a-IR3 monoclonal antibodies, fol-
lowed by Western blotting with antibodies to the IGFRI b-sub-
unit (arrow). (b) Retroviral gene transfer was used to express
ETV6-NTRK3 (EN), Ha-Ras (Ras), and vector alone (Vec) con-
structs in R7 and R+ cells. To confirm that levels of EN protein
expression were similar in these cells and in EN transformed
NIH3T3 cells, whole cell lysates were subjected to immunopreci-
pitation with a-ETV6 antibodies followed by Western blotting
with a-NTRK3 antibodies. Arrows demonstrate the 68/73 kDa
doublet of EN
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5686
Oncogene
ability to grow under anchorage independent condi-
tions. While R7EN cells formed occasional
macroscopic soft agar colonies, this property was
significantly enhanced for R+EN cells (P=0.0001;
see Figure 4a). As well, the size of the colonies
formed by R+EN were greater in diameter as
compared to R7EN (see Figure 4a). In fact, EN
and Ha-Ras both failed to induce significant colony
formation in R7 cells but this property was rescued
by expression in R+ cells (see Figure 4b). Even
though R+ cells alone formed small but reproducible
colony numbers, rates were significantly higher for
R+ cells exp ressing EN (P50.01 for R+ versus
R+EN) or Ha-Ras. These studies indicate that
IGFRI is required for full EN-mediated transforma-
tion.
Mek1/2 and Akt activation are attenuated in EN-
expressing R7 cells
We recently demonstrated that EN transformation is
dependent on constitutive activation of the Ras-
MAPK and PI3K- Akt pathways (Tognon et al.,
2001). We therefore hypothesized that the failure of
EN to fully transform cells lacking IGFR I might be
due to defects in the activation of these pathways.
To assess this possibility, we examined the activation
states of Mek1/2 and Akt molecules in R7EN and
R+EN cells. Under low serum conditions R7EN
and R+EN cells demonstrated equivalent levels of
basal activation of Mek1/2 and Akt (see Figure 5).
Both were greater than those observed in R7 cells
alone, consistent with previous findi ngs in EN-
expressing NIH3T3 cells (Tognon et al., 2001).
However, while serum stimulation led to a marked
increase in activation of Mek1/2 and Akt in R+EN
cells, this was not observed in R7EN cells where
levels of phosphorylat ed Mek1/2 and Akt remained
at basal levels even after serum stimulation (Figure
5). This was peculiar to R7EN cells, as R7 cells
alone (see Figure 5) as well as R+ cells (data not
shown), responded to serum stimulation as expected
by maximally activating Mek1/2 and Akt. Therefore
cellular mechanisms for activating Ras-MAPK and
PI3K-Akt pathways (e.g. through other growth
factor receptors) are intact in R7 cells. These
observations were consistent over many independent
experiments, and persisted even after longer term
serum stimulation (data not shown). Therefore
Figure 3 Morphology of R7 and R+ cells expressing ETV6-
NTRK3 (EN). R7 cells and R+ were engineered to stably
express EN using retroviral gene transfer (R7EN and R+EN,
respectively). R7, R+ (not shown), and R7EN display a
flattened, contact-inhibited, non-transformed phenotype, while
R+EN cells display a transformed phenotype with increased
refractility, loss of contact inhibition and focus formation in
monolayer cultures
Figure 4 Comparison of soft agar colony formation in R7EN
and R+EN cells. Anchorage-independent growth was assessed
by the ability of cells to form macroscopic colonies (greater than
0.1 mm in size) when plated in soft agar. After appropriate single
or double antibiotic selection, cells were seeded in 0.2% agarose
and 10% FBS at a density of 2000 cells/mL of media placed on
an underlayer of 0.4% agarose/10% FBS in DMEM as described
in Materials and methods. All macroscopic colonies and single
cells were counted in 10 high-powered fields per well. (a) Typical
results of three separate infection experiments are shown for
R7EN cells (top row) and R+EN (bottom row). (b) Histogram
comparing colony formation in R7 and R+ cells infected with
vector alone (R7 and R+), or constructs containing ETV6-
NTRK3 (EN) or activated Ha-Ras (Ha-Ras). Results are ex-
pressed as the ratio of colonies formed per number of cells seeded.
Each cell line was assessed in triplicate in 35 mm wells, and each
experiment was performed at least seven times. Statistical analysis
was performed using the Student’s t-test: *P-value comparing
R7EN and R+EN cells (P=0.0001); {P-value comparing R+
and R+EN cells (P50.01)
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5687
Oncogene
IGFRI signaling may be involved in full activation
of the Ras-MAPK and PI3K-Akt pathways in EN-
expressing cells.
