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Feedback upregulation of HER3 (ErbB3) expression and
activity attenuates antitumor effect of PI3K inhibitors
Anindita Chakrabarty
a
, Violeta Sánchez
a
, María G. Kuba
b
, Cammie Rinehart
a
, and Carlos L. Arteaga
a,c,d,1
Departments of
a
Medicine,
b
Pathology, and
c
Cancer Biology and
d
Breast Cancer Research Program, Vanderbilt–Ingram Cancer Center, Vanderbilt University
School of Medicine, Nashville, TN 37232
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved February 4, 2011 (received for review December 2, 2010)
We examined the effects of an inhibitor of PI3K, XL147, against
human breast cancer cell lines with constitutive PI3K activation.
Treatment with XL147 resulted in dose-dependent inhibition of
cell growth and levels of pAKT and pS6, signal transducers in the
PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition
of PI3K was followed by up-regulation of expression and phos-
phorylation of multiple receptor tyrosine kinases, including HER3.
Knockdown of FoxO1 and FoxO3a transcription factors suppressed
the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon
inhibition of PI3K. In HER2
+
cells, knockdown of HER3 with siRNA
or cotreatment with the HER2 inhibitors trastuzumab or lapatinib
enhanced XL147-induced cell death and inhibition of pAKT and
pS6. Trastuzumab and lapatinib each synergized with XL147 for
inhibition of pAKT and growth of established BT474 xenografts.
These data suggest that PI3K antagonists will inhibit AKT and re-
lieve suppression of receptor tyrosine kinase expression and their
activity. Relief of this feedback limits the sustained inhibition
of the PI3K/AKT pathway and attenuates the response to these
agents. As a result, PI3K pathway inhibitors may have limited clin-
ical activity overall if used as single agents. In patients with HER2-
overexpressing breast cancer, PI3K inhibitors should be used in
combination with HER2/HER3 antagonists.
signaling
|
targeted therapy
PI3K transmits signals from ligand-activated receptor tyrosine
kinases (RTKs) to intracellular molecules that regulate growth,
metabolism, cell size, motility, and survival. In turn, PI3K cata-
lyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate
to produce the second messenger phosphatidylinositol-3,4,5-
trisphosphate (PIP3) (1, 2). Several pleckstrin homology domain-
containing proteins, including AKT and PDK1, bind to PIP3 at the
plasma membrane. Phosphorylation of AKT at T308 by PDK1
and at S473 by a complex involving mTOR/Rictor (i.e., TORC2)
results in the full activation of this enzyme. AKT facilitates survival
and cell cycle entry by phosphorylation of proteins including
GSK3α/β, FoxO transcription factors, MDM2, BAD, and p27
KIP1
(3). In addition, AKT regulates protein synthesis and cell growth
via activation of the mTOR/Raptor (i.e., TORC1) complex (4, 5).
PI3K/AKT is arguably the most commonly altered pathway in
human cancers (6, 7). Gain-of-function mutations in PIK3CA,
the gene encoding the class I
A
PI3K catalytic subunit p110α, are
frequently present in multiple human tumors (8). Second, the
PIP3 phosphatase PTEN is a tumor suppressor frequently inac-
tivated by mutation, gene deletion, and promoter methylation
(9). Further, PI3K is potently activated by oncogenes like mutant
Ras and tyrosine kinases such as Bcr-Abl, HER2 (ErbB2), MET,
and KIT, among others (1). Therefore, a large group of tumors
with molecular alterations in the PI3K/AKT pathway is thera-
peutically targetable with PI3K inhibitors.
Several PI3K pathway antagonists have been developed. These
include ATP mimetics that bind reversibly in the ATP pocket of
the kinase domain of WT and mutant p110 (10, 11). Herein, we
examined the effect of XL147 (an ATP-competitive reversible
PI3K inhibitor; Exelixis) against a panel of human breast cancer
cell lines harboring molecular alterations indicative of PI3K
dependence, such as HER2 gene amplification, PIK3CA muta-
tion, and/or loss of PTEN. XL147 has recently completed phase I
clinical development; it exhibits an IC
50
against WT and mutant
p110αof approximately 40 nM (12).
