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Small Molecule Therapeutics
Inhibition of Insulin-like Growth Factor–Binding Protein-3
Signaling through Sphingosine Kinase-1 Sensitizes Triple-
Negative Breast Cancer Cells to EGF Receptor Blockade
Janet L. Martin, Hasanthi C. de Silva, Mike Z. Lin, Carolyn D. Scott, and Robert C. Baxter
Abstract
The type I EGF receptor (EGFR or ErbB1) and insulin-like growth factor–binding protein-3 (IGFBP-3) are
highly expressed in triple-negative breast cancer (TNBC), a particularly aggressive disease that cannot be
treated with conventional therapies targeting the estrogen or progesterone receptors (ER and PR), or HER2. We
have shown previously in normal breast epithelial cells that IGFBP-3 potentiates growth-stimulatory signaling
transduced by EGFR, and this is mediated by the sphingosine kinase-1 (SphK1)/sphingosine 1-phosphate
(S1P) system. In this study, we investigated whether cotargeting the EGFR and SphK1/S1P pathways in TNBC
cells results in greater growth inhibition compared with blocking either alone, and might therefore have novel
therapeutic potential in TNBC. In four TNBC cell lines, exogenous IGFBP-3 enhanced ligand-stimulated EGFR
activation, associated with increased SphK1 localization to the plasma membrane. The effect of exogenous
IGFBP-3 on EGFR activation was blocked by pharmacologic inhibition or siRNA-mediated silencing of SphK1,
and silencing of endogenous IGFBP-3 also suppressed EGF-stimulated EGFR activation. Real-time analysis of
cell proliferation revealed a combined effect of EGFR inhibition by gefitinib and SphK1 inhibition using SKi-II.
Growth of MDA-MB-468 xenograft tumors in mice was significantly inhibited by SKi-II and gefitinib when
used in combination, but not as single agents. We conclude that IGFBP-3 promotes growth of TNBC cells by
increasing EGFR signaling, that this is mediated by SphK1, and that combined inhibition of EGFR and SphK1
has potential as an anticancer therapy in TNBC in which EGFR and IGFBP-3 expression is high. Mol Cancer Ther;
13(2); 316–28. 2013 AACR.
Introduction
Approximately, 15% of breast tumors are classified as
triple-negative breast cancers (TNBC), a term that denotes
their lack of estrogen receptor (ER) and progesterone
receptor (PR), and nonamplification of the HER2. These
tumors, which are particularly aggressive and tend to
occur with higher frequency in young women, cannot be
targeted by therapies that depend on the expression of
functional ER, PR, and HER2. Expression of type I EGF
receptor (EGFR), a receptor tyrosine kinase that trans-
duces potent proliferative and cell survival signals in
many malignancies including breast cancer (1, 2), is typ-
ically upregulated in TNBC (3), but clinical trials have not
shown significant benefit from single-line targeting of
EGFR in TNBC (4).
Insulin-like growth factor–binding protein-3 (IGFBP-3)
is one of six proteins that bind the growth factors IGF-I and
-II with high affinity, and modulate their potent prolifer-
ative and cell-survival effects mediated by the type I IGF
receptor (IGF-IR). IGFBP-3 also exerts growth-inhibitory
activity independent of modulating IGF-IR activation by
IGFs, and in some tissues this results in suppression of
tumor growth and metastasis (5, 6). In contrast, studies
from a number of groups have demonstrated growth-
promoting activity of IGFBP-3 in vitro (7–11) and elevated
gene expression of IGFBP3 occurs in a range of malignan-
cies (12–14). In human breasttumors, expression of IGFBP-
3 is correlated with markers of poor prognosis such as ER
and PR negativity, S-phase fraction, and tumor size (15–
18). These clinical observations were recapitulated in a
xenograft tumor model in which T47D cells expressing
IGFBP-3 as a result of cDNA transfection developed larger
tumors than control cells in nude mice (19).
Several mechanisms underlying the stimulation of
breast cancer cell growth by IGFBP-3 have been described
by our laboratory, including the prevention of inhibitory
nuclear receptor signaling (20) and the promotion of cell
survival by autophagy (21). Our studies have also
revealed that in MCF-10A mammary epithelial cells
Authors' Affiliation: Hormones and Cancer Division, Kolling Institute of
Medical Research, University of Sydney, Royal North Shore Hospital, St.
Leonards, New South Wales, Australia
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Janet L. Martin, Kolling Institute of Medical
Research, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia .
Phone: 61-2-9926-4716; Fax: 61-2-9926-4034; E-mail:
janet.martin@sydney.edu.au
doi: 10.1158/1535-7163.MCT-13-0367
2013 American Association for Cancer Research.
Molecular
Cancer
Therapeutics
Mol Cancer Ther; 13(2) February 2014
316
IGFBP-3 potentiates ligand-stimulated activation of EGFR
(11, 22). The effects of IGFBP-3 on EGFR activation
required sphingosine kinase-1 (SphK1), which catalyzes
the conversion of sphingosine to sphingosine 1-phosphate
(S1P), and were mimicked by exogenous S1P, suggesting
that S1P mediates potentiation of the EGFR signaling
pathway by IGFBP-3 in breast epithelial cells. These find-
ings imply that functional blockade of SphK pathway
signaling has the potential to block IGFBP-3 stimulatory
bioactivity.
Breast cancer cell lines that exhibit molecular features of
TNBC, such as the Hs578T and MDA-MB-231 cell lines,
reflect the clinical disease with high expression of EGFR
and IGFBP-3 (23, 24). In view of this, we have investigated
the role of IGFBP-3 and SphK1 in EGFR signaling in TNBC
cells, and the potential efficacy of cotargeting the IGFBP-
3/SphK and EGFR pathways as a novel therapy for the
treatment of these cancers.
Materials and Methods
Materials
Tissue culture reagents and plasticware were from
Trace Biosciences and Nunc. Bovine insulin, EGF, hydro-
cortisone, and a-tubulin antibody were purchased from
Sigma. Antibodies against phospho-Tyr1068 EGFR and
total EGFR, phospho-Ser473 AKT and total AKT, and
phospho-Thr202/Tyr204 extracellular signal–regulated
kinase (ERK)1/2 and total ERK1/2 were purchased from
Cell Signaling Technology and their specificity verified
previously (22). SphK1 antibody (ab16491) was from
Abcam, ER-aantibody was from Epitomics, and aqua-
porin 1 (AQP1) was from Santa Cruz Biotechnology.
Recombinant human IGFBP-3 was expressed in human
911 retinoblastoma cells using an adenoviral expression
system and purified as previously described (25). SphK
inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole
(SKi-II; ref. 26) was from Calbiochem. Gefitinib was pur-
chased from LC Laboratories. Electrophoresis and enh-
anced chemiluminescence (ECL) reagents were purchased
from Bio-Rad, Amrad-Pharmacia, and Pierce.
Cell culture
The following breast cancer cell lines were purchased
from the American Type Culture Collection (ATCC):
BT549, MDA-MB-231, MDA-MB-436, MDA-MB-468,
Hs578T, MCF-7, T47D, and ZR-75-1. Identity of Hs578T,
T47D, and ZR-75-1 cells, which were obtained from ATCC
between 2001 and 2005, was confirmed by short-tandem
repeat profiling by CellBank Australia in December 2012.
