Lack of interaction between ErbB2 and insulin receptor substrate signaling in breast cancer
Farabaugh et al. Cell Communication and Signaling
Lack of interaction between ErbB2 and insulin receptor substrate signaling in breast cancer
Susan M. Farabaugh 1
Bonita T. Chan 0
Xiaojiang Cui 0
Robert K. Dearth 0
Adrian V. Lee 0 1
0 Lester and Sue Smith Breast Center, Department of Molecular and Cellular Biology, Baylor College of Medicine , Houston, TX 77030 , USA
1 Women's Cancer Research Center, Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, Magee Women's Research Institute , 204 Craft Avenue, Room A412, Pittsburgh, PA 15213 , USA
Background: ErbB2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/Neu) is amplified in breast cancer and associated with poor prognosis. Growing evidence suggests interplay between ErbB2 and insulin-like growth factor (IGF) signaling. For example, ErbB2 inhibitors can block IGF-induced signaling while, conversely, IGF1R inhibitors can inhibit ErbB2 action. ErbB receptors can bind and phosphorylate insulin receptor substrates (IRS) and this may be critical for ErbBmediated anti-estrogen resistance in breast cancer. Herein, we examined crosstalk between ErbB2 and IRSs using cancer cell lines and transgenic mouse models. Methods: MMTV-ErbB2 and MMTV-IRS2 transgenic mice were crossed to create hemizygous MMTV-ErbB2/MMTVIRS2 bigenic mice. Signaling crosstalk between ErbB2 and IRSs was examined in vitro by knockdown or overexpression followed by western blot analysis for downstream signaling intermediates and growth assays. Results: A cross between MMTV-ErbB2 and MMTV-IRS2 mice demonstrated no enhancement of ErbB2 mediated mammary tumorigenesis or metastasis by elevated IRS2. Substantiating this, overexpression or knockdown of IRS1 or IRS2 in MMTV-ErbB2 mammary cancer cell lines had little effect upon ErbB2 signaling. Similar results were obtained in human mammary epithelial cells (MCF10A) and breast cancer cell lines. Conclusion: Despite previous evidence suggesting that ErbB receptors can bind and activate IRSs, our findings indicate that ErbB2 does not cooperate with the IRS pathway in these models to promote mammary tumorigenesis.
IRS; ErbB2; Breast cancer
Insulin receptor substrate (IRS) proteins are cytoplasmic
adapters which function as signaling intermediates
downstream of cell surface receptors. Although IRS
proteins are common intermediates of multiple growth and
hormone receptors [1–6], they are most well known as
signaling intermediates for the insulin receptor (InsR)
and the insulin-like growth factor I receptor (IGF1R) [2,
7–9]. IRS1 and IRS2 are expressed in normal and
cancerous breast epithelium [6, 8, 10]. IRS1 and IRS2
contain high homology and activate common signaling
pathways, such as PI3K/Akt and MAPK/ERK kinases
, yet these two proteins have distinct functions .
Generally, IRS1 is associated with tumor initiating
programs such as growth and survival while IRS2 is associated
more closely with progression and metastasis [13–15]. As
IRS2 is the more definitive mediator of tumor progression
and metastasis, we focused our studies on IRS2.
It is becoming increasingly evident that the IRS proteins
are regulated by epidermal growth factor receptor (EGFR)
and Erb-B2 Receptor Tyrosine Kinase 2 (ErbB2, HER2/
Neu) [15–18]. For example, IRS2 levels are increased by
EGFR and ErbB2 [15, 18]. Further, stimulation of cells
with EGF enhances IRS phosphorylation and downstream
PI3K signaling in the absence of IGF signaling [15, 19, 20].
In tamoxifen-resistant breast cancer cells, EGFR and
ErbB3 recruit and phosphorylate IRS1 [16, 17].
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Given the emerging evidence for interaction between
EGFR/ErbB2 and IGF/IRS signaling in breast cancer, we
investigated crosstalk between ErbB2 and IRSs. Herein, we
demonstrate that overexpression of IRS2 in MMTV-ErbB2
transgenic mice did not alter mammary tumorigenesis or
metastasis. Consistent with this, overexpression or
knockdown of IRS1 and IRS2 had little or no affect upon ErbB2
action in both mouse and human mammary epithelial and
breast cancer cells. Taken together, our data indicates little
to no role for IRSs in ErbB2 action in breast cancer.