EN expression leads to constitutive IRS-1 tyrosine
phosphorylation in NIH3T3 fibroblasts
Adapter proteins linking EN to activation of Ras-
MAPK and PI3K-Akt pathways remain to be
determined (Wai et al., 2000). Recent studies suggest
that the major IGFRI substrate, insulin-receptor
substrate-1 (IRS-1), may serve as a molecular bridge
between the NTRK1 receptor and activation of the
Ras-MAPK and PI3K-Akt pathw ays (Miranda et al.,
2001). Moreover, Yamada and co-workers have
reported that NTRK2 activation is associated with
increased IRS-1 tyrosine phosphorylation and recruit-
ment of the p85 regulatory subunit of PI3K (Yamada
et al., 1997). We therefore hypothesized that IRS-1
might be acting as an adapter linking EN to down-
stream signaling pathways. To assess this possibility,
we first analysed the tyrosine phosphorylation status of
IRS-1 in the presence and absence of EN. NIH3T3 cell
lines grown under serum-free conditions showed a
tyrosine phosphorylated band at 185 kDa by Western
blotting in EN-expressing cells but not in vector
controls (see top panel, Figure 6a). This band became
tyrosine phosphorylated in vector controls only after
serum or IGF2 stimulation (top panel, Figure 6a). Re-
probing of the blot with an IRS-1 specific antibody
revealed that the 185 kDa differentially phosphorylated
band corresponds to IRS-1 (bottom panel, Figure 6a).
This was confirmed by immunoprecipitation of lysates
from serum-starved NIH3T3 cells using an IRS-1
specific antibody, which revealed markedly increased
levels of tyrosine phosphorylation of IRS-1 in EN-
expressing cells compared with vector controls (see
Figure 6b). Therefore IRS-1 undergoes constitutive
tyrosine phosphorylation in NIH3T3 fibroblasts
expressing EN.
Constitutive IRS-1 tyrosine phosphorylation is maintained
in EN-expressing R7 cells
We ne xt tested whether the constitutive tyrosine
phosphorylation of IRS-1 observed in EN-expressing
cells is dependent on the presence of IGFRI. R7EN
and R+EN cells along with R7 and R+ vector
controls were subjected to Western blotting using a-
phosphotyrosine antibodies, which demonstrated
equivalent levels of a 185 kDa tyrosine phosphorylated
band in the EN-expressing cells even in the absence of
serum or IGF2 (see Figure 7a). This band was present
only at basal levels in serum- or IGF2-deprived R7
and R+ cells. Its tyrosine phosphorylation was
induced by serum or IGF2 stimulation in R+ cells
but not in R7 cells (as expected given that they do not
express IGFRI). Blots were re-probed with an a-IRS-1
antibody to verify the identity of the 185 kDa band as
IRS-1 (data not shown). As shown in Figure 7b, these
findings were confirmed by immunoprecipitation
studies using an IRS-1 specific antibody. While R7
cells demonstrated only very low basal levels of
tyrosine phosphorylated IRS-1, R+EN and R7EN
Figure 5 Differential Mek1 and Akt activation in ETV6-
NTRK3-expressing R7 and R+ cells. R7 cells (designated as
IGFRI7) or R+ cells (designated as IGFRI+) infected with
(+) or without (7) ETV6-NTRK3 constructs were grown in
35 mm dishes to 75% confluence and then serum-deprived in
0.5% serum DMEM for 18 h. Cells were then stimulated with
or without 9% serum for the indicated times. Whole cell lysates
were collected and used for Western blot analysis with antibodies
to activated Akt (phospho-Akt) or activated Mek1/2 (phospho-
Mek). Western blotting using Grb2 antibodies was used to
demonstrate equal protein loading
Figure 6 IRS-1 is constitutively tyrosine phosphorylated in EN-
expressing NIH3T3 cells. (a) Western blot analysis. NIH3T3 in-
fected with (+) or without (7) ETV6-NTRK3 retroviral con-
structs were grown in 35 mm dishes to 75% confluence and
then serum-deprived for 18 h in 0.5% serum. Cells were then sti-
mulated with or without 9% serum or 50 ng/ml of IGF2 for
30 min. Whole cell lysates were prepared and separated by
SDS – PAGE and probed with PY20 anti-phosphotyrosine antibo-
dies (P-Tyr). The top panel shows a differentially tyrosine phos-
phorylated band at approximately 185 kDa, which was
confirmed to be IRS-1 by re-probing with a-IRS-1 antibodies
(IRS-1). (b) Immunoprecipitation analysis. NIH3T3 infected with
(+) or without (7) ETV6-NTRK3 retroviral constructs were
grown in 9% calf serum to 75% confluence. Whole cell lysates
were then prepared (1000 mg total protein) and subjected to im-
munoprecipitation using a-IRS-1 antibodies. Immunoprecipitates
were separated by SDS – PAGE and probed with PY20 anti-phos-
photyrosine antibodies (top panel), followed by re-probing with a-
IRS-1 antibodies (bottom panel). Arrows show the position of the
185 kDa IRS-1 band
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5688
Oncogene
cells showed equivalently elevated level s of IRS-1
tyrosine phosphorylation even in the ab sence of serum.
These findings indicate that there is constitutive
tyrosine phosphorylation of IRS-1 in EN-expressing
cells, and that this does not depend on the presence of
IGFRI.