Cryopreserved stocks of other cell lines (purchased in
2010 from ATCC) were established within 1 month of
receipt, and fresh cultures for use in experiments were
established from these stocks every 2 to 3 months. All lines
were maintained in RPMI-1640 medium containing 5%
FBS and 10 mg/mL bovine insulin in a humidified 5% CO
2
atmosphere at 37C, and were negative for Mycoplasma
contamination.
siRNA-mediated protein knockdown
Knockdown of protein expression was achieved by
electroporation using the following siRNA duplexes from
Qiagen: SphK1, Hs_SPHK1_6, and Hs_SPHK1_7; IGFBP-
3, Hs_IGFBP3_8, and Hs_IGFBP3_3. To achieve protein
silencing, cells were harvested by trypsinization and
resuspended at 2.5 10
6
cells in 100 mL Amaxa transfec-
tion reagent (Lonza Australia Pty Ltd.), then mixed with
1.5 mg silencing siRNA or AllStars negative control siRNA
(Qiagen). Nucleoporation was carried out using an Amaxa
electroporation unit (Lonza), and cells were transferred to
complete medium then plated for specific endpoints as
indicated below. Knockdown of protein expression was
confirmed by quantitative real-time PCR (qRT-PCR), in-
house IGFBP-3 radioimmunoassay (RIA; ref. 24), and
Western blot analysis.
qRT-PCR
IGFBP3 and expression was monitored by qRT-PCR
using TaqMan Gene Expression Assays (Applied Biosys-
tems). Total RNA was isolated from breast cancer cells
using TRIzol reagent (Life Technologies) and reverse-
transcribed using SuperScript III First Strand Synthesis
SuperMix (Invitrogen) according to the manufacturer’s
protocols. TaqMan assays for IGFBP-3 (Hs00181211_m1)
and SphK1 (Hs00184211_m1) were performed using a
Rotor-Gene 3000 thermal cycler (Corbett Research), with
hydroxymethylbilane synthase (HMBS; Hs00609297_m1)
amplification used as internal control. Results were ana-
lyzed using the Rotor-Gene 6 software.
EGFR activation assays
Cells were plated into 12-well plates at 2.5 10
5
cells per
well, and maintained in growth medium for 48 hours,
then medium without insulin for 24 hours. Fresh medium
containing IGFBP-3 with or without inhibitors was added
for 16 hours, then EGF was added directly to cells to give
final concentrations as indicated for individual experi-
ments. Incubations were continued at 37C for 10 minutes,
then cells were washed with ice-cold PBS and lysed dir-
ectly into Laemmli sample buffer [62.5 mmol/L Tris–HCl
pH 6.8, containing 20 g/L SDS, 100 mL/L glycerol, 1 g/L
bromphenol blue, and 50 mmol/L dithiothreitol (DTT)] at
4C for 10 minutes. Lysates were transferred to ice-cold
Eppendorf tubes, and stored at 80C until Western blot
analysis.
Plasma membrane isolation
Fractions containing plasma membranes were isolated
to monitor redistribution of SphK1 in response to treat-
ment with IGFBP-3. Briefly, treated cells were washed two
to three times with cold PBS, and then harvested in
homogenization buffer [20 mmol/L HEPES pH 7.6 con-
taining 250 mmol/L sucrose, 2 mmol/L DTT, 2 mmol/L
EDTA, 2 mmol/L EGTA, and protease inhibitor cocktail
(Roche)]. Cells were homogenized in a Teflon-glass
homogenizer using 50 to 60 strokes and then incubated
on ice for 20 minutes. Cellular debris and other organelles
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 317
and nuclei were pelleted by centrifugation at 10,000 gat
4C for 20 minutes, and the supernatant containing cyto-
solic and membrane fractions was then centrifuged at
100,000 gfor 1 hour. The final membrane pellet was
resuspended in radioimmunoprecipitation assay buffer
(RIPA) lysis buffer (50 mmol/L Tris–HCl pH 7.4, 150
mmol/L NaCl, 1 mmol/L EDTA) containing 0.1% Triton
X-100 and stored at 80C until Western blot analysis.
Western blotting
Cell lysates were prepared for SDS-PAGE analysis by
sonication for 15 seconds on ice, heating at 95C for 8
minutes, and centrifugation for 1 minute at 12,000 rpm.
Samples were fractionated by 7.5% SDS-PAGE and then
proteins were transferred to Hybond C nitrocellulose
(Amersham) at 115 mA for 2 hours. Filters were blocked
in 50 g/L skim milk powder in TBS-T (TBS with Tween-20:
10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4 containing 1
mL/L Tween-20) and probed with primary antibodies
diluted in TBS-T containing 10 g/L BSA at 4C for 16
hours. Filters were washed in cold TBS-T, and incubated
with the appropriate horseradish peroxidase–labeled sec-
ondary antibody for 1 to 2 hours at room temperature.
Washed filters were developed by ECL using SuperSignal
West Pico Chemiluminescent Substrate (Pierce Biotech-
nology). Total and phosphorylated proteins were ana-
lyzed on replicate blots, and filters were reprobed with
a-tubulin antibody as a loading control. Bands were
visualized using a FUJIFILM Luminescent Image Ana-
lyzer LAS-300, and quantified using Image Guage soft-
ware (Science Lab 2004).
Cell proliferation assays
Real-time assessment of cell proliferation over 4 to 6
days was carried out using the IncuCyte Imaging System
(Essen Biosciences). Cells (1 10
3
/well for MDA-MB-231,
MDA-MB-436, and Hs578T, and 5 10
3
cells/well for
MDA-MB-468) were dispensed into 96-well plates in
complete medium and incubated overnight before chang-
ing to fresh medium containing 5% FBS and inhibitors.
Plates were transferred to the IncuCyte apparatus, and
incubations were continued over 72 to 136 hours, depend-
ing on the cell line. Images (4/well) were collected every 2
hours over this time. Cell proliferation over 5 days was
also measured using the CyQUANT NF Cell Proliferation
Assay (Molecular Probes, Life Technologies). Resus-
pended cells (5 10
3
) were dispensed into 96-well plates
in 200 mL complete medium and allowed to adhere for 24
hours at 37C. Media were changed to 100 mL RPMI
containing 5% FBS and inhibitors, and incubations were
continued for 5 days before quantitation.
Tumor growth in vivo
Animal studies were approved by the Institutional
Animal Care and Ethics Committee (ACEC protocol
1105-010A). Tumors were established in 8-week-old
female athymic BALB/c-Foxn1nu/Arc mice (Animal
Resources Centre) by injecting MDA-MB-468 cells (5
10
6
in 100 mL mixed with 50 mL Matrigel) subcutaneously
between the scapulae. Tumor growth was monitored by
caliper measurement weekly until tumors reached a vol-
ume of approximately 150 mm
3
(calculated as LW
2
/2),
when drug treatment was initiated. Groups of 10 animals
were injected intraperitoneally three times weekly
with vehicle [dimethyl sulfoxide (DMSO)], gefitinib (75
mg/kg), SKi-II (50 mg/kg), or combined SKi-II and gefi-
tinib at these doses, in a volume of 50 mL. Treatment and
tumor measurements were continued until the volume of
tumors in control (vehicle) mice was 400 to 500 mm
3
.
Animals were euthanized, and tumors removed, weig-
hed, and snap-frozen in liquid nitrogen. Tumors were
processed for Western blot analysis by homogenization in
lysis buffer (10 mmol/L Tris, pH 8.0 containing 137
mmol/L NaCl, 10 g/L Triton X-100, 10% glycerol, and
protease, and phosphatase inhibitors) using 10 strokes of a
Teflon-glass homogenizer. Samples were sonicated and
centrifuged, and the supernatant transferred to fresh
tubes for storage at 80C. Protein was quantified and
200 mg loaded onto replicate gels for immunoblot analysis
of total and phosphorylated EGFR, AKT, and ERK1/2 as
described above.
Statistical analysis
All in vitro experiments were performed a minimum of
three times and are shown as quantified data (mean
SEM) pooled from the three experiments, unless indicated
otherwise. Statistical analysis (ANOVA with Bonferroni
post hoc test) was performed using Prism 4 for Macintosh
(GraphPad Software, Inc.).
Results
Molecular features of TNBC and ER
þ
cell lines
We have previously shown high levels of IGFBP-3
expression in ER
tumors and an ER
breast cancer cell
line, Hs578T, and low levels in ER
þ
tumors and cells (24).