The BRI-JM04 mouse cell line was maintained in
DMEM (with glutamine, glucose, sodium pyruvate) with
10 % serum. The MCF-10A human mammary epithelial
cell was maintained in DMEM/F-12 supplemented with
5 % horse serum, EGF, chlorea toxin, hydrocortisone and
insulin according to ATCC standards.
BRI-JMO4 cells were plated at 25–30 % density one day
before transfection in antibiotic free complete medium.
3.6x105 cells were plated per well of a 6 well plate. For
overexpression experiments, the next day, cells were
transfected with pcDNA3.1 plasmids containing either
HA-IRS1, HA-IRS2, or an empty vector control with
Lipofectamine 2000 (Thermo Fisher Scientific). For
knockdown experiments, the next day, cells were transfected with
50uM of siRNA against IRS1, IRS2, or both IRS1 and IRS2
together using DharmaFECT 1 (Dharmacon). Transfections
were performed following the instructions provided by the
Twenty-four hours after siRNA transfection, BRI-JO4
cells were washed and left to rest for four hours. 2,000
cells per well were then seeded into 96-well plates and
starved overnight. After starvation, cells were treated
with or without 10 % fetal bovine serum. Plates were
washed with PBS and frozen on days 1 and 4. When all
of the plates were collected, cell growth was examined
by CyQuant (Thermo Fisher Scientific).
Signaling #3724S), pIGF-IR (Biosource #44804), IGF1R
(Cell Siganling #9750), pY877-ERBB2 (Cell Signaling
#2241), ERBB2 (Cell Signaling #2248), IRS1 (Upstate
#06248), IRS2 (Upstate #06-506), pAKT (Cell Signaling 9272),
AKT (Cell Signaling 4060), pERK1/2 (Cell Signaling 4377),
ERK1/2 (Cell Signaling 9102S) were diluted in 5 % BSA
blocking buffer. Generation of ErbB2/IRS2 Bigenic Mice.
All procedures were conducted in accordance with the
NIH Guide for the Care and Use of Laboratory Animals
and were approved by the IACUC at Baylor College of
Medicine. MMTV-ErbB2 mice were received from
Jackson Laboratories . MMTV-IRS2 mice have been
previously reported . MMTV-ErbB2 mice were
maintained homozygous and MMTV-IRS were hemizygous.
Mice were maintained on a 12-h light, 12-h dark schedule
with ad libitum access to laboratory chow and water. To
generate bigenic mice, homozygous MMTV-ErbB2 male
mice were bred with hemizygous MMTV-IRS2 female
mice to generate hemizygous MMTV-ErbB2 and
hemizygous MMTV-ErbB2/MMTV-IRS2 bigenic mice. To study
the effect of parity, hemizygous MMTV-ErbB2 and
hemizygous MMTV-ErbB2/MMTV-IRS2 bigenic female mice
were bred with outbred CD1 male mice and allowed to go
through a full pregnancy, lactation, and involution. At
weaning pups were removed and euthanized.
Analysis of median time to tumor formation (MTTF)
Median times to tumor formation (MTTF) was
determined by weekly palpation. Tumor formation was
recorded when tumors were first palpable. When tumors
reached 1,000 mm3, tumors were harvested for
molecular analysis. Tumor volumes were measured with
calipers and volumes were calculated using the formula
Volume = (Length × Width × Width)/2.
Five um serial tumor sections were deparaffinized,
gradually hydrated, and stained for hematoxylin and eosin
(H&E). Sections were then examined and scored by a
pathologist to determine histological phenotypes as well
as stromal, inflammatory, and lactation properties.
For the detection of lung metastases, 5 um sections
were cut at intervals of 100 um through one half of the
lung, all sections were stained by H&E, and then
examined microscopically. Lungs were scored positive for
lung metastases if they contained lesions of more than
100 cells. All lesions were stained for HA (HA-IRS2) by
Cells were lysed in RIPA lysis buffer 48 h after transfection.