IRS-1 co-immunoprecipitates with EN
It is possible that IRS-1 functions in EN signaling as
part of a molecular complex with EN. To begin to test
this hypothesis we carried out co-immunoprecipitation
experiments to investigate potential interactions
between these two molecules. R+EN cells were lysed
after serum stimulation and subjected to immunopre-
cipitation using either an a-HLH-ETV6 antibody or a
control IgG antibody. As shown in lane 1 of Figure 8a,
a-ETV6 antibodies were able to co-immunoprecipitate
a 68/73 kDa doublet (confirmed to be EN by re-
probing with a-NTRK3 antibodies; not shown) along
with a tyrosine-phos phorylated band at 185 kDa,
neither of which were observed using non-specific
IgG (Figure 8a, lane 2). The latter band was identical
in size to a 185 kDa tyrosine-phosphorylated protein
immunoprecipitated from serum-stimulated parental
R+ cells (which contain high IRS-1 levels) using a-
IRS-1 antibodies (Figure 8a, lane 3). This band was
not observed when a-ETV6 antibodies were used for
immunoprecipitation in R+ (or R7 cells) lacking EN
expression (data not shown). To show that this
185 kDa protein represented IRS-1, the blot was re-
probed with the a-IRS-1 antibody (see lanes 1 and 3 of
Figure 8b). Identical results were found using R7EN
cells, in which we also observed co-immunoprecipita-
tion of EN and the 185 kDa tyrosine phosphorylated
band (data not shown). This indicates that IGFRI is
not required for the EN/IRS-1 interaction.
To independe ntly confirm these findings using a
different cell line, we performed the reverse experiment
in EN-transf ormed NIH3T3 cells using either a-ETV6
or a-IRS-1 antibodies for the immunoprecipitation
step. This showed that the characteristic EN doublet
could be pulled down with both antibodies (see Figure
9a). The identity of this doublet was confirmed to be
EN by Western blotting using a-NTRK3 antibodies,
and its tyrosine phosphorylation was verified by prior
blotting with a-phosphotyrosine antibodies (data not
shown). Co-immunoprecipitation of EN and IRS-1 was
not affected by mutating tyrosine 594 of EN to
phenylalanine (see Figure 9a). This amino acid
corresponds to tyrosine 464 of NTRK1, mutation of
Figure 7 IRS-1 is constitutively tyrosine phosphorylated in both
R7 and R+ cells expressing ETV6-NTRK3. (a) Western analy-
sis: R7 (IGFRI7; ETV6-NTRK37), R+ (IGFRI+; ETV6-
NTRK37), R7EN (IGFRI7; ETV6-NTRK3+), and R+EN
cells (IGFRI+; ETV6-NTRK3+) were grown in 35 mm dishes
to 75% confluence and then serum-deprived for 18 h in 0.5% ser-
um. Cells were then stimulated with or without 9% FBS/DMEM
or 50 – 100 ng/ml IGF2 as indicated for 3 h. Whole cell lysates
were prepared for Western blotting using PY20 anti-phosphotyr-
osine antibodies (P-Tyr) (top panel), which demonstrated differen-
tial tyrosine phosphorylation of a 185 kDa band (arrow). This
band was confirmed to be IRS-1 by stripping and re-probing with
a-IRS-1 antibodies (data not shown). Grb2 blotting was used to
control for equal protein loading of samples. (b) Immunoprecipi-
tation analysis: R7,R7EN and R+EN cells were serum-de-
prived as in (b) and then stimulated with or without 9% FBS/
DMEM for 30 min. Equal protein amounts (1000 mg) from whole
cell lysates were subjected to immunoprecipitation using a-IRS-1
antibodies, followed by Western blotting with PY20 anti-phos-
photyrosine antibodies (P-Tyr) (top panels). This revealed immu-
noprecipitation of a 185 kDa tyrosine phosphorylated band,
which was confirmed to be IRS-1 by re-probing with a-IRS-1
antibodies (bottom panels)
Figure 8 ETV6-NTRK3 co-immunoprecipitates with IRS-1. (a)
Immunoprecipitation was carried out from R+EN whole cell ly-
sates (lanes 1 and 2) using a-ETV6 antibodies (lane 1) or normal
rabbit IgG (lane 2), or from control R+ lysates (lane 3) using a-
IRS-1 antibodies, followed by Western blotting with PY20 anti-
phosphotyrosine antibodies (P-Tyr). The arrow shows the posi-
tion of a 185 kDa tyrosine phosphorylated protein in lanes 1
and 3. P68/p73 refers to the immunoprecipitated EN doublet.
(b) Reprobing of the blot with a-IRS-1 antibodies shows that
the p185 band represents IRS-1
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5689
Oncogene
which was previously shown to abolish the interaction
between IRS-1 with NTRK1 (Miranda et al., 2001).
We did observe, however, that the EN/IRS-1 associa-
tion requires a functional EN PTK domain. The non-
transforming EN-K380N kinase dead mutant (Wai et
al., 2000) could not be pulled down using a-IRS-1
antibodies even though it was expressed at similar
levels as EN (Figure 9a). Moreover, IRS-1 tyrosine
phosphorylation was markedly diminished in NIH3T3
cells expressing this mutant compared with EN (see
Figure 9b). In view of the findings of Miranda et al.