To extend this, we screened eight human breast cancer cell
lines, five ER
and representative of TNBC, and three
ER
þ
, for IGFBP-3 gene and protein expression. The cell
lines were selected for analysis based on their documen-
ted lack of expression of both ER and PR, and no ampli-
fication of HER2 (23). Both IGFBP-3 gene expression
(measured by qRT-PCR; Fig. 1A, top) and secreted protein
levels (measured by RIA; Fig. 1A, bottom) were markedly
higher in ER
cell lines than ER
þ
cell lines, which had
levels of IGFBP-3 protein below the limit of detection of
the assay. The highest expressing line, MDA-MB-468, had
1,000-fold higher levels of IGFBP-3 mRNA than the lowest
ER
þ
cell line, ZR-75-1.
The expression of proteins involved in EGFR and
IGFBP-3 signaling was characterized in these cell lines
maintained in 5% FBS (Fig. 1B). Expression of EGFR was
readily apparent, although variable, in the five TNBC cell
lines, but was virtually undetectable in the ER
þ
cell lines
MCF-7, T47D, and ZR-75-1. Like IGFBP-3 expression,
EGFR expression was highest in the MDA-MB-468 cell
line, and phosphorylation of EGFR at tyrosine 1068
Martin et al.
Mol Cancer Ther; 13(2) February 2014 Molecular Cancer Therapeutics
318
(Y1068) was apparent in only these cells under serum-
containing (Fig. 1B) and serum-free (data not shown)
conditions. Expression and activation (phosphorylation)
of AKT and ERK1/2, two key signaling intermediates
downstream of EGFR, were apparent in all cell lines under
serum-replete (Fig. 1B) and serum-free (data not shown)
conditions.
Our previous study demonstrated that IGFBP-3 poten-
tiation of EGFR activation requires SphK1 (22). Analysis
of SphK1 by immunoblot of cell lysates revealed its
Figure 1. Protein expression and activation in breast cancer cell lines. A, IGFBP-3 mRNA expression (top) and secreted protein levels in 24 hours conditioned
medium (bottom) from cells maintained in 5% FBS were measured using qRT-PCR and RIA, respectively, as described in the Materials and Methods. IGFBP-3
mRNA expression in each line is expressed relative to that in ZR-75-1 cells, which had the lowest level of IGFBP-3 expression (arbitrarily set as 1). B,
confluent cultures of breast cancer cells were incubated for 24 hours in medium containing 5% FBS. Conditioned media were collected for Western blot
analysis of secreted IGFBP-3, and cells were lysed in Laemmli buffer. Lysates and media were analyz ed using 7.5% SDS-PAGE and Western blotting using the
indicated antibodies (as specified in the Materials and Methods), with a-tubulin as a loading control. Replicate blots were used for phosphorylated
and total proteins. Migration points of molecular mass markers are shown on the right of each blot. The white, black, and gray arrows shown to the left of the
SphK1 immunoblot indicate SphK1a (42 kDa), SphK1b (51 kDa), and SphK1c (44 kDa), respectively, determined in TNBC cells as shown in C.
C, qRT-PCR (top) and Western blot analysis (bottom) of SphK1 gene and protein expression in TNBC cell lines after siRNA-mediated SphK1 silencing as
described in Materials and Methods, using either of two SphK1 siRNA (SK#1 andSK#2), or nonsilencing siRNA (ctl). The level of SphK1 mRNA for each cell line
is expressed relative to nonsilencing control siRNA. For Western blot analysis, the migration of molecular mass standards is shown on the left, and
the white, black, and gray arrows indicate SphK1a (42 kDa), SphK1b (51 kDa), and SphK1c (44 kDa), respectively. Two predominant nonspecific bands that
were not decreased when SphK1 was silenced (of 45 and 53 kDa) are indicated by asterisks to the left of each panel. All blots have been cropped to
highlight relevant bands.
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 319
expression by all cell lines, with multiple bands ranging in
mass from approximately 40 to 70 kDa variably expressed
in the different cell lines (Fig. 1B). In view of literature
suggesting the existence of only three SphK1 forms (27),
the presence of additional bands suggested that the anti-
body used to detect SphK1 (Abcam #16419) was also
reacting nonspecifically with unrelated proteins in the
cell lysates. To determine the bands corresponding spe-
cifically to SphK1 in TNBC cell lines, and thereby the
expression profile of this protein in TNBC, we applied
siRNA-mediated knockdown of SphK1 in the TNBC cell
lines, reasoning that bands which were reduced in inten-
sity represented proteins immunologically related to
SphK1. qRT-PCR confirmed that two siRNA constructs
targeting SphK1 reduced SphK1 gene expression approx-
imately 90% in MDA-MB-231, MDA-MB-436, and MDA-
MB-468 cells, and by approximately 75% in Hs578T (Fig.
1C, top). Western blot analysis of SphK1 in lysates from
the cells (Fig. 1C, bottom) revealed that a number of bands
were reduced or lost in the TNBC cell lines in siRNA-
transfected cells, including bands of approximately 42
kDa (white arrows in Fig. 1C), 51 kDa (black arrows), and
44 kDa (gray arrows), which likely represent SphK1a, and
86- and 14-amino acid N-terminally extended forms of
SphK1 (SphK1b and SphK1c), respectively (27). These
were differentially expressed in the different cell lines,
with SphK1a expressed by all cell lines, SphK1b expressed
by MDA-MB-436, MDA-MB-468, and Hs578T, and
SphK1c expressed in the MDA-MB-231, MDA-MB-436,
and Hs578T cell lines. An uncharacterized 65-kDa protein
was also reduced by SphK1 silencing in MDA-MB-436,
MDA-MB-468, and Hs578T. Two proteins of approxi-
mately 45 and 53 kDa, indicated by asterisks in Fig. 1C,
were not decreased by SphK1 knockdown. There was no
correlation between either the form of SphK1 expressed or
its total levels, and ER status of the cell lines (Fig. 1B).
Exogenous IGFBP-3 potentiates EGFR activation in
TNBC lines and induces SphK1 translocation to the
plasma membrane
To determine whether IGFBP-3 enhances the effects
of EGF in TNBC cell lines, EGFR phosphorylation at
Y1068 was assessed in four TNBC cell lines treated
overnight with IGFBP-3 before stimulation with EGF
for 10 minutes. As shown in Fig. 2A, preincubation with
IGFBP-3 enhanced EGF-stimulated EGFR phosphory-
lation in MDA-MB-231, MDA-MB-436, and Hs578T cell
lines, with the greatest enhancement (2- to 2.5-fold) in
the MDA-MB-231 and Hs578T lines. Although a trend
toward slightly increased EGFR phosphorylation in
response to IGFBP-3 was apparent in the MDA-MB-
468 cells, this was not statistically significant. In the
absence of EGF, IGFBP-3 did not stimulate EGFR phos-
phorylation or expression in any cell line (Supplemen-
tary Fig. S1).
Phosphorylation-induced translocation of SphK1 to the
plasma membrane is essential for generation of S1P (28).
To assess activation of SphK1 in response to IGFBP-3, we
analyzed SphK1 in membranes isolated from cells treated
with IGFBP-3, using the transmembrane water channel
protein AQP1 as a loading control (Fig. 2B). Western blot
analysis of membrane fractions revealed a transient
increase in SphK1a (42 kDa), which is expressed by all
the TNBC cell lines, peaking 30 to 60 minutes after addi-
tion of IGFBP-3, and declining thereafter in MDA-MB-231
and MDA-MB-436 cells. A slightly delayed time-course
and blunted response was apparent for Hs578T cells,
with a significant increase apparent only after 2 hours.
The very low level of SphK1 expression in MDA-MB-468
cells (as shown in Fig. 1) precluded this analysis in that
cell line. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), a cytoplasmic protein, was not detected in
these fractions, indicating no cytoplasmic contamination
of the membrane fractions (data not shown). There was no
clear evidence of translocation of either SphK1b (51 kDa)
or SphK1c (44 kDa) in response to IGFBP-3 (Supplemen-
tary Fig. S2), although both are expressed in MDA-MB-436
and Hs578T. The increase in membrane SphK1 over this
time-course was dependent on IGFBP-3, as shown for
MDA-MB-231 cells in Supplementary Fig. S2, with similar
results obtained for MDA-MB-436 cells (data not shown).