Protein concentration was determined using the BCA kit
(Pierce). Twenty-five to 50 ug of protein was loaded onto
SDS-PAGE gels for analysis. Primary antibodies of HA (Cell
IRS2 overexpression does not affect ErbB2-mediated
tumorigenesis and metastasis in transgenic mice
Recent evidence suggests that ErbB2 may utilize IRSs for
signaling. To determine potential cooperation between
these pathways, we analyzed the effect of elevated IRS2 on
ErbB2-mediated breast tumorigenesis. We compared
mammary tumorigenesis between MMTV-ErbB2 and
MMTV-ErbB2/MMTV-IRS2 bigenic mice. Under parous
(p) and nulliparous (np) conditions, median time to tumor
formation (MTTF) was similar for both ErbB2 (p = 214
and np = 244 days) and ErbB2/IRS2 bigenic mice (p = 211
and np = 264 days) (Fig. 1a and b). Histological analysis
revealed that the bigenic ErbB2/IRS2 tumors closely
mirrored the phenotype of ErbB2 tumors with 88 % solid
adenocarcinoma in ErbB2 and 66 % solid adenocarcinoma
in ErbB2/IRS2 bigenic tumors (Fig. 1c and Table 1).
Although some bigenic tumors (21 %) did show features of
MMTV-IRS2 mouse tumors such as squamous
differentiation , most bigenic tumors recapitulated ErbB2 tumor
phenotypes, suggesting that the ErbB2 pathway may be
the primary driver of tumorigenesis in these bigenic mice.
We also noted no difference in lung metastasis in ErbB2
and bigenic ErbB2/IRS2 mice with nodules observed at 53
and 50 % for ErbB2 and bigenic mice, respectively (Fig. 1d
and Table 1). Protein analysis of the tumors confirmed
overexpression of HA-tagged IRS2 in the bigenic ErbB2/
IRS2 tumors (Fig. 1e). No difference in IRS1 levels were
noted. Interestingly, ErbB2 levels were decreased in bigenic
Modulation of IRS1 and IRS2 levels in ErbB2-expressing
mouse cells has little effect upon ErbB2 signaling and cell
As overexpression of IRS2 showed an unexpected lack
of influence on ErbB2-driven tumorigenic phenotypes
(Fig. 1), we further evaluated the interaction using in
Fig. 1 ErbB2 and ErbB2/IRS2 mice had similar time to tumor formation, tumor histology, and lung metastases. Kaplan-Meier plots of age-matched
MMTV-ErbB2/IRS2 bigenic (black line) and MMTV-ErbB2 transgenic (gray line) (a) parous and (b) nulliparous mice. Mean time to tumor formation (MTTF) is
shown in days and was measured by weekly palpation. Formation was recorded when first palpable. c H&E of tumors from nulliparous mice representing
the histological phenotypes presented. d Immunoblotting of protein lysates from ErbB2 and ErbB2/IRS2 bigenic tumors for expression of IRS1, IRS2, HER2,
and downstream signaling pathways. e Lungs of tumor-bearing mice were sectioned, stained with H&E, and analyzed for lung metastases. Numbers
represent percentage of mice containing lung lesions to total number of mice analyzed
Table 1 Histological analysis of the ErbB2/IRS2 bigenic tumors
closely mirror ErbB2 single transgenic tumors
Adenosquamous Carcinoma 8 % (2/25)
Histological analysis of nulliparous and parous tumors were combined for a
total of 24 ErbB2/IRS2 bigenic tumors (18 mice) and 25 age-matched ErbB2
transgenic tumors (17 mice). All tumor phenotypes were scored by a pathologist
based on H&E staining
vitro culture models. As the functions and roles for IRS1
and IRS2 are still unclear, we analyzed the effect of both
IRSs. To determine modulation of ErbB2 signaling upon
overexpression of IRS1 and IRS2 in vitro, HA-tagged
IRS1 or HA-tagged IRS2 were transiently transfected
into the MMTV-ErbB2 mouse cell line BRI-JM04.