(2001) on the interaction between IRS-1 and NTRK 1,
we also tested whether mutations of any of the three
most C-terminal tyrosines of EN affected IRS-1
tyrosine phosphorylation. However, none of these
mutants showed diminished IRS-1 tyrosine phosphor-
ylation (Figure 9b), consistent with each mutant
retaining full transformation activity (data not shown).
These studies provide preliminar y evidence that IRS-1
is involved in complex formation with EN, although it
is not yet known whether this is a direct or indirect
interaction. We also attempted to pull down EN using
a-IGFRI b-subunit antibodi es (or IGFRI with a-
NTRK3 antibodies) but have been unable to repro-
ducibly show an association between EN and IGFRI
(data not shown). Therefore it remains unclear whether
IGFRI is also part of this complex. It also remains to
be determined whether EN is directly responsible for
the constitut ive tyrosine phosphorylation of IRS-1
observed in EN-expressing cells.
Recruitment of Grb2 and p85 by IRS-1 is maintained in
EN-expressing R7 cell s
The constitutive tyrosine phosphorylation of IRS-1 in
R7EN cells suggested that the attenuated levels of
Mek1/2 and Akt activation in response to serum-
stimulation observed in these cells (see Figure 5) was
not due to a defect in IRS-1 tyrosine phosphorylation
secondary to the absence of IGFRI. IRS-1 is an
important adapter protein for the IGF and insulin
signaling cascades (Myers and W hite, 1996). In
response to IGF or insulin stimulation IRS-1 becomes
tyrosine phosphorylated and recruits SH2-containing
proteins such as Grb2, SHP-2, and the PI3K p85
regulatory subunit, in turn activating downstream Ras-
MAPK, PI3K-Akt and other pathways (Myers and
White, 1996). It is therefore possible that IRS-1 is not
able to recruit downstream signaling molecules in the
absence of the IGFRI protein. To test if this might be
the basis for the observed lack of maximal Ras-MAPK
and PI3K-Akt activation in R7EN cells and poten-
tially why these cells are not transformed, we wished to
assess whet her there were differences in IRS- 1 recruit-
ment of Grb2 and p85 in R7EN versus R+EN cells.
We therefore performed immunoprecipitation from
lysates of EN-expressing R7 and R+ cells before
and after serum stimulation using antibodies to IRS-1.
As before, the levels of tyrosine phosphorylated IRS- 1
were very low in R7 (see Figure 10) and R+ cells (not
shown), but were markedly increased in R7EN and
R+EN cells even in the absence of serum (see Figure
10). However, the EN-induced increase in IRS-1
tyrosine phosphorylation correlated with enhanced
recruitment of Grb2 and p85 in both R7EN and
R+EN cells. There was no significant difference in the
levels of Grb2 and p85 recruited by IRS-1 in R7EN
versus R+E N cells. Therefore the presence of IGFRI
does not appear to influence the ability of IRS-1 to
recruit these signaling adapters to the putative EN/
IRS-1 complex, and other mechanisms must underlie
the complementary ro le of IGFRI in EN-mediated
transformation.
Discussion
In this study we evaluated the role of the IGFRI
signaling axis in transformation of fibroblasts by the
CFS and CMN-associated ETV6-NTRK3 chimeric
oncoprotein. We have previously shown that EN
transformation involves links between known NTRK3
signaling pathways and aberrant cell cycle progression,
Figure 9 Co-immunoprecipitation of IRS-1 and ETV6-NTRK3
using a-IRS-1 antibodies. (a) NIH3T3 cells expressing vector
alone (Vec), ETV6-NTRK3 (EN), the kinase dead EN-K380N
non-transforming mutant (KD), or the EN-Y594F transforming
mutant (Y594F) were grown to near confluence in 9% FBS/
DMEM. Whole cell lysates (1000 mg) were collected and subjected
to immunoprecipitation with a-ETV6 as well as with a-IRS-1
antibodies. Immunoprecipitates were separated by SDS – PAGE
and subjected to PY20 anti-phosphotyrosine immunoblotting. A
tyrosine phosphorylated doublet at 68 and 73 kDa is seen in ly-
sates from EN and EN-Y594F expressing cells, which is con-
firmed to represent EN by re-probing with a-NTRK3 antibodies
(data not shown). (b) NIH3T3 cells expressing ETV6-NTRK3
(EN), EN-K380N (KD), or the three C-terminal tyrosine mutants
EN-Y620F, EN-Y594F, or EN-Y560F (all of which retain trans-
formation activity) were cultured in 9% FBS/DMEM to near
confluence. Whole cell lysates were then prepared and a 1000 mg
protein aliquot was immunoprecipitated using a-IRS-1 antibodies,
followed by Western blotting with PY20 anti-phosphotyrosine
antibodies. This revealed immunoprecipitation of a 185 kDa band
(arrow in top panel) which re-probed with a-IRS-1 antibodies
(arrow in bottom panel)
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5690
Oncogene
and that the Ras-MAPK and PI3K-Akt pathways act
synergistically to mediate these effects (Tognon et al.,
2001; Wai et al., 2000). A possible role for IGF
signaling in this process was first suggested to us by the
striking correlati on found between the presence of the
ETV6-NTRK3 gene fusion and trisomy 11 (Knezevich
et al., 1998a; Rubin et al., 1998). We hypothesized that
trisomy 11, well known to occur in both CFS and
CMN (Bernstein et al., 1994; Dal Cin et al., 1998;
Kaneko et al., 1991; Mascarello et al., 1994; Schofield
et al., 1993, 1994), leads to over-expression of a
chromosome 11 gene or genes impor tant for EN
oncogenesis. A prime candidate was the 11p15.5
IGF2 gene whose expression is elevated in numerous