These data suggest that IGFBP-3 is activating SphK1 in
these cells at least in part by inducing its relocation to the
plasma membrane.
Silencing of IGFBP-3 in mammary epithelial cells
reduces the ability of EGF to stimulate EGFR phosphor-
ylation, indicating an influence of endogenous IGFBP-3 on
receptor activation in these cells (22). To investigate
whether endogenous IGFBP-3 is also required for optimal
EGFR activation in breast cancer cells that highly express
the protein, IGFBP-3 was silenced using either of two
siRNAs, and EGFR phosphorylation was determined
after stimulation with EGF. RIA and Western blot analysis
of conditioned medium and cell lysates confirmed >90%
knockdown of IGFBP-3 protein expression in each cell line
(data not shown). As shown in Fig. 2C, the ability of EGF
to stimulate EGFR phosphorylation was significantly
reduced in all cell lines when IGFBP-3 was silenced
compared with nonsilencing control. We also found that
basal EGFR phosphorylation in MDA-MB-468 was red-
uced in IGFBP-3 knockdown cells in the absence of exog-
enous EGF (Fig. 2C). Thus, the sensitivity of EGFR to
stimulation by EGF in these TNBC cell lines is enhanced
by endogenous IGFBP-3.
SphK1 inactivation or silencing blocks IGFBP-3
potentiation of EGFR activation in TNBC cell lines
To demonstrate that SphK1 is involved in the effect of
IGFBP-3 on EGFR activation in TNBC cells, downregula-
tion of its expression and activity was achieved by siRNA-
mediated silencing and pharmacologic inhibition, respec-
tively. As shown in Fig. 3A, transfection with an siRNA
construct targeting SphK1, which reduced its mRNA and
protein levels by 75% to 90% (shown in Fig. 1C) was
accompanied by a loss in the potentiation of EGF-stimu-
lated EGFR phosphorylation elicited by preincubation
Martin et al.
Mol Cancer Ther; 13(2) February 2014 Molecular Cancer Therapeutics
320
with IGFBP-3. Similar results were seen for a second
SphK1 siRNA (data not shown). Consistent with this,
SKi-II, an inhibitor of SphK activity (26), blocked the
enhancement of EGF-stimulated EGFR phosphorylation
induced by IGFBP-3 in MDA-MB-231, MDA-MB-436, and
Hs578T cells (Fig. 3B). Taken together, these data indicate
Figure 2. Endogenousand exogenous IGFBP-3 enhanceEGFR activation in TNBC cell lines. A, cellswere incubated with the indicatedconcentration of IGFBP-3
for 16 hours, then stimulated with EGF (10 ng/mL final concentration) for 10 minutes, lysed in Laemmli buffer, and analyzed by Western blotting for
phosphorylatedEGFR (Y1068) and total EGFR, with a-tubulinas a loading control. Quantificationof pooled data from four experimentsis shown as mean SEM
with 100% representing EGF-stimulated EGFR phosphorylation in the absence of IGFBP-3. Bars having different letters are significantly different from
each other (P<0.05). B, cells were treated with 100 ng/mL recombinant IGFBP-3 for 0, 30, 60, or 120 minutes, as indicated. Membranes were isolated as
described in the Materials and Methods, and subjected to Western blotting to detect SphK1 and AQP1, a plasma membrane marker. A representative blot of
three for each cell line is shown. The histogram depicts quantified data as mean SEMfrom three experiments and indicates the calculated ratio of SphK1to
AQP1, determined by densitometric analysis of immunoblots. Bars with different letters are significantly different from each other (P<0.05). Uncropped
images are shown in Supplementary Fig. S2. C,endogenous IGFBP-3 expression was reduced by transfection with either of two siRNA constructs (BP#1 and
BP#2) or nonsilencing control (ctl). Two days later, cells were stimulated with EGF (10 ng/mL) for 10 minutes, then harvested into Laemmli buffer for
analysis of phosphorylated EGFR (Y1068)and total EGFR by Western blotting,with a-tubulin as loading control. EGFR phosphorylation without EGF stimulation
is also shown for MDA-MB-468cells transfected with controlor IGFBP-3 siRNA. Blots are representativeof three experiments. Quantification of pooled data for
BP#1 siRNA from three experiments is shown as mean SEM with 100% representing EGF-stimulated EGFR phosphorylation in ctl siRNA samples.
All blots have been cropped to highlight relevant bands. Migration points of molecular mass markers are shown on the right of each blot.
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 321
that SphK1 expression and activity are required for
IGFBP-3 to enhance EGFR signaling in TNBC cells.
SKi-I induces degradation of SphK1a in TNBC cell
lines
Gefitinib has been reported to inhibit expression of
SphK1 in glioblastoma cells (29), and SKi-II has been
shown in other cell types to induce degradation of SphK1
by targeting it to the ubiquitin-proteasomal degradation
pathway (27). In TNBC cells, SKi-II (1–10 mmol/L) alone
significantly reduced SphK1a in all cell lines (P<0.001)
with the greatest decrease in MDA-MB-436 cells (80%)
and smallest in Hs578T (50%; Fig. 4A). Gefitinib alone
had a significant inhibitory effect on SphK1a expression in
MDA-MB-436 cells (to 77% 6.3% of control levels; P<
0.001), but there was no combined effect of gefitinib and
SKi-II in this or any other cell line (Fig. 4A). To demon-
strate that the loss of SphK1a induced by SKi-II was due to
its proteolysis, treatment with SKi-II was carried out in the
presence of the proteasome inhibitor MG132. As shown
in Fig. 4B, MG132 increased SphK1a in the absence of SKi-
II, and prevented its SKi-II–induced loss in all cell lines.
Similarly, SKi-II decreased, and MG132 increased,
SphK1b and SphK1c in those cells expressing these forms
of the protein, although the changes were not as marked as
for SphK1a (data not shown). The slight decrease in
SphK1a elicited by gefitinib in MDA-MB-436 (shown
in Fig. 4A) was also reversed by MG132 (data not shown).
Figure 3. Potentiation of EGFR
activation by IGFBP-3 requires
SphK1. A, 1 day after nonsilencing
control (ctl) or SphK1 siRNA
transfection, IGFBP-3 was added
and incubations were continued for
a further 16 hours. Cells were
stimulated with EGF (10 ng/mL) for
10 minutes, then harvested into
Laemmli buffer for analysis of
phosphorylated and total EGFR by
Western blotting, with a-tubulin as
loading control. Graphs below
each blot show quantification of
EGFR phosphorylation relative to
that stimulated by EGF in the
absence of exogenous IGFBP-3,
with data derived from three
experiments (mean SEM). B,
cells were incubated in medium
without or with 10 mmol/L SKi-II for
4 hours, then IGFBP-3 was added
to final concentrations of 10 or 100
ng/mL, as indicated, for 16 hours.
EGF was added (10 ng/mL final),
incubations were continued for 10
minutes, and then cells were
harvested into Laemmli buffer for
analysis of phosphorylated EGFR
(Y1068) and total EGFR by Western
blot analysis. Representative blots
of three in total are shown, and the
data depicted in histograms are
mean SEM derived from the
pooled results for IGFBP-3 at 100
ng/mL in three experiments. All
blots have been cropped to
highlight relevant bands. Migration
points of molecular mass markers
are shown on the right of each blot.
Martin et al.
Mol Cancer Ther; 13(2) February 2014 Molecular Cancer Therapeutics
322
This implies that SphK1 turnover involves proteasomal
degradation, and that this is increased by SKi-II.
Effect of combined EGFR and SphK1 inhibition of
proliferation of TNBC cells
The SphK and EGFR signaling systems act both inde-
pendently and cooperatively to stimulate cell prolifera-
tion and survival in many normal and malignant cell types
(30), raising the possibility that targeting these two sys-
tems together in TNBC cells, which express IGFBP-3
highly, will have greater effect than blocking either alone.