Knowlden et al. demonstrated that, in ER+ cells,
heregulin (HRG) stimulates the recruitment of IRS1 to ErbB3
[16, 17] and thus we examined the effect of HRG in
BRI0-JM04 cells with or without IRS overexpression. As
shown in Fig. 2a, overexpression of IRS1 or IRS2 in
BRIJM04 cells in SFM (vehicle) conditions did not affect
phosphorylation of IGF1R, Akt, or ERK1/2. HRG and
IGF1 treatment increased phosphorylation of Akt and
ERK1/2 in BR10-JM04 cells, but overexpression of IRS1 or
IRS2 (noted by HA expression) had little to no effect upon
HRG or IGF1 induced Akt or ERK1/2 phosphorylation. If
anything, we noted a reproducible but very small decrease
in HRG-induced Akt activity.
To determine if IRS1 or IRS2 are required for ErbB2
signaling, we knocked-down each IRS individually and
together in ErbB2 expressing BRI-JM04 cells and then
stimulated cells with HRG or EGF. siRNA knockdown of
IRS1 and IRS2 were both efficient and specific. In
control cells, HRG induced p-Akt, whereas EGF induced
p-ERK1/2. Knockdown of IRS1, IRS2 or the combination
caused a slight decrease in HRG-induced p-Akt. No
major differences were observed with EGF treatments
with or without IRS1/IRS2 knockdown. We next tested
whether the minor effect of IRS knockdown on p-Akt
may reduce cell growth; however, knockdown of IRS1 or
IRS2 followed by a growth assay showed no significant
effect on cells grown in SFM or 10 % serum (Fig. 2c).
Elevated IRS1 and IRS2 levels in human breast cells does
not affect EGFR or ErbB2 signaling
As the previous experiments were all performed using
murine models, we explored the effect of IRS1 and IRS2
on ErbB2 signaling in human immortalized
nontransformed human mammary epithelial cells (MCF10A)
which overexpress IRS1 or IRS2 and have previously
been reported to have enhanced IGF signaling .
Stimulation of MCF10A cells with HRG or EGF resulted
in phosphorylation of ErbB2, Akt and ERK1/2. Cells
overexpressing IRS1 or IRS2 had increased basal
phosphorylation of Akt that was not affected by HRG or EGF
treatment. The basal phosphorylation of ERK1/2 was
also elevated by IRS overexpression, but in this case the
induction (both fold induction and total phosphorylated
Fig. 2 Modulation of IRS1 and IRS2 levels in ErbB2 expressing mouse cells alters ErbB2 signaling but not cell growth. a BRI-JM04 mouse cells were
starved overnight and then treated in the morning with either 60 ng/ul of heregulin (HRG) or 20 ng/ul of IGF1 for 15 mins and then harvested.
Expression of ErbB2 signaling proteins was determined by immunoblotting. Blots are representative of 3 experiments. b BRI-JM04 cells were transfected
with siRNA against IRS1, IRS2, or both IRS1 and IRS2. At 48 h post-transfection, cells were stimulated with 20 ng/ul heregulin (H) or 60 ng/ul EGF (E) for
15 min. Cells were then harvested and analyzed for ErbB2 signaling by immunoblotting. c After IRS1 or IRS2 knockdown, BRI-JMO4 cells were plated
and maintained with or without serum for 4 days. Cell growth was determined by CyQuant. Graph is representative of 3 experiments. One-way Anova
was applied. N.S. = not significant
The IGF and ErbB pathways are involved in tumor
initiation, tumor progression and resistance to therapy [22–25].
These pathways overlap in their signal transduction
pathways, sharing PI3K/Akt and MEK/ERK signaling, and
several studies suggest crosstalk between the pathways may be
at least partially responsible for resistance to both
ErbB2and IGF1R-targetered therapies [25–31]. Several studies
have previously demonstrated that EGFR/ErbB recruits and
activates IRSs [16–18], and in some cases suggests a process
for therapeutic resistance to drugs such as tamoxifen. In this
report we examined the role of IRSs in ErbB2 action in vitro
and in vivo. Contrary to expected, we demonstrate that
increased IRS1 or IRS2 expression does not enhance the
ability of ErbB2 to further stimulate downstream signaling
pathways and enhance tumorigenic phenotypes.