pediatric malignancies (reviewed in Toretsky and
Helman, 1996), including CMN (Sharifah et al.,
1995). Consistent with our hypothesis, we confirmed
that IGF2 transcripts are markedly increased in EN-
expressing primary CFS and CMN cases with trisomy
11. IGF2 is an imprinted gene that is normally
expressed only from the paternal allele (reviewed in
Polychronakos et al. (1995)). Analysis of two of the
CFS and CMN primary tumors from the current study
revealed that the extra copy of chromosome 11 was
paternally derived (data not shown), thus providing a
possible mechanism for the observed over-expression of
IGF2 in such lesions. These observations prompted us
to characterize the pathophysiologic role of the IGF/
IGFRI axis in cells transformed by ETV6-NTRK3. EN
was incapable of fully transforming R7 mouse
fibroblasts lacking IGFRI expression; this was also
observed with Ha-Ras, as has been previously reported
(Sell et al., 1994). Interestingly, R7EN and R7Ras
cells did show increased saturation densities compared
with R7 cells alone (Figure 3), suggesting that they
were partially transformed as observed for R7 cell
expressing SV40 large T antigen (Sell et al., 1993). Full
transformation activity was restored for both oncopro-
teins by re-expression of IGFRI in these cells. When
we assessed Ras-MAPK and PI3K-Akt activation in
R7EN cells, we found that both were markedly
attenuated compared with R+EN cells, consistent
with the lack of transformation of the former by EN.
These findings indicate that EN, like several other
dominantly-acting oncoproteins, requires a functional
IGF/IGFRI axis for transformation. In fact, IGFRI
signaling axis has been found to be essential for
transformation mediated by other pediatric tumor
fusion genes, including EWS/FLI1 of Ewing tumors
(Toretsky et al., 1997) and PAX3-FKHR of alveolar
rhabdomyosarcoma (Wang et al., 1998). This, coupled
with widespread over-expression of IGFs in pediatric
neoplasms, suggests that the IGF/IGFRI axis plays a
generalized role in childhood cancer (Toretsky and
Helman, 1996).
Since transformation is blocked in R7 cells
expressing EN and these cells show attenuation of
Ras-MAPK and PI3-Akt pathways essential for EN
transformation, we hypothesized that IGFRI may
somehow link EN to these pathways. Previous studies
in our laboratory confirmed that because of the
position of the t(12;15) breakpoints, the wild-typ e
NTRK3 binding sites for SHC, GRB2, p85 and other
known adaptors are not present in EN and the
chimeric oncoprotein does not bind these molecules
(Knezevich et al., 1998b; Tognon et al., 2001; Wai et
al., 2000). Therefor e adaptor proteins for EN remain
unknown. Insulin receptor substrate-1 (IRS-1) serves
an important adaptor function for IGF and insulin
signaling cascades (Myers and White, 1996). IRS-1
becomes tyrosine phosphorylated with IGF stimulation
and recruits SH2-containing proteins such as Grb2/
mSos, SHP-2, and p85 to IGFRI (Giovannone et al.,
2000). These events in turn activate downstream
pathways including Ras-MAPK and PI3K-Akt. Both
IRS-1 and the related protein IRS-2 have been
reported to act as direct substrates for other NTRK
family member s, namely NTRK1 (Miranda et al.,
2001) and NTRK2 (Yamada et al., 1997). This led us
to assess whether IRS-1 functions as an adaptor
molecule in EN transformed cells. We found that EN
expression leads to constitutive IRS-1 tyrosine phos-
phorylation in NIH3T3 cells, and that IRS-1 can be
pulled down with antibodies to EN or vice versa in
these cells. This suggests that EN and IRS-1 associate
with each other in EN-expr essing cells. It is not yet
known whether EN directly phosphorylates IRS-1,
although we did find that both IRS-1 tyrosine
phosphorylation and its association with EN were
blocked if the kinase domain of EN was inactivated by
site-directed mutagenesis. Although our data implicate
IRS-1 as an EN interactor and possibly as a link
between EN and oncogenic signaling pathways,
Figure 10 Recruitment of Grb2 and PI3K p85 subunit by IRS-1
is retained in both R+EN and R7EN cells. R7,R7EN and
R+EN cells were cultured to 75% confluency in 100 mm dishes
and then placed in 0.5% serum for 18 h. Cells were then stimu-
lated with 9% FBS/DMEM for the indicated times and whole cell
lysates were prepared using standard methods. Equivalent
amounts of protein (1000 mg) were subjected to immunoprecipita-
tion using a-IRS-1 antibodies, followed by Western blotting with
the indicated antibodies. The top two rows represent a 185 kDa
protein immunoblotted with anti-phosphotyrosine antibodies (P-
Tyr) or a-IRS-1 antibodies (IRS-1). The bottom two rows show
immunoblotting results using antibodies against the PI3K p85
subunit (p85) or Grb2 (Grb2), respectively
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5691
Oncogene
tyrosine phosphorylation of IRS-1 and its association
with EN could also be detected in non-transformed
R7EN cells. Moreover, we found that tyrosine
phosphorylated IRS-1 associated with Grb2 and p85
equally well in R+EN and R7EN cells (Figure 10),
even though activation of Ras-MAPK and PI3K-Akt is
attenuated in R7EN cells (Figure 5). Therefore it
appears that tyrosine phosphorylation of IRS-1 and
association with EN are not sufficient for EN
transformation, and that IGFRI is still required for
this process and for maximal activation of oncogenic
pathways.