Therefore, the functional consequences of single and
combined blockade of the EGFR and SphK1 signaling
pathways was determined in the four TNBC cell lines.
Initially, the sensitivity to gefitinib and SKi-II in medium
containing 5% FBS was determined using CyQUANT, a
cell proliferation assay that measures cellular DNA con-
tent. As shown in Supplementary Fig. S3A, SKi-II alone
inhibited proliferation of all cell lines over a range of
doses, with MDA-MB-468 exhibiting greatest sensitivity,
and Hs578T least sensitivity, to its effects. Gefitinib alone
was strongly inhibitory to MDA-MB-436 and MDA-MB-
468 cells, and had an additive effect with low concentra-
tions of SKi-II in all cell lines. The pattern of inhibition in
response to gefitinib alone was similar, and a clear addi-
tional inhibitory effect was apparent when SKi-II was
included at low gefitinib concentrations (Supplementary
Fig. S3B).
To study the effects of the EGFR kinase and SphK1
inhibitors in more detail, real-time proliferation experi-
ments were conducted using low gefitinib and SKi-II
doses, selected according to the relative sensitivity of
the individual cell lines to these agents determined by
using the CyQUANT assay. Cells were sparsely plated
in 96-well plates, changed to medium containing inhi-
bitors 16 hours later, and proliferation over the follow-
ing 72 to 140 hours was imaged using an IncuCyte
apparatus. As shown in Fig. 5A, at the low doses chosen
gefitinib had no significant inhibitory effect when used
alone in any TNBC cell line. SKi-II significantly inhib-
ited growth of MDA-MB-231 (from 76 hours onwards)
and MDA-MB-436 (at the latest time point, 128 hours).
Remarkably, in view of these modest effects when the
inhibitors were used separately, the combination of
gefitinib and SKi-II virtually abolished cell proliferation
in all cell lines, with a significant effect compared with
Figure 4. SKi-II induces degradation of SphK1 in TNBC cell lines. A, cells were incubated with SKi-II (0, 1, 3, and 10 mmol/L) without or with 10 mmol/L gefitinib
(gef) for 24 hours. Harvested lysates were analyzed for SphK1 expression by Western blot analysis, with the band representing SphK1a (42 kDa)
shown. One representative blot of three is shown. Bar graphs in the bottom depict quantification of data for gefitinib (10 mmol/L) and SKi-II (10 mmol/L) used
alone and in combination. B, cells were incubated for 24 hours with SKi-II (10 mmol/L) in the absence or presence of the proteasome inhibitor MG132
(1 mmol/L), and lysates were analyzed for SphK1a. For A and B, densitometric quantification of the SphK1a data is expressed as a percent age of
control (no gefitinib, SKi-II, or MG132). Quantification of pooled data from three experiments is shown as mean SEM, with bars having different letters being
significantly different from each other (P<0.05 by ANOVA). All blots have been cropped to highlight relevant bands. Migration points of molecular
mass markers are shown on the right of each blot.
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 323
control apparent from 28 hours for Hs578T, 52 hours for
MDA-MB-468, 56 hours for MDA-MB-231, and 68 hours
for MDA-MB-436 (P<0.01).
In view of the profound combined effect of gefitinib and
SKi-II on the proliferation of TNBC cells, we investigated
the effects of these agents on EGF-stimulated activation of
EGFR, and two key survival and proliferative signaling
pathways, AKT and ERK1/2. As shown in Fig. 5B, EGFR
phosphorylation at Y1068 was markedly increased by
exogenous EGF in MDA-MB-231, MDA-MB-436, and
Hs578T cell lines, with no apparent increase in EGFR
phosphorylation above the high basal level seen under
unstimulated conditions in the MDA-MB-468 line (Fig.
5B). Gefitinib alone inhibited EGFR phosphorylation in all
cell lines, but the inclusion of SKi-II further inhibited
EGFR phosphorylation only in MDA-MB-468 at the high-
est dose (3 mmol/L) of gefitinib (P<0.05). The four cell
lines showed differing patterns of activation of ERK1/2
and AKT in response to EGF (Fig. 5B). In MDA-MB-231,
EGF stimulated phosphorylation of AKT with very little
Figure 5. Effect of combined
gefitinib and SKi-II on TNBC cell
proliferation and activation of
signaling intermediates. A, TNBC
cells were plated in 96-well plates
in complete medium, then changed
24 hours later to medium
containing 5% FBS alone (*), or
with SKi-II (&), gefitinib (&), or a
combination of the two (*). The
concentrations of inhibitors used
were: MDA-MB-231, 1 mmol/L
gefitinib, 1 mmol/L SKi-II; MDA-
MB-436, 2 mmol/L gefitinib,
1mmol/L SKi-II; MDA-MB-468, 1
mmol/L gefitinib, 0.3 mmol/L SKi-II;
Hs578T, 4 mmol/L gefitinib, 2.5
mmol/L SKi-II. Cell proliferation was
measured in real time over 3 to 5
days using an IncuCyte apparatus,
as described in the Materials and
Methods. ns, not significant at final
time point; ,P<0.05 and
,P<0.001 at final time point, by
repeated measures ANOVA. B,
cells were incubated with the
indicated concentration of gefitinib
in the absence or presence of 10
mmol/L SKi-II for 4 hours, then
stimulated with EGF (10 ng/mL
final) for 10 minutes. Harvested
lysates were analyzed by Western
blot analysis for phosphorylated
EGFR (Y1068), AKT (S473), and
ERK1/2 (T202/Y204) on single
filters probed sequentially for these
proteins. A replicate filter was
probed with antibodies to detect
total proteins. The first lane for each
cell line contains samples that have
not been stimulated with EGF, and
blots shown are representative of
three to six experiments. All blots
have been cropped to highlight
relevant bands. Migration points of
molecular mass markers are shown
on the right of each blot.
Martin et al.
Mol Cancer Ther; 13(2) February 2014 Molecular Cancer Therapeutics
324
effect on ERK1/2, the opposite pattern of activation was
apparent in MDA-MB-436 and MDA-MB-468, and the two
proteins showed a similar degree of activation of Hs578T
cells. Gefitinib alone had the greatest inhibitory effect on
those pathways most sensitive to EGF stimulation: AKT in
MDA-MB-231, pERK1/2 in MDA-MB-436 and MDA-MB-
468, and similar inhibition of these pathways in Hs578T
(Fig. 5B). SKi-II did not enhance the inhibitory effect of
gefitinib on AKT or ERK1/2 phosphorylation in any cell
line. The quantified data from three to six similar experi-
ments are given in Supplementary Fig. S4. Collectively
these data indicate that the remarkable combined inhib-
itory effect of SKi-II and gefitinib on growth of these TNBC
cell lines shown in Fig. 5A, cannot be explained by effects
on these signaling intermediates.
The combination of gefitinib and SKi-II inhibits
growth of MDA-MB-468 xenograft tumors
The profound combined effect of EGFR and SphK1 on
inhibition of TNBC cells in vitro provided proof-of-prin-
ciple that cotargeting these pathways may have therapeu-
tic potential for the treatment of aggressive TNBC breast
tumors. To investigate this in vivo, MDA-MB-468 cells
were established as xenograft tumors in immunocompro-
mised mice, then treated with vehicle (50 mL DMSO),
gefitinib alone (75 mg/kg), SKi-II alone (50 mg/kg), or
a combination of the two at these doses given as a single
injection intraperitoneally three times weekly. Tumor
volumes were measured immediately before treatment
was initiated and then at 5 and 11 days of treatment.
