Oncogene (2011) 30, 3222–3233
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
www.nature.com/onc
ORIGINAL ARTICLE
PIK3CA mutation, but not PTEN loss of function, determines
the sensitivity of breast cancer cells to mTOR inhibitory drugs
B Weigelt, PH Warne and J Downward
Signal Transduction Laboratory, Cancer Research UK London Research Institute, London, UK
The phosphatidylinositol 3-kinase (PI3K) pathway is
commonly activated in breast cancers due to frequent
mutations in PIK3CA, loss of expression of PTEN or
over-expression of receptor tyrosine kinases. PI3K pathway activation leads to stimulation of the key growth and
proliferation regulatory kinase mammalian target of
rapamycin (mTOR), which can be inhibited by rapamycin
analogues and by kinase inhibitors; the effectiveness
of these drugs in breast cancer treatment is currently
being tested in clinical trials. To identify the molecular
determinants of response to inhibitors that target mTOR
via different mechanisms in breast cancer cells, we
investigated the effects of pharmacological inhibition of
mTOR using the allosteric mTORC1 inhibitor everolimus
and the active-site mTORC1/mTORC2 kinase inhibitor
PP242 on a panel of 31 breast cancer cell lines. We
demonstrate here that breast cancer cells harbouring
PIK3CA mutations are selectively sensitive to mTOR
allosteric and kinase inhibitors. However, cells with
PTEN loss of function are not sensitive to these drugs,
suggesting that the functional consequences of these two
mechanisms of activation of the mTOR pathway are quite
distinct. In addition, a subset of HER2-amplified cell lines
showed increased sensitivity to PP242, but not to
everolimus, irrespective of the PIK3CA/PTEN status.
These selective sensitivities were confirmed in more
physiologically relevant three-dimensional cell culture
models. Our findings provide a rationale to guide selection
of breast cancer patients who may benefit from mTOR
inhibitor therapy and highlight the importance of
accurately assessing the expression of PTEN protein
and not just its mutational status.
Oncogene (2011) 30, 3222–3233; doi:10.1038/onc.2011.42;
published online 28 February 2011
Keywords: PIK3CA; PTEN; mTOR; everolimus; PP242;
breast cancer
Correspondence: Dr J Downward, Signal Transduction Laboratory,
Cancer Research UK London Research Institute, 44 Lincoln’s Inn
Fields, London WC2A 3PX, UK.
E-mail: [email protected]
Received 21 July 2010; revised and accepted 26 January 2011; published
online 28 February 2011
Introduction
Breast cancers frequently harbour molecular/genomic
aberrations that increase activation of the phosphatidylinositol 3-kinase (PI3K)–AKT–mammalian target of
rapamycin (mTOR) signalling pathway including the
(over-) expression of receptor tyrosine kinases (for
example HER2, IGF1R), loss of PTEN function or
activating PIK3CA mutations (Yuan and Cantley, 2008;
Engelman, 2009). Given the pivotal role of this pathway
in cell growth, proliferation and survival (Engelman
et al., 2006), PI3K, AKT and mTOR have emerged as
promising therapeutic targets for subgroups of breast
cancer patients (Engelman, 2009; Liu et al., 2009).
mTOR is a highly conserved Ser/Thr kinase that
integrates nutrient and growth factor signals through
two distinct protein complexes: (1) mTORC1, which is
inhibited by rapamycin, responsive to growth factors,
amino acids, energy and oxygen levels, and regulates
translation initiation and protein synthesis through
S6K1 and 4E-BP1 and (2) mTORC2, which is rapamycin insensitive and modulates growth factor signalling
by phosphorylating various kinases including AKT at
serine 473 (Guertin and Sabatini, 2007).
Rapamycin and rapamycin analogues (‘rapalogues’)
are potent allosteric inhibitors of mTORC1, which act
by forming a complex with the intracellular receptor
FKBP12 (Brown et al., 1994; Sabatini et al., 1994). The
rapalogues everolimus (RAD001) and temsirolimus
(CCI-779) have received Food and Drug Administration
(FDA) approval for the treatment of patients with
advanced renal cell carcinoma (Atkins et al., 2009;
Kwitkowski et al., 2010). In breast cancer, phase II
clinical trials revealed that pre-treated patients with
locally advanced or metastatic disease had a modest
response when receiving rapalogues as single agent
(12% and 9.2% complete or partial clinical response to
everolimus (Ellard et al., 2009) and temsirolimus (Chan
et al., 2005), respectively). Thus, for the introduction of
mTOR inhibitors into clinical practice, it is germane to
identify markers to guide selection of patients who
would benefit from these drugs and to define mechanisms of de novo resistance (that is patients who would
not derive any benefit from these targeted agents).