In a panel of HER2-overexpressing human breast cancer cell
lines, treatment with XL147 abrogated AKT and S6 phosphor-
ylation but also induced the expression and phosphorylation of
HER3 and other RTKs. The increase in mRNA of these RTKs
depended on the Forkhead transcription factors FoxO1 and
FoxO3a, which are negatively regulated by AKT (13). In HER2
+
cells, phosphorylation of HER3 was maintained by the HER2
tyrosine kinase, resulting in partial recovery of phosphorylated
AKT (pAKT) and thereby limiting the antitumor action of
XL147. Knockdown of HER3 or treatment with the anti-HER2
agents trastuzumab or lapatinib sensitized HER2
+
breast cancer
cells to XL147 in vitro and in vivo. These data suggest that be-
cause of relief of FoxO-mediated feedback, therapeutic inhib-
itors of PI3K will have limited clinical activity if used as single
agents. Thus, to maximally disable PI3K/AKT signaling, thera-
pies targeted against HER2/HER3 should be added to PI3K
inhibitors in HER2-dependent cells.
Results
Inhibition of PI3K Is Associated with Induction of HER3 and pHER3.
We treated with XL147 a panel of breast cancer cell lines with
dysregulated PI3K activity. As XL147 binds to serum proteins
with high affinity, we conducted most studies in 2.5% FBS-
containing media. Treatment with XL147 inhibited the mono-
layer growth of all cell lines in a dose-dependent manner (Fig.
1A). A similar IC
50
of 6 μM or less was observed in cells tested in
3D (Fig. 1B). Analysis of the growth curves by using the initial
amount of cells at the start of treatment as baseline indicated
that at the IC
50
of approximately 6 μM, the main effect of XL147
was inhibition of cell proliferation. At 20 μM, however, XL147
induced cell death as revealed by the reduction in cell number
below the baseline (Fig. S1A) (14). This was further confirmed by
immunoblot of biomarkers of cell death and G
1
–S transition in
cells treated with XL147 for 24 h (Fig. S1B). In all cell lines,
treatment with XL147 resulted in dose-dependent inhibition of
PI3K as measured by pAKT
S473
. Consistent with the inhibition of
cell proliferation, XL147 induced a reduction in cyclin D1 and
pRB and an increase in levels of the CDK inhibitor p27
KIP1
but
no detectable change in levels of total or cleaved poly (ADP-
ribose) polymerase (PARP), a biomarker of cell death (Fig.
S1B). Focusing on molecules in the PI3K and mTOR pathways,
treatment with XL147 resulted in a dose-dependent reduction in
pAKT
S473/T308
and pS6
S240/244
. Surprisingly, in six of seven cell
lines, XL147 also caused up-regulation of total HER3 and/or
pHER3
Y1289
levels. In five of six HER2-overexpressing cell lines,
total HER2 and/or pHER2
Y1248
were also modestly up-regulated
upon inhibition of PI3K (Fig. 2).
Author contributions: A.C. and C.L.A. designed research; A.C., V.S., and C.R. performed
research; A.C., M.G.K., and C.L.A. analyzed data; and A.C. and C.L.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: carlos.arteaga@vanderbilt.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1018001108/-/DCSupplemental.
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XL147-Induced Up-Regulation of HER3 Depends on FoxO-Mediated
Transcription. We next determined the half-life of the HER3
protein by using cycloheximide, an inhibitor of protein synthesis.
The rate of decay of HER3 was not different in BT474 cells with
or without 6 μM XL147. We then quantified the levels of HER3
mRNA upon inhibition of PI3K over a time course in XL147-
treated cells. BT474, SKBR3, and MDA453 cells depicted a
maximal increase in HER3 mRNA at 6 h, which was maintained
as long as 48 h after the addition of the inhibitor (Fig. 3 Aand
C). The allosteric inhibitor of AKT1/2 5J8 (15) and the PI3K
inhibitor LY294002 (16), but not the mTOR inhibitor rapamy-
cin, also up-regulated HER3 mRNA (Fig. 3B).
To delineate the mechanisms of HER3 transcriptional acti-
vation, we focused on the FoxO transcription factors FoxO1,
FoxO3a, and FoxO4. AKT regulates the subcellular localization
of these molecules by phosphorylation, thereby preventing them
from translocating to the nucleus and regulating transcription
(13). In absence of AKT activity, FoxO factors are predomi-
nantly nuclear and therefore can activate transcription (13). Of
note, by using the PROMO database, we identified multiple
putative FoxO-binding sites in the HER3 promoter (up to 5,000
bp upstream of the transcription start site) (17).