In this study, we hypothesized that increasing both
ErbB2 and IRSs would result in enhanced downstream
signaling (through Akt and ERK1/2). However, in our in
vitro cell line data, we observed that increased or
decreased IRS levels had little to no effect upon ErbB2
signaling. It should be noted that our studies were
performed in the presence of overexpressed ErbB2 which
was developed to mimic the amplification seen in
human breast cancer. Other studies have noted ErbB2-IRS
interaction and cooperation in antiestrogen resistance
where the ErbB2 is simply elevated and not amplified. It
is possible that in our model systems, ErbB2 is
overexpressed at a level where it no longer requires IRS
expression beyond what is endogenously expressed or the level
at which ErbB2 is able to induce. This does not rule out
a role for the ErbB2-IRS cooperation in antiestrogen
resistance, but suggests that ErbB2 amplified human breast
cancers may have sufficient ErbB2-induced regulation
and signaling. It is also possible that the overexpression
of ErbB2 has disrupted normal signaling networks and
interactions. Recent studies have shown an intricate
network of feedback mechanisms with PI3K/S6K regulating
IGF1R/IRSs and MEK negatively feeding back to the
ErbB receptors [32, 33]. There is thus a fine balance of
receptor signaling components with interference
possible when one is overexpressed. Limited amounts of
substrates can also cause squelching. For example,
recruitment of IRSs to alternate receptors such as the
EGFR family limits the amount of IRS available for
association with IGF1R which in turn limits downstream
IGF1R signaling . Inhibition of the EGFR pathway
can then direct signaling back through the IGF1R-IRS
In vivo, overexpression of IRS2 did not alter ErbB2
mediated tumorigenesis. Interestingly, ErbB2 tumors showed
similar histologic phenotypes (solid adenocarcinomas)
compared to bigenic ErbB2/IRS2 tumors, suggesting that
even in the presence of elevated IRS2, the ErbB2 signaling
pathway remains the major driver of tumorigenesis. We
did identify a small amount of squamous metaplasia in
bigenic tumors, something that is rarely seen in ErbB2
tumors, but widely observed in IRS2 tumors; however, this
effect was minimal and rarely seen.
In conclusion, we find little evidence that increased IRS
expression enhances ErbB2 mediated signaling and
tumorigenesis. This does not corroborate or conflict
with other studies as our models may have sufficient IRS
Fig. 3 Modulation of IRS1 and IRS2 expression in human MCF10A breast cells alters ErbB2 signaling. MCF10A cells stably overexpressing HA-IRS1
or HA-IRS2 were serum starved for 24 h and then treated with 20 ng/ul heregulin (H) or 60 ng/ul EGF (E) for 15 mins and harvested. Immunoblotting
was performed to determine ErbB2 signaling
expression to support ErbB2 signaling. We show this
phenotype in both human and murine models, in vitro
and in vivo. A better understanding of this complex
systems network will be critical to optimize response to
anti-growth factor receptor and signaling intermediate
(e.g. PI3K, mTOR) inhibitors in breast cancer.
Research reported in this publication was supported in part by the National Cancer
Institute of the National Institutes of Health under award numbers R01CA94118 and
P30047904. The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of Health. AVL is a
recipient of a Susan G. Komen Scholar award (SAC150021). SMF is a recipient of a
Department of Defense Breast Cancer Research Postdoctoral Fellowship award
(W81XWH-14-1-0063). The authors acknowledge support from the University of
Pittsburgh Cancer Institute (UPCI) and UPMC.
XC developed the transgenic mice and initiated the study. RD carried out mouse
breeding, harvesting, and analysis of tumors. BC performed in vitro experiments
and drafted the original manuscript. SF performed in vitro experiments and
re-drafted and revised the manuscript. AL conceived the study, participated in its
design and coordination, and revised the manuscript. All authors read and approve
the final manuscript.
Ethics approval and consent to participate
All animal procedures were conducted in accordance with the NIH Guide for
the Care and Use of Laboratory Animals and were approved by the IACUC at
Baylor College of Medicine. MMTV-ErbB2 mice were received from Jackson
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