Second-generation mTOR small molecule inhibitors,
which unlike rapalogues inhibit both mTORC1 and
mTORC2 by directly targeting the ATP-binding site of
mTOR, have recently entered clinical trials (Dancey,
PIK3CA mutation predicts mTOR inhibitor response
B Weigelt et al
3223
2010). Although the clinical benefit of these agents is yet
to be determined, in vitro and in vivo pre-clinical models
using the mTOR kinase inhibitor PP242 have demonstrated that this compound inhibits proliferation more
completely than the mTORC1 inhibitor rapamycin
(Feldman et al., 2009; Janes et al., 2010). This
emphasises the complexity of the PI3K–AKT–mTOR
signalling pathway and the distinct contributions of
mTORC1 and mTORC2 to cell proliferation and
survival.
We set out to define the molecular determinants of
response to inhibitors that target mTOR via different
mechanisms in breast cancer cells. To address this, we
investigated the effects of pharmacologic inhibition of
mTOR using the rapalogue everolimus and the kinase
inhibitor PP242 in a panel of 31 breast cancer cell lines,
and showed that activating PIK3CA mutations but not
PTEN loss of function determine response of breast
cancer cells to allosteric mTORC1 and active-site
mTORC1/2 inhibitors. This suggests that the functional
consequences in the tumour of these two mechanisms of
activation of the mTOR pathway are quite distinct and
are differentially affected by drugs in clinical usage.
In addition, a subset of cell lines harbouring HER2
amplification showed increased sensitivity to PP242 but
not everolimus irrespective of the PIK3CA mutation
status. The predictive nature of the PIK3CA mutations
and HER2 amplification for response to everolimus and/
or PP242 was independent of the cell culture environment, as this association was observed in breast cancer
cell lines grown in conventional two-dimensional (2D)
monolayers as well as in more physiologically relevant
three-dimensional (3D) culture models.
Results
PIK3CA mutations determine response to everolimus and
PP242 in breast cancer cells
The effects of pharmacologic mTOR inhibition were
tested in a panel of 31 breast cancer cell lines grown in
2D cultures. Cells were treated for 72 h with serial
dilutions of the allosteric mTORC1 inhibitor everolimus
or the mTOR kinase inhibitor PP242. Cell viability was
determined by CellTiter-Blue assay, and a wide range of
SF50s (that is surviving fraction of 50% relative to
untreated cells; Turner et al. (2010)) from 1.7 nM to
6.6 mM was observed (Figure 1). The SF50s of everolimus and/or PP242-sensitive cell lines were in the
nanomolar range (Figure 1); however, for many of the
breast cancer cell lines studied, the SF50 was not
reached when treated with everolimus (SF50s 410 mM;
Figure 1a).
To identify genomic alterations linked to PI3K–
AKT–mTOR pathway dependency and mTOR inhibitor sensitivity (that is SF50o1 mM), the mutation status
of the 31 breast cancer cell lines was correlated with
their mTOR inhibitor response. Given the contradictory
reports on the pattern of PIK3CA and PTEN mutations
in these cell lines in previous publications (Hollestelle
Figure 1 PIK3CA mutant breast cancer cell lines are selectively
sensitive to allosteric mTORC1 and mTOR kinase inhibitors.
Thirty-one breast cancer cell lines with wild-type PIK3CA/PTEN
(grey), mutant PIK3CA (white), PTEN loss of function (black), or
concurrent PIK3CA and PTEN mutations (striped) were treated
with serial dilutions of (a) the rapalogue everolimus or (b) the
active-site inhibitor PP242. The surviving fraction of 50% (SF50)
relative to untreated cells was assessed 72 h after treatment using
the CellTiter-Blue assay. *HER2 amplification; **PIK3R1 mutation; mut, mutant; null, loss of function; wt, wild type.