We next determined the subcellular distribution of FoxO
proteins following inhibition of PI3K and AKT with XL147 and
5J8, respectively. FoxO4 was almost undetectable; thus, we fo-
cused on FoxO1 and FoxO3a. Treatment with XL147 and 5J8
resulted in accumulation of both FoxO factors in the nucleus of
BT474 and MDA453 cells, sometimes accompanied by a re-
duction in the baseline levels in the cytosol (Fig. 3D). This was
not observed with rapamycin (Fig. S2A). To determine if FoxO is
involved in the increase of HER3 transcription, we transfected
BT474, SKBR3, and MDA453 cells with FoxO1- and FoxO3a-
specific siRNA duplexes. In all three cell lines, the siRNAs re-
duced both FoxO mRNAs by 70% to 80% (Fig. 3F) and abro-
gated the XL147-induced increase in HER3 mRNA as measured
by quantitative PCR (qPCR; Fig. 3E). To further confirm this
FoxO-mediated effect, we transfected BT474 cells with FoxO1
and FoxO3a siRNA duplexes and then treated them with DMSO
or XL147 for 24 h. Cell lysates were then hybridized to arrays
containing probes for 42 different phosphorylated receptor ty-
rosine kinases (pRTKs). There was a decrease in pHER3 signal
in XL147-treated cells in which FoxO1 and FoxO3a had been
knocked down compared with cells transfected with control oli-
gonucleotides (Fig. 3G).
Knockdown of Compensatory Feedback to HER3 Sensitizes to PI3K
Inhibition. A time course of BT474 cells treated with XL147 for
0 to 72 h revealed time-dependent up-regulation of total HER3,
HER3 phosphorylated at Y1197 and Y1289 (two PI3K binding
sites (18)], T308 and S473 pAKT, and pS6 levels (Fig. 4A), im-
plying partial recovery of PI3K/AKT/mTOR signaling. Although
pHER3 is known to activate ERK/MAPK via its interaction with
Shc (18), we did not detect consistent recovery of pERK upon
reactivation of HER3 (Fig. 4A). Even though recovery of pAKT
was less with a suprapharmacological dose of 20 μM, feedback
up-regulation of total HER3 and pHER3 was more noticeable
with this dose of XL147, further suggesting inhibition of PI3K
was causal to the reactivation of HER3 (Fig. 4B). These data
imply that, upon inhibition of PI3K, cells partially restore HER3
phosphorylation to maintain membrane-bound p85/p110 and
some level of PIP3 which, in turn, may limit the net inhibitory
effect of the PI3K inhibitor.
In HER2-overexpressing cells, the major mechanism of PI3K
activation is the coupling of pHER3 to an N-terminal SH2 domain
in p85, the regulatory subunit of PI3K (19, 20). In these cells, the
main tyrosine phosphorylated protein precipitated with p85
antibodies is pHER3. This HER3 and p85 association de-
pends on the catalytic activity of HER2 as it is disrupted by HER2
tyrosine kinase inhibitors (TKIs) (21, 22). Thus, we examined if,
upon inhibition of PI3K, there was maintenance or recovery of the
HER3/p85 association. BT474 cells were treated with increasing
concentrations of XL147 followed by pull-down assay with p85
antibodies and subsequent pTyr and HER3 immunoblot. After
XL147 treatment, there was a dose-dependent increase of an
approximately 200-kDa major p85-associated pTyr band as well
as other smaller and less abundant pTyr proteins (Fig. 4C,
arrows). The p85-associated approximately 200-kDa band was
Fig. 1. XL147 inhibits cell growth in a dose-dependent manner. (A) Breast
cancer cell lines with different lesions in the PI3K pathway (as noted within
parentheses) were treated with 0 to 20 μM XL147 and counted on the days
indicated. Each bar represents the mean ±SE of six replicates (
a,b,c
P<0.05 vs.
0μM XL147, paired ttest). (B) Cells were cultured in Matrigel with or without
0to20μM XL147 and photographed (magnification 10×) on indicated days.
Fig. 2. XL147-mediated
inhibition of PI3K asso-
ciates with induction of
HER3 and pHER3. Cells
lines were harvested af-
ter overnight treatment
with 0 to 20 μM XL147
in serum-free medium
followed by immunoblot
analysis with indicated
antibodies.
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also detected with HER3 and pHER3
Y1197
antibodies (Fig. 4 D
and F). Knockdown of HER3 with siRNA eliminated the HER3
and pHER3
Y1197
band in the p85 pull-downs of lysates from
XL147-treated cells, further confirming HER3 at least as a com-
ponent of the approximately 200-kDa pTyr band (Fig. 4 Eand F).
The compensatory up-regulation of total HER3 and partial
maintenance of pHER3 and pAKT upon inhibition of PI3K sug-
gested that combined inhibition of HER3 and PI3K would syner-
gistically inhibit tumor cell viability. Therefore, we transfected
BT474 cells with control or HER3 siRNA duplexes, treated them
with XL147, and measured cell growth. Cell proliferation was sig-
nificantly reduced by a combination of HER3 knockdown and
XL147 compared with either intervention alone (Fig. 4 Gand H).