Experiments were terminated after 11 days due to the
largest tumors in control mice reaching the maximum size
stipulated in the animal ethics protocol (500 mm
3
). Before
starting treatment, mice were randomized into treatment
groups of 9 to 10 mice per group. The mean SEM of
tumor volumes in the groups before treatment were:
control, 146.1 20.2 mm
3
; gefitinib, 138.5 18.6 mm
3
;
SKi-II, 151.6 20.7 mm
3
; and combination, 142.1 21.5
mm
3
(P¼ns). As shown in Fig. 6A, which depicts the
mean SEM change in tumor volume of mice after 5
and 11 days of treatment, the combination of SKi-II and
gefitinib markedly inhibited tumor growth within 5
days treatment, and this inhibitory effect was main-
tained until the termination of the experiment (P¼
0.011, by repeated measures). Under these conditions,
neither SKi-II (P¼0.477) nor gefitinib (P¼0.82) alone
significantly inhibited tumor growth compared with
control. There was no significant effect of any treatment
on body weight at any time point (data not shown). At
the conclusion of the experiment, mean SEM of tumor
volumes of the combination group was 243.3 22.84
mm
3
, compared with control of 398.1 48.24 mm
3
(P<
0.05). The tumor volumes of the gefitinib (331.8 49.7
mm
3
)andSKi-II(309.735.4 mm
3
)groupsdidnot
differ significantly from control.
Western blot analysis of activation of EGFR, AKT, and
mitogen-activated protein kinase (MAPK) in excised
tumors (Fig. 6B) revealed that, similar to the effects of
gefitinib and SKi-II on MDA-MB-468 cells in vitro,EGFR
and ERK1/2 phosphorylation in tumors was markedly
inhibited by gefitinib alone, with little effect on AKT
phosphorylation. SKi-II alone did not significantly affect
phosphorylation of any of these proteins. Although the
addition of SKi-II to the gefitinib treatment reduced
EGFR phosphorylation further as compared with gefi-
tinib alone, this did not reach statistical significance
(P¼0.06).
Figure 6. Growth of xenograft MDA-MB-468 tumors is inhibited by
combined blockade of SphK1 and EGFR pathways. Tumors were
established from MDA-MB-468 cells in female athymic BALB/c-Foxn1
nu
mice as described in the Materials and Methods, then animals were
randomly allocated to groups of nine to 10 for treatment. Gefitinib
(75 mg/kg), SKi-II (50 mg/kg), combination, or vehicle control (50 mL
DMSO) were administered intraperitoneally three times weekly, then
animals were euthanized and tumors collected. A, change in tumor
volume from day 0 (first day of treatment) to days 5 and 11 was calculate d
for each animal, and data were pooled for each treatment. Shown are
mean SEM with significance determined by repeated measures
ANOVA and Bonferroni post hoc test. B, Western blot analysis of EGFR,
AKT, and ERK1/2 in excised tumors (3/treatment). Tumor lysates were
prepared as described in the Material and Methods, and subjected to
Western blotting for total and phosphorylated EGFR, AKT, and ERK1/2.
Bar graphs show quantified data (mean SEM) from five tumors for each
treatment analyzed in the same way, with the effect of each treatment
expressed relative to control. Bars in each graph having different letters
are significantly different from each other (P<0.05 by ANOVA). All blots
have been cropped to highlight relevant bands. Migration points of
molecular mass markers are shown on the right of each blot.
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 325
Discussion
This study sought to investigate the potential efficacy of
cotargeting growth-stimulatory signaling pathways of
two proteins that are highly expressed in TNBC: EGFR
and IGFBP-3. Because many such cancers express EGFR
highly (3, 31, 32), there has been a major interest in drugs
that target this pathway, for use either as a single agent or
in combination with conventional chemotherapy. In gen-
eral, EGFR inhibition as a monotherapy for TNBC has
been disappointing (33, 34) and a number of combined
targeted treatments are currently under investigation.
The decision to combine EGFR blockade with inhibition
of an IGFBP-3 signaling pathway drew on observations
made by us and others of high IGFBP-3 and EGFR expres-
sion in ER
breast tumors and cancer cells compared with
ER
þ
(3, 15, 16, 24, 35, 36) and our previous studies showing
potentiation of EGFR signaling by IGFBP-3 in untrans-
formed breast epithelial cells (11, 22). Although targeting
IGFBP-3 itself would arguably be a more direct means of
blocking its growth-stimulatory signaling, its function as a
key regulator of the IGF axis makes this approach tech-
nically implausible in vivo. Having identified the SphK1/
S1P system as the mediator of IGFBP-30s stimulatory
effects on EGFR signaling in normal breast cells (22), we
regarded this system as a logical target for inhibiting
IGFBP-3 stimulatory bioactivity in breast cancer cells.
Three of the four TNBC cell lines studied responded to
exogenous IGFBP-3 with significantly increased EGF-
stimulated EGFR phosphorylation and, importantly,
endogenous IGFBP-3 was also shown to modulate EGFR
activation in TNBC cells, as demonstrated by a reduction
in EGF-stimulated EGFR phosphorylation when IGFBP-3
was silenced. This observation alone places IGFBP-3 clear-
ly in the pathway of EGFR action in TNBC cells. The
effects of IGFBP-3 on EGFR phosphorylation in TNBC cell
lines required SphK activity as SKi-II, a dual SphK1 and
SphK2 inhibitor, blocked enhancement of EGFR phos-
phorylation by IGFBP-3. SphK1 mediates these effects of
IGFBP-3 in TNBC cells, because when SphK1 expression
was silenced using siRNA, IGFBP-3 no longer potentiated
EGF-stimulated EGFR phosphorylation. A role for SphK2
in the effects of IGFBP-3 on EGFR signaling seems unlike-
ly, as siRNA-mediated silencing of SphK2 did not prevent
IGFBP-3–potentiating EGFR activation in phenotypically
normal breast epithelial cells (22).
Exogenous IGFBP-3 upregulates both SphK1 expres-
sion and activity in breast epithelial cells (22), but here we
found no clear correlation between IGFBP-3 expression
and either the total amount of SphK1 or the isoforms
expressed in breast cancer cells. This suggests that endog-
enous IGFBP-3 is not a dominant regulator of SphK1
expression in breast cancer cells and that its effects on
EGFR signaling relate to its modulation of SphK1 activity
rather than expression. Supporting this, exogenous
IGFBP-3 increased SphK1 in the membrane fractions of
TNBC cells, demonstrating that in these cells IGFBP-3
increases the activity of SphK1. Translocation of SphK1 to
the plasma membrane is a process clearly linked with the
generation of S1P (37). Although formation of S1P in
response to IGFBP-3 was not explicitly shown in the
present study, we and others have previously shown
induction of SphK activity and increased S1P levels in
response to IGFBP-3 in breast and other cell types
(22, 38, 39). The mechanism by which it does so remains
unknown.
Analysis of SphK1 expression by the TNBC cell lines
revealed a number of bands by Western blot analysis,
ranging in size from approximately 42 to 65 kDa. Silencing
of SphK1 revealed that some of these bands represented
proteins not immunologically related to SphK1, presum-
ably reacting nonspecifically with the antibody. However,
proteins of mass consistent with SphK1a, SphK1b, and
SphK1c were expressed by TNBC, though not all species
were detected in all cell lines. The biologic significance of
different SphK1 forms is not known, although it has been
suggested that they exhibit different subcellular localiza-
tion (40) and may therefore have functional specificity.
The SphK1 variants were all decreased by SKi-II and, as in
other cell types (27, 41, 42), this was reversed by the
proteasome inhibitor MG132, implying their degradation
via ubiquitin-proteasomal pathways. MG132 also
increased various SphK1 forms to levels above those in
untreated cells, suggesting proteasome regulation of the
enzyme under basal conditions. The functional conse-
quence of SphK1 degradation in TNBC cells is not clear,
but SKi-II–induced degradation of SphK1 has been
reported to increase apoptosis of androgen-sensitive pros-
tate cancer cells (27), perhaps by shifting the sphingolipid
rheostat toward the accumulation of proapoptotic pre-
cursors of S1P, such as sphingosine and ceramide (43).