et al., 2007; Hu et al., 2009) and the COSMIC database
(Forbes et al., 2008), and the possibility of crosscontamination and misidentification of cell lines (ASN0002, 2010), we re-sequenced in all our breast cancer cell
lines the entire PTEN transcript and exons 2, 4–11 and
19–21 of the PIK3CA transcript (Supplementary Table 1;
Figure 2a; Table 1), covering the high-frequency
mutations in the p85, C2, helical and kinase domains of
the PIK3CA gene (Samuels et al., 2004; Gymnopoulos
et al., 2007). In addition, as PTEN function is regulated
also by post-translational modifications (Wang and
Jiang, 2008; Huse et al., 2009; Poliseno et al., 2010),
PTEN protein levels were assessed in these cells by
western blotting (Figure 2b). Sequencing analysis reOncogene
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Figure 2 PTEN transcript and protein levels in a panel of 31 breast cancer cell lines. (a) Agarose gel electrophoresis of PCR products
amplified from cDNA using primers specific for exons 5–7 of the PTEN transcript (628 bp product). (b) Whole-cell lysates were
analysed by western blotting for PTEN and b-actin as loading control. MDA, MDA-MB.
Table 1 Mutations identified in PTEN and PIK3CA transcript sequences in 31 human breast cancer cell lines studied
Cell line
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
a
AU565
BT20
BT474
BT483
BT549
CAMA1
HCC1187
HCC1395
HCC1419
HCC1428
HCC1500
HCC1569
HCC1806
HCC1937
HCC1954
HCC202
HCC38
HCC70
HS578T
MCF7
MDA-MB-157
MDA-MB-231
MDA-MB-361
MDA-MB-415
MDA-MB-436
MDA-MB-453
MDA-MB-468
SKBR3
T47D
ZR751
ZR7530
PIK3CA transcript
PIK3CA amino acid
3140A4G, 1616C4G
333G4C
1624G4A
H1047R, P539R
K111N
E542K
3075C4T
3140A4G
1633G4A
PTEN protein
823delG
274G4C
V275fs*1
D92H
No expression
Weak expression
Not detectable
(N212fs*1)a
No expression
800het_delA
K267fs
No expression
Not detectable
(Homozygous deletion)b
No expression
Not detectable
270delT
F90fs*9
No expression
No expression
407G4A
C136Y
919G4A
Not detectable
E307K
(L70fs*7d; A72fs*5e)
No expression
323T4G
L108R
Weak expression
H1047R
E545K
E545K
1633G4A
E545K
H1047R
c
3140A4G
PTEN amino acid
T1025T
1633G4A
3140A4G
PTEN transcript
Weak expression
No expression
H1047R
Reported in COSMIC database (Forbes et al., 2008).
Reported in Tomlinson et al. (1998).
c
PIK3CA mutation (E545A) reported in COSMIC database; no PIK3CA mutation was identified here consistent with recent studies (Hollestelle
et al., 2007; Hu et al., 2009).
d
Reported in COSMIC database.
e
Reported in Hollestelle et al. (2007).
b
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vealed 10 breast cancer cell lines harbouring activating
PIK3CA mutations (BT20, BT474, BT483, HCC1500,
HCC1954, HCC202, MCF7, MDA-MB-361, T47D,
MDA-MB-453), of which one cell line had a concurrent
PTEN mutation (MDA-MB-453) (Table 1). Additional
11 cell lines showed defective PTEN; in six cell lines a
mutation in the PTEN transcript was identified (BT549,
CAMA1, HCC1569, HCC70, MDA-MB-415, ZR75-1),
whereas five cell lines did not express PTEN protein and/
or PTEN transcript (HCC1395, HCC1937, HCC38,
MDA-MB-436, MDA-MB-468) (Table 1; Figure 2).
Correlation of the PIK3CA and PTEN status revealed
that in this setting, the response of breast cancer cells
to the allosteric mTORC1 inhibitor everolimus and to
the active-site inhibitor PP242 was determined by the
presence of activating PIK3CA mutations (Figure 1).
Only one out of nine cell lines with mutated PIK3CA
(and wild-type PTEN), MDA-MB-361, was resistant
to everolimus treatment. Noteworthy was the observation that PP242 treatment induced a more efficient
response in a subset of HER2-amplified cell lines
(that is HCC1419, SKBR3, HCC1569, MDA-MB-453;
Figure 1b) and in the PIK3R1-mutated cell line HS578T,
irrespective of the PIK3CA mutation status. In contrast,
10/12 PTEN null cell lines were resistant to everolimus
or PP242 treatment, with exception of CAMA1 and
ZR75-1 (Figure 1). The cell line with concurrent PTEN
and PIK3CA mutations (MDA-MB-453) was resistant to everolimus. Cells with wild-type PIK3CA and
functional PTEN were resistant to everolimus or PP242
treatment. Taken together, our findings provide strong
circumstantial evidence that activating mutations of
PIK3CA but not PTEN loss of function may predict that
breast cancer cells will be responsive to allosteric mTORC1
and active-site mTORC1/2 inhibitors.