There are several possible explanations for the
above findings. First, it is possible that EN mediated
tyrosine phosphorylation of IRS-1 occurs at different
tyrosine residues in the presence and absence of
IGFRI. Even though tyrosine phosphorylated IRS-1
can associate with Grb2 and p85 in R7EN cells, this
interaction may be limited in its ability to activate
Ras-MAPK and PI3K-Akt, respectively, due to
conformational or other issue s related to the specific
IRS-1 residues that are phosphorylated in R7EN
cells. We are currently mapping the phosphorylation
sites in IRS-1 to test this hypothesis. Second, in the
absence of IGFRI one or more phosphatases may be
recruited to putative EN/IRS-1 complexes which
might compromise Mek and Akt activation. It should
be noted that the inability of serum (or IGFs) to
maximally activate Mek and Akt in R7EN versus
R+EN cells was found over a wide range of
incubation times (data not shown), suggesting that
the absence of IGFRI may lead to a defect in the
ability to activate Ras-MAPK and PI3K-Akt in a
sustained manner. Third, IGFRI might be necessary
to localize the EN/IRS-1 signaling complexes in such
a way as to be able to maximally activate pathways
required for trans formation. There is increa sing
evidence that the subcellular localization of IRS-1 is
important for its ability to effectively acti vate down-
stream pathways (Kriauciunas et al., 2000). IRS
proteins exist in a dynamic equilibrium between the
cytoplasm and inner cell membrane compartments
(Myers and White, 1996). The N-terminal pleckstrin
homology (PH) domain of IRS-1 is not only essential
for efficient tyrosine phosphorylation of IRS-1 upon
IGF stimulation (Yenush et al., 1998), but also
appears to be ne cessary for the translocation of IRS-
1 from the cytoplasm to the inner cell membrane
during insulin and IGF stimulation (Razzini et al.,
2000). Recent studies with a myristoylated form of
IRS-1 in insulin-expressing cells demonstrated that
constitutive membrane localization of IRS-1 correlated
with decreased p85 binding but with increased Ras-
MAPK and PI3K-Akt activation in response to
insulin (Kriauciunas et al., 2000). It is therefore
possible that IGFRI, like the insulin receptor, may
play a role in localizing IRS-1 (and associated Grb2
and p85) to the inner cell membrane in EN-expressi ng
cells where it can efficiently activate components of
the Ras-MAPK and PI3K-Akt pathways required for
EN transformation. We are currently analysing
whether there is differential localization of IRS-1 in
R7EN compared with R+EN cells.
A fourth possible explanation for the discrepancy
between IRS-1 tyrosine phosphorylation in R7EN
cells and the block in transformation of these cells is
that IGFRI is activating one or more IRS-1-indepen-
dent pathways essential for EN transformation. It was
previously found that IGFRI mutants with C-terminal
deletions starting at residue 1229 retain mitogenic and
survival functions but lose their transformation activity
(Hongo et al., 1996; O’Con nor et al., 1997; Surmacz et
al., 1995). This suggests that the IGFRI C-terminus
may activate an unknown transformation-associated
pathway (Baserga, 1999). R7 cells expressing the C-
terminal mutant showed no defects in IRS-1 tyrosine
phosphorylation (Valentinis et al., 1997), indicating
that this pathway may be IRS-1-independent. While
over-expression of IRS-1 alone is capable of transform-
ing NIH3T3 fibroblasts expressing physiologic levels of
IGFRI, over-expression in R7 cells does not result in
transformation (D’Ambrosio et al., 1995; Fei et al.,
1995). Several recent studies have shed further light on
these observations. Using differential display, a notch
family protein, DBI-1, was found to be specifically
down-regulated in R7 cells re-expressing full length
IGFRI compared to those expressing the C-terminal
108 mutant (Hoff et al., 1998). DBI-1 was subsequently
found to be deleted in lung cancer under the name
DICE-1, and may represent a tumor suppressor gene
down-regulated by IGFRI signaling in transformed
cells (Wieland et al., 1999). The importance of such a
pathway in EN transformation remains to be deter-
mined. However, unless it is somehow linked to
activation of the Ras-MAPK and PI3K-Akt cascades,
its absence in R7 cells does not explain our
observation that pathways essential for EN transfor-
mation are attenuated in R7EN cells and enhanced in
R+EN cells.