Notably, IGFBP-3 has been shown to enhance the apo-
ptotic effects of C2 ceramide in breast cancer cells (44),
suggesting that there may be increased apoptotic activity
in breast cancer cells in which there is accumulation of
ceramide if those cells also express IGFBP-3. We showed
previously that functional blockade of EGFR restored
sensitivity to growth-inhibitory effects of ectopically
expressed IGFBP-3 in ER
þ
breast cancer cells (19), which
would be another reason to target the SphK1 pathway
rather than IGFBP-3 itself in TNBC in which IGFBP-3 is
highly expressed.
Although the sensitivity of the four TNBC cell lines to
gefitinib and SKi-II was variable, the combination of
gefitinib and SKi-II almost completely blocked prolifera-
tion of all lines when the two inhibitors were used at
concentrations that alone had little or no inhibitory effect.
Importantly, when the combination of SKi-II and gefitinib
was tested in an MDA-MB-468 xenograft tumor model, it
significantly inhibited tumor growth under conditions in
which neither gefitinib nor SKi-II alone had a significant
effect. The mechanisms underlying these combined
effects are still to be identified, with no apparent involve-
ment of the key growth-regulatory ERK1/2 or AKT path-
ways either in vitro or in vivo.
Compared with EGFR, interest in SphK1 as a molecular
target in cancer is relatively recent but an increasing
Martin et al.
Mol Cancer Ther; 13(2) February 2014 Molecular Cancer Therapeutics
326
number of pharmacologic SphK inhibitors are being
evaluated in preclinical settings. The growth of MDA-
MB-468 xenograft tumors was shown to be inhibited by
ABC294640, an SphK2-specific inhibitor, when used as a
single-line agent (45). Acquired resistance to chemo-
therapeutic drugs, hormonal therapies, and growth
factor receptor inhibitors, including EGFR inhibitors,
has been linked with overexpression of SphK1 in a
number of malignancies (46–50), providing a clear ratio-
nale for the use of SphK1 inhibitors in the clinic as
adjuvant therapies. Our study has shown potent anti-
proliferative effects of dual inhibition of EGFR and
SphK1 in cell lines representing a subset of breast
cancers for which there is currently a paucity of treat-
ments, and has demonstrated in a preclinical study
proof-of-principle that this combination has therapeutic
potential. Further optimization of dosing schedules may
improve the observed effects. We propose that a regi-
men of combining SphK1 inhibition with EGFR inhibi-
tion constitutes a novel therapeutic option in TNBC that
could be rapidly implemented in the clinic.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J.L. Martin, R.C. Baxter
Development of methodology: M.Z. Lin, C.D. Scott
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J.L. Martin, H.C. de Silva, M.Z. Lin, C.D. Scott, R.
C. Baxter
Analysis and interpretation of data (e.g., statistical analysis, biostatis-
tics, computational analysis): J.L. Martin, H.C. de Silva, M.Z. Lin, R.C.
Baxter
Writing, review, and/or revision of the manuscript: J.L. Martin, M.Z. Lin,
C.D. Scott, R.C. Baxter
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): H.C. de Silva, M.Z. Lin
Study supervision: J.L. Martin, R.C. Baxter
Grant Support
R.C. Baxter and J.L. Martin received a grant from the Cancer Council
NSW (RG 11–09).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received May 14, 2013; revised November 15, 2013; accepted December
1, 2013; published OnlineFirst December 13, 2013.
References
1. Mendelsohn J, Baselga J. The EGF receptor family as targets for
cancer therapy. Oncogene 2000;19:6550–65.
2. Prenzel N, Zwick E, Leserer M, Ullrich A. Tyrosine kinase signalling in
breast cancer. Epidermal growth factor receptor: convergence point
for signal integration and diversification. Breast Cancer Res 2000;2:
184–90.
3. Kobayashi S. Basal-like subtype of breast cancer: a review of its unique
characteristics and their clinical significance. Breast Cancer 2008;15:
153–8.
4. Press MF, Finn RS, Cameron D, Di Leo A, Geyer CE, Villalobos IE,
et al. HER-2 gene amplification, HER-2 and epidermal growth factor
receptor mrna and protein expression, and lapatinib efficacy in
women with metastatic breast cancer. Clin Cancer Res 2008;14:
7861–70.
5. Liu B, Lee KW, Anzo M, Zhang B, Zi X, Tao Y, et al. Insulin-like growth
factor-binding protein-3 inhibition of prostate cancer growth involves
suppression of angiogenesis. Oncogene 2007;26:1811–9.
6. Torng PL, Lee YC, Huang CY, Ye JH, Lin YS, Chu YW, et al. Insulin-like
growth factor binding protein-3 (IGFBP-3) acts as an invasion-metas-
tasis suppressor in ovarian endometrioid carcinoma. Oncogene
2008;27:2137–47.
7. De Mellow JS, Baxter RC. Growth hormone-dependent insulin-like
growth factor (IGF) binding protein both inhibits and potentiates IGF-I-
stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys
Res Commun 1988;156:199–204.
8. Conover CA. Potentiation of insulin-like growth factor (IGF) action by
IGF-binding protein-3: studies of underlying mechanism. Endocrinol-
ogy 1992;130:3191–9.
9. Cohen P, Rajah R, Rosenbloom J, Herrick DJ. IGFBP-3 mediates TGF-
beta 1-induced cell growth in human airway smooth muscle cells. Am J
Physiol Lung Cell Mol Physiol 2000;278:L545–L51.
10. Grill CJ, Sivaprasad U, Cohick WS. Constitutive expression of
IGF-binding protein-3 by mammary epithelial cells alters signaling
through AKT and p70S6 kinase. J Mol Endocrinol 2002;29:
153–62.
11. Martin JL, Weenink SM, Baxter RC. Insulin-like growth factor-
binding protein-3 potentiates epidermal growth factor action in
MCF-10A mammary epithelial cells. Involvement of p44/42 and
p38 mitogen activated protein kinases. J Biol Chem 2003;278:
2969–76.
12. Yamazaki K, Sakamoto M, Ohta T, Kanai Y, Ohki M, Hirohashi S.
Overexpression of Kit in chromophobe renal cell carcinoma. Onco-
gene 2003;22:847–52.
13. Kettunen E, Anttila S, Seppanen JK, Karjalainen A, Edgren H,
Lindstrom I, et al. Differentially expressed genes in nonsmall
cell lung cancer: expression profiling of cancer-related genes in
squamous cell lung cancer. Cancer Gen Cytogenet 2004;149:
98–106.
14. Chuang ST, Patton KT, Schafernak KT, Papavero V, Lin F, Baxter RC,
et al. Over expression of insulin-like growth factor binding protein 3 in
clear cell renal cell carcinoma. J Urol 2008;179:445–9.
15. Yu H, Levesque MA, Khosravi MJ, Papanastasiou DA, Clark GM,
Diamandis EP. Associations between insulin-like growth factors and
their binding proteins and other prognostic indicators in breast cancer.
Br J Cancer 1996;74:1242–7.
16. Rocha RL, Hilsenbeck SG, Jackson JG, Lee AV, Figueroa JA, Yee D.
Correlation of insulin-like growth factor-binding protein-3 messenger
RNA with protein expression in primary breast cancer tissues: detec-
tion of higher levels in tumors with poor prognostic features. J Natl
Cancer Inst 1996;88:601–6.
17. Probst-Hensch NM, Steiner JHB, Schraml P, Varga Z, Zurrer-Hardi U,
Storz M, et al. IGFBP2 and IGFBP3 protein expressions in human
breast cancer: association with hormonal factors and obesity. Clin
Cancer Res 2010;16:1025–32.
18. Sheen-Chen SM, Zhang H, Huang CC, Tang RP. Insulin-like growth
factor-binding protein-3 in breast bancer: analysis with tissue micro-
array. Anticancer Res 2009;29:1131–5.