Everolimus and PP242 elicit a G1 cell-cycle arrest
in PIK3CA-mutated cell lines
To test whether everolimus and PP242 exert antiproliferative and/or apoptotic effects, a panel of cell
lines was treated with everolimus, PP242, and etoposide
or staurosporine as positive controls in 2D cell cultures.
Neither the rapalogue everolimus nor the mTOR kinase
inhibitor PP242 induced apoptosis in mTOR inhibitor
sensitive cell lines as assessed by poly (ADP-ribose)
polymerase cleavage using western blotting (Figure 3a)
and by Caspase-3/7 activation using a fluorescent enzymatic assay (Figure 3b). Instead, both everolimus
and PP242 induced a G1 cell-cycle arrest in PIK3CAmutated mTOR inhibitor sensitive cell lines but not in
resistant PTEN null or PIK3CA/PTEN wild-type cells
(Figure 3c). These results imply that, in this setting, the
allosteric mTORC1 inhibitor everolimus and the mTOR
kinase inhibitor PP242 elicit cytostatic responses in the
breast cancer cell lines studied.
Figure 3 Everolimus and PP242 exert cytostatic effects in breast
cancer cells. (a) The PIK3CA mutant mTOR inhibitor sensitive cell
lines BT474, T47D and HCC202 were treated either for 48 h with
everolimus or PP242 at indicated concentrations or for 3 h with
1 mM Staurosporin. Whole-cell lysates were analysed by western
blotting for poly (ADP-ribose) polymerase and a-tubulin as
loading control. (b) Indicated PIK3CA/PTEN wild-type, PIK3CA
mutant and PTEN null cell lines were treated for 48 h with
everolimus, PP242 or Etoposide at indicated concentrations.
Apoptosis induction is depicted as ratio of Caspase-3/7 activation
determined using the Apo-ONE assay over cell viability determined
using CellTiter-Blue, both relative to control-treated cells. (c) DNA
content measured by flow cytometry in PIK3CA/PTEN wild-type,
PIK3CA mutant and PTEN null cells incubated for 48 h with 10 mM
everolimus or 1 mM PP242 to determine the percentage of cells in
G1 (dark grey), S (light grey) and G2 (off-white) phases of the cell
cycle. Representative of two independent experiments. Evero,
everolimus; mut, mutant; null, loss of function; wt, wild type.
Effects of everolimus and PP242 on signal transduction
pathways in breast cancer cells
To understand the response of signal transduction
pathways to mTOR inhibition, we examined AKT,
mTORC1 downstream effectors, and MAPK pathway
activation in a panel of cell lines. As expected, we
observed efficient reduction of the mTORC2 substrate
S473-AKT phospho-levels upon treatment with the
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active-site mTORC1/2 inhibitor PP242 (Feldman et al.,
2009; Janes et al., 2010) but not with everolimus
(Figure 4; Supplementary Figure 1). In addition,
continued activation of 4E-BP1 was detected in everolimus but not PP242-treated cells.
Furthermore, we observed a difference in activated
ERK1/2 levels between mTOR inhibitor sensitive and
resistant breast cancer cell lines (Figures 4a–d; Supplementary Figures 1 and 2). Quantification of the
phospho- and total ERK1/2 protein bands (on the same
western blot using the Odyssey Infrared Imaging System)
revealed that, as a group, both mTOR inhibitor resistant
PTEN null and PIK3CA/PTEN wild-type cell lines
displayed sustained levels of ERK1/2 activation when
treated with everolimus or increased levels of ERK1/2
activation when treated with PP242, as compared with
untreated cells. This phenomenon was generally not
observed in PIK3CA mutant cell lines, in the outliers
(that is PTEN null mTOR inhibitor sensitive CAMA1
and ZR75-1 cells) and in PTEN mutant HER2-amplified
PP242 responsive cells (Figure 4; Supplementary Figures
1–3). The relative expression levels of phospho-ERK1/2
(that is phospho/total ERK1/2 ratios between treated and
untreated cells) in resistant breast cancer cell lines were
significantly higher than those in sensitive cell lines upon
everolimus (P ¼ 0.0103) or PP242 treatment (P ¼ 0.0262;
two-tailed heteroscedastic t-test).