In summary, our findings indicate that like man y
other dominantly-acting oncoproteins, EN transforma-
tion requires a functional IGFRI signaling axis. While
many chromosome 11 genes may be affe cted by
trisomy 11, our results are consistent with a model in
which this abnormality contributes to increased IGF2
stimulation of IGFRI. IGFRI stimulation appears to
be necessary for full activation of pathways essential
for EN transformation, namely the Ras-MAPK and
PI3K-Akt cascades. EN can associate with IRS-1 and
IRS-1 is constitutively tyrosine phosphorylated in EN-
expressing cells. Therefore IRS-1 may be the adaptor
that links EN to key mitogenic and survival pathways
necessary for its transformation activity. It remains
unclear whether the EN/IRS-1 interaction is direct or
indirect. Attempts to pull down EN using a-IGFRI
antibodies (or the reverse) have so far been unsuccess-
ful in demonstrating an association between EN and
IGFRI or whether IGFRI is part of the putative EN/
IRS-1 complex. However, this may be a weak
interaction that is not maintained under the conditions
we utilized for immunoprecipitation. What is clear
from our studies is that EN-mediated IRS-1 tyrosine
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5692
Oncogene
phosphorylation is insufficient for EN transformation
in the absence of IGFRI. This contrasts with a
previous report that not only demonstrated an
interaction between IRS-1 and the SV40 T-antigen in
R7 cells, but that co-transfection of T-antigen and
IRS-1 was sufficient to transform these cells (Fei et al.,
1995). Additional studies are necessa ry to determine if
the role of IGFRI in EN transform ation is to enhance
EN/IRS-1 mediated signaling (such as by juxta-
positioning active signaling complexes next to down-
stream effector cascades), or whether IGFRI is
activating novel transformation-associated pathways.
Our findings also provide further evidence for overlap
between NTRK and IGFRI signaling. An increasing
number of reports highlight potential roles for NTRK
receptors in oncogenesis (Bongarzone et al., 1989;
Miknyoczki et al., 1999; Pahlman and Hoehner, 1996;
Reinach and MacLeod, 1986; Reuther et al., 2000;
Weeraratna et al., 2000). Therefore elucidation of
pathways activated during EN transformation, such
as those involving the IGFRI axis, may provide novel
insights into how NTRK signaling contributes to
oncogenesis.
Materials and methods
Tissue culture
IGFRI knockout mouse embryo fibroblasts (R7 cells) and
P6 cells, a fibroblast cell line engineered to over-express IGF-
IR, were kind gifts from Dr Renato Baserga (Kimmel Cancer
Center, PA, USA). Cells were maintained in Dulbecco’s
modified Eagle’s medium with high glucose (Gibco/BRL)
with 9% fetal bovine serum (Gibco/BRL) supplemented with
penicillin/streptomycin antibiotics (Gibco/BRL). The 9%
concentration of FBS resulted from addition of 50 ml of
filtered serum to 500 ml of DMEM to a total volume of
550 ml. R7 cells were maintained in media containing 34 mg
G418/litre (Sigma). Cells were cultured at 378C with 5%
carbon dioxide. NIH3T3 cells were obtained from the
American Type Culture Collection (ATCC 1658) and were
cultured in DMEM high glucose, with 9% calf serum. BOSC
23 murine retroviral packaging cells used to produce
ecotropic viruses were a kind gift of Dr Rob Kay (Terry
Fox Laboratories, Vancouver, Canada), and were cultured in
9% fetal bovine serum in DMEM.
Retroviral constructs
Infections of R7 and NIH3T3 fibroblasts were carried out
using the Murine Stem Cell Virus (MSCV) retroviral
expression system (Clontech). Briefly, the BOSC23 packaging
cell line was transfected with target plasmids using the
calcium phosphate method as previously described (Pear et
al., 1993). The R7EN and NIH3T3 EN cell lines were
generated using MSCV puromycin vectors containing the full
length ETV6-NTRK3 cDNA (accession #AF041811) as
described (Wai et al., 2000). The R+ cell line was generated
by infection of R7 cells using the MSCV hygromycin vector
containing full length IGFRI cDNA. A SalI/EcoRI fragment
of pGR13 (a gift of Dr Renato Baserga) containing the full
length cDNA for IGFRI was ligated into the XhoI/EcoRI
sites in the MSCV hygromycin vector. Verification of the
identity and orientation of the IGFRI cDNA in each vector
was carried out using sequencing and restriction enzyme
digest analysis. Cells expressing activated Ha-Ras were made
by transfection of pCTV 2.11 containing activated V12 Ha-
Ras (a gift of Dr Rob Kay) in conjunction with the BOSC 23
packaging system and puromycin selection as described (Wai
et al., 2000).