19. Butt AJ, Martin JL, Dickson KA, McDougall F, Firth SM, Baxter RC.
Insulin-like growth factor binding protein-3 expression is associated
with growth stimulation of T47D human breast cancer cells: the role of
altered epidermal growth factor signaling. J Clin Endocrinol Metab
2004;89:1950–6.
20. Schedlich LJ, O'Han MK, Leong GM, Baxter RC. Insulin-like growth
factor binding protein-3 prevents retinoid receptor heterodimerization:
implications for retinoic acid-sensitivity in human breast cancer cells.
Biochem Biophys Res Commun 2004;314:83–8.
21. Grkovic S, O'Reilly VC, Han S, Hong M, Baxter RC, Firth SM. IGFBP-3
binds GRP78, stimulates autophagy and promotes the survival of
breast cancer cells exposed to adverse microenvironments. Onco-
gene 2013;32:2412–20.
IGFBP-3 and EGFR Inhibition in Triple-Negative Breast Cancer
www.aacrjournals.org Mol Cancer Ther; 13(2) February 2014 327
22. Martin JL, Lin MZ, McGowan EM, Baxter RC. Potentiation of growth
factor signaling by insulin-like growth factor-binding protein-3 in
breast epithelial cells requires sphingosine kinase activity. J Biol Chem
2009;284:25542–52.
23. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A
collection of breast cancer cell lines for the study of functionally distinct
cancer subtypes. Cancer Cell 2006;10:515–27.
24. Martin JL, Baxter RC. Expression of insulin-like growth factor binding
protein-2 by MCF-7 breast cancer cells is regulated through the
phosphatidylinositol 3-kinase/akt/mammalian target of rapamycin
pathway. Endocrinology 2007;148:2532–41.
25. Firth SM, Ganeshprasad U, Poronnik P, Cook DI, Baxter RC. Adeno-
viral-mediated expression of human insulin-like growth factor-binding
protein-3. Protein Expr Purif 1999;16:202–11.
26. French KJ, Upson JJ, Keller SN, Zhuang Y, Yun JK, Smith CD.
Antitumor activity of sphingosine kinase inhibitors. J Pharmacol Exp
Ther 2006;318:596–603.
27. Loveridge C, Tonelli F, Leclercq T, Lim KG, Long JS, Berdyshev E, et al.
The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chloro-
phenyl)thiazole induces proteasomal degradation of sphingosine
kinase 1 in mammalian cells. J Biol Chem 2010;285:38841–52.
28. Pitson SM, Xia P, Leclercq TM, Moretti PA, Zebol JR, Lynn HE, et al.
Phosphorylation-dependent translocation of sphingosine kinase to
the plasma membrane drives its oncogenic signalling. J Exp Med
2005;201:49–54.
29. Estrada-Bernal A, Lawler S, Nowicki M, Ray Chaudhury A, Van Brock-
lyn J. The role of sphingosine kinase-1 in EGFRvIII-regulated growth
and survival of glioblastoma cells. J Neuro-Oncol 2011;102:353–66.
30. Pyne NJ, Pyne S. Sphingosine 1-phosphate and cancer. Nat Rev
Cancer 2010;10:489–503.
31. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, et al.
Immunohistochemical and clinical characterization of the basal-like
subtype of invasive breast carcinoma. Clin Cancer Res 2004;10:
5367–74.
32. Ryden L, Jirstrom K, Haglund M, Stal O, Ferno M. Epidermal growth
factor receptor and vascular endothelial growth factor receptor 2 are
specific biomarkers in triple-negative breast cancer. Results from a
controlled randomized trial with long-term follow-up. Breast Cancer
Res Treat 2010;120:491–98.
33. Duffy MJ, McGowan PM, Crown J. Targeted therapy for triple-negative
breast cancer: where are we? Int J Cancer 2012;131:2471–77.
34. Masuda H, Zhang D, Bartholomeusz C, Doihara H, Hortobagyi G, Ueno
N. Role of epidermal growth factor receptor in breast cancer. Breast
Cancer Res Treat 2012;136:331–45.
35. Clemmons DR, Camacho HC, Coronado E, Osborne CK. Insulin-like
growth factor binding protein secretion by breast carcinoma cell lines:
correlation with estrogen receptor status. Endocrinology 1990;127:
2679–86.
36. Pekonen F, Nyman T, Ilvesmaki V, Partanen S. Insulin-like growth
factor binding proteins in human breast cancer tissue. Cancer Res
1992;52:5204–7.
37. Pitson SM, Moretti PA, Zebol JR, Lynn HE, Xia P, Vadas MA, et al.
Activation of sphingosine kinase 1 by ERK1/2-mediated phosphory-
lation. EMBO J 2003;22:5491–500.
38. Granata R, Trovato L, Garbarino G, Taliano M, Ponti R, Sala G, et al.
Dual effects of IGFBP-3 on endothelial cell apoptosis and survival:
involvement of the sphingolipid signaling pathways. FASEB J 2004;
18:1456–8.
39. Granata R, Trovato L, Lupia E, Sala G, Settanni F, Camussi G, et al.
Insulin-like growth factor binding protein-3 induces angiogenesis
through IGF-I- and SphK1-dependent mechanisms. J Thromb Haem
2007;5:835–45.
40. Venkataraman K, Thangada S, Michaud J, Oo ML, Ai Y, Lee Y-M, et al.
Extracellular export of sphingosine kinase-1a contributes to the vas-
cular S1P gradient. Biochem J 2006;397:461–71.
41. Ren S, Xin C, Pfeilschifter J, Huwiler A. A novel mode of action of the
putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlor-
ophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1
degradation. Cell Physiol Biochem 2010;26:97–104.
42. Lim KG, Tonelli F, Berdyshev E, Gorshkova I, Leclercq T, Pitson SM,
et al. Inhibition kinetics and regulation of sphingosine kinase 1 expres-
sion in prostate cancer cells: functional differences between sphin-
gosine kinase 1a and 1b. Int J Biochem Cell Biol 2012;44:1457–64.
43. Spiegel S, Milstien S. Functions of the multifaceted family of sphin-
gosine kinases and some close relatives. J Biol Chem 2007;282:
2125–29.
44. Perks CM, Bowen S, Gill ZP, Newcomb PV, Holly JM. Differential IGF-
independent effects of insulin-like growth factor binding proteins (1–6)
on apoptosis of breast epithelial cells. J Cell Biochem 1999;75:652–64.
45. Antoon JW, White MD, Driver JL, Burow ME, Beckman BS. Sphingo-
sine kinase isoforms as a therapeutic target in endocrine therapy
resistant luminal and basal-A breast cancer. Exp Biol Med 2012;237:
832–44.
46. Guillermet-Guibert J, Davenne L, Pchejetski D, Saint-Laurent N, Bri-
zuela L, Guilbeau-Frugier Cl, et al. Targeting the sphingolipid metab-
olism to defeat pancreatic cancer cell resistance to the chemothera-
peutic gemcitabine drug. Mol Cancer Therap 2009;8:809–20.
47. Baran Y, Salas A, Senkal CE, Gunduz U, Bielawski J, Obeid LM, et al.
Alterations of ceramide/sphingosine 1-phosphate rheostat involved in
the regulation of resistance to imatinib-induced apoptosis in K562
human chronic myeloid leukemia cells. J Biol Chem 2007;282:
10922–34.
48. Sukocheva O, Wang L, Verrier E, Vadas MA, Xia P. Restoring endoc rine
response in breast cancer cells by inhibition of the sphingosine kinase-
1 signaling pathway. Endocrinology 2009;150:4484–92.
49. Madhunapantula SV, Hengst J, Gowda R, Fox TE, Yun JK, Robertson
GP. Targeting sphingosine kinase-1 to inhibit melanoma. Pigment Cell
Melanoma Res 2012;25:259–74.
50. Rosa R, Marciano R, Malapelle U, Formisano L, Nappi L, D'Amato C,
et al. Sphingosine kinase 1 overexpression contributes to cetuximab
resistance in human colorectal cancer models. Clin Cancer Res
2013;19:138–47.
Martin et al.
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