Consistent with a synergistic proapoptotic effect, the combination
induced a greater proportion of cells in the sub-G
1
phase DNA
fraction as well as PARP cleavage (Fig. S3 Aand B) compared with
either treatment alone. Similar results were obtained with MDA453
and SKBR3 cells (Fig. S3 Dand F). In both these cell lines, the
combination of HER3 knockdown and XL147 inhibited pAKT and
pS6 more effectively than each treatment alone (Fig. S3 Cand E).
Recovery of HER3 Phosphorylation Depends on HER2. In breast can-
cer cells with HER2 gene amplification, HER2 is the main kinase
that phosphorylates HER3 (19, 22). As XL147 does not affect the
catalytic activity of HER2 (Fig. 2), it is logical to speculate that, in
HER2-overexpressing cells, HER2 remains as the kinase main-
taining pHER3 upon inhibition of PI3K. Therefore, we examined
the effect of XL147 in combination with the HER2 antibody
trastuzumab or the HER2 TKI lapatinib. In BT474 cells, either of
these combinations was significantly more effective at inhibiting
cell proliferation (Fig. 5 Aand B) or PARP cleavage (Fig. 5C) than
XL147 or each HER2 antagonist alone. Similar results were ob-
served with MDA453 and SKBR3 cells (Fig. S4).
We speculated that the synergistic action of XL147 in com-
bination with lapatinib or trastuzumab on cell growth was caused
by a reduction in the recovery of pHER3 but not inhibition
of HER3 mRNA transcription. Thus, we performed qPCR for
HER3 in RNA from BT474 cells treated with XL147 plus lapa-
tinib, trastuzumab, or both. Treatment with trastuzumab alone
did not have any significant effect on HER3 mRNA levels, but in
combination with XL147, it enhanced the up-regulation of HER3
RNA mediated by the PI3K inhibitor (Fig. 5D). As shown pre-
viously, lapatinib induced HER3 mRNA (23). However, the ef-
fect of lapatinib was more prominent when used in combination
with XL147 (Fig. 5D), possibly because of a more pronounced
inhibition of PI3K/AKT compared with either agent alone. Treat-
ment with XL147 plus lapatinib or plus trastuzumab attenuated
the recovery of pHER3 compared with cells treated with XL147
alone (Fig. 5E), suggesting that inhibition of the HER2 kinase
with lapatinib or of ligand-independent HER2/HER3 dimers
with trastuzumab limits the HER2-mediated activation of HER3
upon inhibition of PI3K/AKT.
Fig. 3. XL147-induced up-regulation of HER3 transcription is dependent
on FoxO. (A) BT474 cells were treated with 6 μM XL147 for the indicated
times before RNA isolation and real-time qPCR with HER3-specific primers.
(B) BT474 cells were treated with 2 μM5J8,20μM LY294002, and 50 nM
rapamycin for 10 h before RNA isolation and qPCR for HER3. (C) MDA453
and SKBR3 cells were treated with 6 μMXL147foraslongas48hbefore
RNA isolation and qPCR for HER3. For A–C, each bar represents the mean ±
SE of three wells. (D) BT474 and MDA453 cells were treated with DMSO,
6μM XL147, or 2 μM5J8for4h.Nuclearandcytoplasmicextractswere
subjected to immunoblot analysis with FoxO1 and FoxO3a antibodies.
Loading controls for nuclear extracts: HDAC3; for cytoplasmic extracts:
RhoA (BT474) and MEK1/2 (MDA453). Arrow indicates the FoxO3a-specific
band. (EandF) BT474, MDA453, and SKBR3 cells were transfected with
control or FoxO1- and FoxO3a-specific siRNA duplexes followed by treat-
ment with XL147 for 6 h before harvesting, RNA isolation and qPCR for
HER3 (E) or FoxO1 and FoxO3a (F). Each bar represents the mean ±SE of
triplicate wells. (G) BT474 cells were transfected with control or FoxO1 and
FoxO3a siRNA and treated with XL147 for 6 h. Cell lysates were used for
hybridization with pRTK arrays.