Soft agar colony assays
Cells were plated at a density of 2000 cells per milliliter of
0.2% agarose supplemented with 10% FBS over an under-
layer of 0.4% agarose/10% FBS in DMEM. Each experiment
was carried out in triplicate in 35 mm wells for each cell line.
Cell lines were examined in a minimum of seven separate soft
agar experiments. Colony formation was assessed at 2 weeks
by macroscopic colony formation. All colonies greater than
0.1 mm in size were counted along with all single cells in each
high-powered field (406 objective). Ten high powered-fields
per well were counted for a total of 30 counts per experiment.
The results are represented as the ratio of macroscopic
colonies per total cells plated (single cells plus colonies).
Statistical analysis was carried out using the Student’s t-test.
Statistical significance was set at a P-value 50.05.
Northern blotting
Total RNA was extracted from primary frozen tumor
samples and cell lines using the acid guanidinium-phenol/
chloroform method as previously described (Sambrook et al.,
1989). Northern blotting was carried out using 20 mg of total
RNA and following standard protocols (Sambrook et al.,
1989). A 500 base-pair IGF2 probe was generated by RT –
PCR of human RNA using primers specific for IGF2 exon 9,
including 5’-CTTGGATTTGAGTCAAATTGG-3’ (sense
primer) and 5’-CCTCCTTTGGTCTTACTGGG-3’ (antisense
primer). The PCR cycling parameters were as follows: one
30 s denaturation cycle at 958C, followed by 25 cycles at
958C for 30 s, 588C for 1 min, 728C for 1 min, and followed
by a final incubation of 688C for 10 min. The IGF2 PCR
product was purified using a Qiagen gel extraction kit. The b-
actin probe for RNA integrity was obtained from Clontech.
Western blotting and immunoprecipitation
Cells were cultured in either 9% serum or in 0.5% serum for
18 h (to synchronize cells in G
0
) and then stimulated with 9%
serum or differing concentrations of purified IGF1 or IGF2
as indicated in the figure legends. Cells were cultured to
*75% confluence, and either starved or stimulated as
indicated and then rinsed twice with PBS followed by lysis
as previously described (Obermeier et al., 1993). Cell lysates
were cleared by centrifugation and protein concentrations
were determined using Biorad protein quantification kit (Dc
Protein Assay Kit). Immunoprecipitation was carried out
with protein A-conjugated Sepharose beads (Amersham)
overnight at 48C with mixing. The beads were washed three
times with lysis buffer. Samples for both Western blotting
and from immunoprecipitation were mixed with Laemmli
buffer and separated by electrophoresis on 10 – 15% gels
according to standard protocols (Sambrook et al., 1989). The
proteins were transferred to Immobilon-P membrane (Milli-
pore) and blocked in 5% bovine serum albumin. Analysis
was carried out with the indicated antibodies and detection
was achieved with ECL according to the manufacturer’s
protocol (Amersham). The antibodies used were as follows:
a-IR3 a-IGFRI mouse monoclonal (Calbiochem), a-Grb2
ETV6-NTRK3 transformation requires IGF1 receptor signaling
KB Morrison et al
5693
Oncogene
mouse polyclonal (Transduction Laboratories), a-phospho-
Mek1/2 Ser217/221 rabbit polyclonal (New England Biolabs),
a-phospho-Akt Ser473 rabbit polyclonal (New England
Biolabs), a-Cyclin D1/2 mouse monoclonal (Upstate Biotech-
nology), a-IGF-IR b-subunit rabbit polyclonal antibody
(Santa Cruz), a-NTRK3 rabbit polyclonal antibody (Santa
Cruz), a-C-terminal IRS-1 rabbit polyclonal (Upstate
Biotechnology), a-p85 PI-3K (Transduction Laboratories),
a-PLCg (Transduction Laboratories), and a-Tel (ETV6)
rabbit polyclonal (P Marynen, University of Leiden,
Belgium). Purified IGF1 and IGF2 peptides were obtained
from Sigma.
Site-directed mutagenesis
Site-directed mutagenesis was carried out using the Strata-
gene QuikChange Site Directed Mutagenesis Kit. This was
used to create the EN kinase dead (EN-K380N) mutant as
well as the PLCg-binding mutant using methods as previously
described (Wai et al., 2000). The Y560F and Y594F mutants
were created using the same kit with the following primers;
5’-GAGATCTTCACCTTTGGAAAGCAGCC-3’ for the
Y560F mutant, and the 5’-CCCAAAGAGGTGTTC-
GATGTCATGCTG-3’ for the Y594F mutant. The DNA
sequence of the mutants was verified by sequence analysis.
The PCR cycling parameters were as follows: one 30 s
denaturation cycle at 958C, followed by 18 cycles at 958C for
30 s, 558C for 1 min, 728C for 10 min, and followed by a
final incubation of 688C for 10 min.
Acknowledgments
The authors wish to thank Dr Renato Baserga for
supplying R7 cellsandDrsMichaelCoxandRobKay
for helpful discussions. This work was supported by funds
from the Canadian Institutes for Health Research (to PHB
Sorensen) and by funds from the Johal Program in
Pediatric Oncology Basic and Translational Research at
the B C Research Institute for Children’s and Women’s
Health.
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