Fig. 4. Knockdown of compensatory feedback to HER3 sensitizes to PI3K
inhibitor. (A) BT474 cells were treated with 6 μM XL147 for as long as 72 h
and subjected to immunoblot analysis. (B) Immunoblot of lysates from
BT474 cells treated with 6 or 20 μM XL147 for 0 to 48 h. (Cand D) BT474
cells were treated with 0 to 20 μM XL147 for 24 h and lysed. Cell lysates
(0.5 mg) were subjected to immunoprecipitation with a p85 antibody
followed by immunoblot for p85 and pTyr (C) or p85 and HER3 (D). Arrows
in Cindicate p85-associated pTyr bands. (Eand F) BT474 cells were trans-
fected with control or HER3-specific siRNA duplexes and treated with 6 μM
XL147 for 24 h. Cell lysates (0.5 mg) were subjected to immunoprecipita-
tion with a p85 antibody followed by immunoblot with HER3 (E),
pHER3
Y1197
(F), and p85 (Eand F) antibodies. (GandH) BT474 cells were
transfected with HER3-specific siRNA and treated with DMSO or 2 μM
XL147. Cells were harvested for counting (G) or crystal violet staining
(H)onday6.InG, each bar represents the mean ±SE of six replicates
(*P<0.05, paired ttest).
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www.pnas.org/cgi/doi/10.1073/pnas.1018001108 Chakrabarty et al.
Combined Inhibition of HER2 and PI3K Is Synergistic in Vivo. Based on
the in vitro data, we proposed that addition of trastuzumab or
lapatinib would enhance the antitumor effect of XL147 against
HER2-dependent xenografts. Athymic mice bearing BT474 xe-
nografts were randomized to therapy with XL147, lapatinib,
trastuzumab, or XL147 plus each HER2 antagonist. Each mono-
therapy significantly delayed tumor growth with trastuzumab be-
ing the only agent that induced a complete tumor regression in one
of eight mice. Both combinations were superior to the respective
drugs given alone. Of note, the combination of trastuzumab and
XL147, but not lapatinib and XL147, induced a complete tumor
response in three of eight mice (P<0.05; Fig. 6A). There was no
significant drug-related toxicity in any of the treatment arms.
We next examined pharmacodynamic biomarkers of target in-
activation after 28 d of treatment. Treatment with XL147 for 28
d did not change pHER3 levels, whereas lapatinib was more ef-
fective than trastuzumab in reducing pHER3. The combination of
XL147 plus trastuzumab inhibited pHER3 more potently than any
of the other treatments (Fig. 6Band Fig. S5). The oncogenic ac-
tion of AKT has been shown to correlate with cytoplasmic and
nuclear pAKT
S473
levels (24). Therefore, we quantitated pAKT
S473
in both cellular compartments. Consistent with differences in tu-
mor growth among treatment arms, nuclear pAKT was lower in
tumors treated with XL147 plus lapatinib or XL147 plus trastu-
zumab compared with tumors treated with single agents. Of all
three single drugs, XL147 was the only one shown statistically to
inhibit nuclear pAKT levels. There were no detectable changes in
cytoplasmic pAKT levels (Fig. 6Band Fig. S5). These results
suggest that combined inhibition of HER2 and PI3K in HER2-
dependent xenografts is required to maximally inhibit signaling
output of the PI3K/AKT pathway. The levels of total HER3 ob-
served after 28 d of treatment (Fig. 6Band Fig. S5, top row) did
not reflect the up-regulation of HER3 mRNA and protein after
short-term assays in cells in culture. We speculate this could be the
result of the late timing of the analysis of the xenografts.
Inhibition of PI3K Induces RTKs Other than HER3. In Fig. 4Cwe
showed that, after XL147 treatment of BT474 cells, there is an
increase in p85-associated pTyr proteins. We also showed that
a prominent pTyr band of approximately 200 kDa that copreci-
pitates with p85 (PI3K) is recognized by HER3 and pHER3
antibodies (Fig. 4 D–F) and is eliminated upon knockdown of
HER3 with siRNAs (Fig. 4 Eand F). However, the dose-
dependent increase in the lower molecular weight pTyr bands
when PI3K is inhibited (Fig. 4C) suggested an increase in p85-
associated proteins other than HER3. In addition, results in Fig.
7Ashowed that, upon knockdown of HER3, there is still an in-
crease in the p85-associated approximately 200-kDa pTyr band,
leading further to speculation of the presence of other com-
pensatory p85-associated tyrosine kinases and/or adaptors aimed
at partially maintaining PI3K as active. To test this hypothesis
we hybridized to pRTK arrays two different concentrations (high
and low) of lysates from BT474cells treated over a 24-h time course
with XL147. Treatment with XL147 resulted in an increase in
the phosphorylation of HER3 and multiple other RTKs, including
EGFR, ERBB4/HER4, FGFR1, FGFR2, FGFR3, FGFR4, InsR,
IGF1R, EphA1, Tie2, TrkA, Flt3, MER, and macrophage-stimu-
lating protein receptor (Fig. 7 Band C). Several of these RTKs,
such as EGFR and ERBB4, migrate at approximately 200kDa, thus
potentially explaining the persistent high molecular weight pTyr
band associated with p85in cells in which HER3 has been knocked
down (Fig. 7A). We then performed qPCR analysis to determine
whether these changes occurred at the transcriptional level. Fol-
lowing treatment of BT474 cells with XL147, there was an increase
in ERBB4, IGF1R, InsR, EphA1, FGFR2, and FGFR3 mRNAs,
with IGF1R and InsR being the most prominent (Fig. 7D).
Computational analyses revealed multiple putative FoxO-
binding sites in the INSR, IGF1R, and FGFR2 gene promoters
(17). Like for HER3 (Fig.3E), knockdown of FoxO and FoxO3a
with siRNA limited the induction of IGF1R, InsR, and FGFR2
mRNAs (Fig. 7E) in cells treated with XL147. Finally, to de-
termine the potential therapeutic relevance of this feedback, we
examined whether depletion of these RTKs sensitized cells to
Fig. 5. Recovery of HER3 phosphorylation depends on HER2 and is limited
by HER2 inhibitors. (A and B) BT474 cells were treated with 2 μM XL147 alone
or in combination with 0.1 μM lapatinib (Lap) (A)or10μg/mL trastuzumab
(Tras) (B) and counted after 6 d (A)or8d(B). Each bar represents mean ±SE
of six replicates. (C) Immunoblot of biomarkers of apoptosis and G
1
–S phase
transition with lysates from BT474 cells treated with the indicated inhibitors
for 72 h. (D) Real-time qPCR for HER3 mRNA in cells treated with XL147 (6
μM), Lap (1 μM), Tras (10 μg/mL), or the indicated combinations for 10 h. Each
bar represents mean ±SE of triplicate wells. (E) Total HER3 and pHER3 im-
munoblot of lysates from BT474 cells treated over a time course (0–24 h) with
the indicated inhibitors at similar concentrations as in D.
Fig. 6. Combined inhibition of HER2 and PI3K is synergistic in vivo. (A)
Tumor growth curve from nude mice transplanted with BT474 cells and
treated with vehicle (Ctrl), XL147, lapatinib, trastuzumab, or the indicated
drug combinations for 28 d. Each data point represents the mean tumor
volume (in mm
3
)±SE of eight mice per treatment (CR, complete response to
treatment; *P<0.05 for trastuzumab vs. trastuzumab plus XL147 and
lapatinib vs. lapatinib plus XL147, two-way ANOVA). (B) Histoscore (H-score)
analysis of immunohistochemical sections. Each bar represents mean ±SE
(*P<0.05 vs. control, unpaired ttest).
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the PI3K inhibitor. We transfected BT474, MDA453, and (low
HER2) MCF7 cells with IGF1R and InsR siRNAs followed by
treatment with XL147. Knockdown of both RTKs was effective
in all three cell lines (Fig. S6 Aand B) and depletion of either
RTK sensitized all cells to XL147-mediated growth inhibition
(Fig. S6 C–E). Collectively, these data suggest that cancer cells
limit the inhibition of PI3K by feedback mechanisms that up-
regulate multiple RTKs or adaptors capable of engaging p85 and
thus activating PI3K. In turn, these molecules partially maintain
PI3K/AKT signaling and counteract the antitumor effect of
single-agent therapeutic inhibitors of this pathway.
Discussion
We have examined the effects of the PI3K inhibitor XL147 on
a panel of breast cancer cell lines with molecular alterations
indicative of PI3K dependence. In this report, we mainly focused
on breast cancer cells with HER2 gene amplification. In addition
to the inhibition of AKT and mTOR, XL147 treatment resulted
in time-dependent feedback up-regulation of HER3 expression
and phosphorylation. In turn, pHER3 engaged p85, activated
PI3K, and induced partial recovery of pAKT and pS6 (Figs. 2
and 4 and Fig. S3 Cand E). As AKT phosphorylates FoxO
transcription factors, thus preventing their nuclear translocation
(13), the inhibition of AKT by XL147 resulted in accumulation
of FoxO3a and FoxO1 in the nucleus (Fig. 3D). Knockdown of
FoxO1 and FoxO3a suppressed the induction of HER3 mRNA
(Fig. 3E) and pHER3 (Fig. 3G) upon inhibition of PI3K/AKT
with XL147. These results suggest that HER3 is down-regulated
by PI3K/AKT and are consistent a recent observation in ovarian
cancer cells in which the PI3K inhibitor GDC-0941 blocked
down-regulation of the HER3 mRNA upon treatment with the
HER3-activating ligand heregulin (25).
RNAi of HER3 sensitized to XL147-induced cell death (Fig.
S3 Aand B) and enhanced XL147-mediated inhibition of pAKT
and pS6 (Fig. S3 Cand E), suggesting that the up-regulation of
total and pHER3 limited the antitumor effect of XL147. This
result has particular relevance in HER2-overexpressing cells in
which the kinase-deficient HER3 coreceptor, when it has been
dimerized with and activated by HER2, is the key mechanism
engaging p85 and activating PI3K. Indeed, breast cancer cells
with HER2 amplification are particularly sensitive to apoptosis
induced by PI3K inhibitors (14). In addition, this association of
HER2/HER3 dimer with p85 has been found to be essential for
the viability of HER2-dependent cells (22, 26, 27).
As inhibition of PI3K with XL147 did not affect the HER2
kinase, HER3 phosphorylation was maintained and further in-
creased, remaining associated with p85 in XL147-treated cells
(Fig. 4 D–F). Addition of the HER2 antibody trastuzumab or the
HER2 TKI lapatinib attenuated the recovery of pHER3 upon
treatment with XL147 (Fig. 5E) even though HER3 transcription
was further increased by the combinations of inhibitors compared
with each inhibitor alone (Fig. 5D). Also, both combinations of
XL147 with each HER2 antagonist were also more effective than
each inhibitor alone at reducing pHER3 and pAKT and inhibiting
growth of HER2
+
xenografts (Fig. 6). These data suggest that, in
HER2-overexpressing cells, inhibition of the HER2 kinase with
lapatinib or disruption of ligand-independent HER2/HER3
dimers with trastuzumab (27) limits the activating input of HER2
to the up-regulated HER3 coreceptor when PI3K is inhibited.
These data are reminiscent of reports in which inhibition of
mTOR with rapamycin or “rapalogues”relieves suppression of
insulin and IGF-I receptor signaling via up-regulation of IRS-1,
thereby reactivating PI3K/AKT (28, 29). In addition, dual
inhibitors of TORC1 and PI3K have also been shown to induce
HER3 expression (23). However, the TORC1 inhibitor rapa-
mycin did not mirror the effect of XL147 or the AKT inhibitor
5J8 on HER3 mRNA (Fig. 3Band Fig. S2B). This suggests that
the effects of AKT inhibition on FoxO-mediated feedback up-
regulation of HER3 transcription are not mediated by TORC1.
These data have implications that apply to therapeutic inhib-
itors of RTKs that rely on PI3K/AKT, such as HER2. For ex-
ample, trastuzumab, when given alone, is a weak inhibitor of
AKT (26, 27, 30) (Fig. 6Band Fig. S5) and thus does not relieve
AKT-mediated suppression of FoxO-induced HER3 transcrip-
tion. On the contrary, the prompt and strong inhibition of pAKT
with lapatinib results in strong up-regulation of HER3 expres-
sion (Fig. 5D). The combination of lapatinib and XL147 was
synergistic in vitro (Fig. 5Aand Fig. S4 Aand C) and in vivo (Fig.
6A), but was also the most potent at inducing HER3 mRNA
(Fig. 5D). Despite the weaker inhibition of pAKT by trastuzu-
mab, the combination of XL147 and trastuzumab appeared to be
superior to the combination of XL147 and lapatinib at inhibiting
pAKT and tumor growth (Fig. 6 and Fig. S5). These differences
suggest that the inability of trastuzumab to derepress compen-
satory HER3 expression is a possible advantage of trastuzumab
versus lapatinib. Current trials comparing both inhibitors may
shed light on the clinical relevance of these differences.
We detected an increase in p85-associated pTyr proteins on
inhibition of PI3K with XL147. Indeed, a p85-associated P-Tyr
band of approximately 200 kDa that comigrates with HER3 was
still detectable in XL147-treated BT474 cells after HER3 had
been knocked down with siRNA (Fig. 7A), suggesting the pres-
ence of other compensatory RTKs and/or adaptors aimed at
partially maintaining PI3K active. By using RTK arrays and
siRNA knockdown, we found FoxO-dependent up-regulation of
InsR, IGF1R, and FGFR2 after inhibition of PI3K/AKT with
XL147 (Fig. 7 C–E). In several cell lines, knockdown of InsR and
IGF1R sensitized to the PI3K inhibitor (Fig. S6 C–E). One of
these, the MCF-7 cell line, harbors an activating mutation in
Fig. 7. Inhibition of PI3K induces RTKs other than HER3. (A) BT474 cells
were transfected with HER3 siRNA duplexes and treated with XL147 for 24 h.
Cell lysates (0.5 mg) were precipitated with a p85 antibody, followed by
immunoblot analysis with antibodies indicated to the right. Cell lysates from
BT474 cells treated with 1 μM lapatinib for 6 h were used as positive controls
(lanes 1 and 2). (B and C ) BT474 cells were treated with 6 μM XL147 over
a time course to 24 h as indicated. Cell lysates were prepared and 0.2 mg
(lower sensitivity; B) or 0.5 mg (higher sensitivity; C) of total protein were
applied to pRTK arrays. Arrows indicate RTKs whose phosphorylation was
up-regulated on treatment with the PI3K inhibitor. (D) Real-time qPCR
analysis of the indicated RTKs in RNA collected from cells treated with DMSO
or 10 μM XL147 for 6 h. (E) Analysis of IGF1R, InsR, and FGFR2 mRNA by qPCR
in RNA extracted from BT474 cells transfected with FoxO1 and FoxO3a siRNA
and then treated with 10 μM XL147 for 6 h. Each bar represents the mean ±
SE of triplicate wells.
2722
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www.pnas.org/cgi/doi/10.1073/pnas.1018001108 Chakrabarty et al.
PIK3CA but not HER2 gene amplification. These data imply that
PI3K inhibition could be associated with the compensatory in-
duction of a group of RTKs over a range of tumor cells. Similar
changes were observed with a second pan-PI3K small molecule
inhibitor, BKM120,* suggesting these changes are not secondary
to off-target effects of XL147 (Fig. S7).
These findings have several clinical implications. In cancer
cells, therapeutic antagonists of the PI3K pathway will inhibit
AKT and relieve suppression of RTK expression and their ac-
tivity. Relief of this feedback limits the sustained inhibition of
the pathway and attenuates the therapeutic response to these
agents. We propose that, as a result, if used as single agents,
PI3K pathway inhibitors will have limited clinical activity. Relief
of this feedback is commensurate with the magnitude and in-
tensity of inhibition of AKT and cannot be applied to all types of
PI3K pathway antagonists (i.e., trastuzumab vs. lapatinib vs.
XL147). In HER2-overexpressing cells, up-regulated expression
of HER3- and HER2-induced phosphorylation of HER3 are the
main mechanisms that counteract inhibition of PI3K/AKT.
Therefore, PI3K inhibitors should be used in combination with
HER2/HER3 antagonists in HER2
+
breast cancers. The most
appropriate anticancer agents to combine with PI3K/AKT in-
hibitors in other PI3K-dependent cancers without HER2 gene
amplification will depend on the major compensatory feedback
mechanisms that are activated on inhibition of this pathway.
These will require additional investigation in clinical trials.
Materials and Methods
Cell Lines. SI Materials and Methods includes information on cell lines.
Inhibitors. The following inhibitors were used: lapatinib and rapamycin (LC
Laboratories), trastuzumab (Vanderbilt Medical Center Pharmacy), LY294002
(Calbiochem), the AKT1/2 inhibitor 5J8 (provided by Craig Lindsley, Van-
derbilt University, Nashville, TN) (15), BKM-120 (Active Biochemical), and
XL147 (Exelixis).
Growth and Crystal Violet Assay, Immunoprecipitation, Immunoblotting, RNAi,
RNA Isolation, and Real-Time qPCR. Growth and crystal violet assay, immu-
noprecipitation, immunoblotting, RNAi, RNA isolation, and real-time qPCR
were performed as described previously (31, 32). Sequences for mismatch
control and human HER3 siRNA duplexes were described by Wang et al. (32).
Human FoxO1, FoxO3a, IGF1R, and InsR siRNA duplexes were obtained from
Ambion and Qiagen, respectively. Primer sequences can be found in SI
Materials and Methods.
Cell Cycle Analysis. SI Materials and Methods includes information on cell
cycle analysis.
Cytoplasmic, Nuclear Fractionation, and Proteome Profiler Human Phospho-RTK
Arrays. Cytoplasmic and nuclear extracts were prepared using the Nuclear
Extract Kit from Active Motif. The pRTK arrays were performed according to
the manufacturer’s instructions (R&D Systems).
Xenograft Experiments and Immunohistochemistry. Protocols for xenografts
studies and immunohistochemistry can be found in SI Materials and Methods.
Animal experiments were approved by the institutional animal care com-
mittee of Vanderbilt University Medical Center (VUMC).
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grant R01 CA80195, American Cancer Society Clinical Research Pro-
fessorship Grant CRP-07-234, the Lee Jeans Translational Breast Cancer
Research Program (C.L.A.), Breast Cancer Specialized Program of Research
Excellence (SPORE) Grant P50 CA98131, and Vanderbilt–Ingram Cancer Cen-
ter Support Grant P30 CA68485. A.C. is partially supported by postdoctoral
fellowship Award KG091215 from the Susan G. Komen Breast Cancer
Foundation.
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