These data suggest the possibility that MAPK
signalling might contribute to the resistance of breast
cancer cells to mTOR inhibitors. We therefore tested the
effect of combined mTOR and MEK inhibition on cell
viability in 2D and 3D cultures. The MEK inhibitors
U0126 (Favata et al., 1998) or AZD6244 (Yeh et al.,
2007) were used at concentrations at which PIK3CA/
PTEN wild-type and PTEN null cells displayed a
decrease in phospho-ERK1/2 levels (that is 1 mM and
10 mM; Supplementary Figure 4a). Simultaneous inhibition of mTORC1 and MEK did not exert a major effect
on the viability of PIK3CA/PTEN wild-type or PTEN
null cells beyond the effect of the MEK inhibitor alone.
The addition of U0126 or AZD6244 to PP242, however,
resulted in a substantial decrease in the surviving cell
fraction (Supplementary Figures 5 and 6). As expected,
combined treatment with the mTORC1/2 inhibitor
PP242 and U0126 or AZD6244 resulted in a marked
reduction in the levels of phospho-ERK and phosphoAKT, whereas combination of the mTORC1 inhibitor
everolimus and U0126 or AZD6244 led to an effective
reduction of phospho-ERK levels but not phospho-
AKT levels in the breast cancer cells studied (Supplementary Figure 4b). These data suggest that a subset of
breast cancer cell lines that lack PTEN function or are
PIK3CA/PTEN wild-type may be resistant to mTOR
kinase inhibitors in part due to activation of the MAPK
pathway.
PIK3CA mutation and HER2 amplification status
correlates with response to mTOR inhibitors independent
of cell culture environment
Given that 3D cell cultures have been suggested to
resemble their in vivo counterparts more closely than
conventional 2D monolayer models (Pampaloni et al.,
2007; Yamada and Cukierman, 2007), and that drug
response of breast cancer cells has been reported to vary
according to culture conditions (that is 2D vs 3D)
(Serebriiskii et al., 2008; Weigelt and Bissell, 2008; Pickl
and Ries, 2009; Weigelt et al., 2010), we assessed
whether the extracellular matrix (ECM) and tissue
architecture would have an impact on the mTOR
inhibitor response of breast cancer cells. A selection of
cell lines was plated on top of 3D ECM cultures (that is
Matrigel) for 4 days until tumour-like structures were
formed, treated for 72 h with serial dilutions of everolimus or PP242 and cell viability was determined using
CellTiter-Blue. The response of PIK3CA/PTEN wildtype, PIK3CA-mutated and PTEN null cell lines was
remarkably similar between 2D and 3D culture conditions (Figure 5). In a way akin to our findings in 2D, cell
lines with functional PI3K/PTEN or PTEN loss were
resistant to everolimus and PP242 treatment, whereas
PIK3CA mutant cells were sensitive when grown in a 3D
culture environment (Figure 5; Supplementary Figure 7).
This was also observed for the HER2-amplified PP242sensitive PIK3CA/PTEN wild-type cell lines, and the
outliers CAMA1, a PTEN null mTOR inhibitor
sensitive, and MDA-MB-361, a PIK3CA mutant everolimus-resistant cell line (Supplementary Figure 8).
Taken together, our results suggest that PIK3CA
mutations determine response of breast cancer cells to
everolimus and/or PP242 independent of the culture
environment.
Discussion
In this study, we assessed the molecular determinants of
response to inhibitors that target mTOR via different
Figure 4 Effects of everolimus and PP242 on signal transduction pathways in breast cancer cells. (a) PIK3CA/PTEN wild-type
mTOR inhibitor resistant, (b) PTEN null mTOR inhibitor resistant cells, (c) PIK3CA mutant mTOR inhibitor sensitive, and (d) the
outlier PTEN null mTOR inhibitor sensitive CAMA1 and ZR75-1, and the HER2-amplified PTEN mutant everolimus-resistant
PP242-sensitive MDA-MB-453 cells were treated for 3 h with 10 mM everolimus or 1 mM PP242 and whole-cell lysates were analysed by
western blotting for total and activated levels of AKT, RPS6, 4E-BP1 and ERK1/2, and a-tubulin as loading control.
(e) Quantification of ERK activation in these breast cancer cells using an infrared imaging system (for western blots see
Supplementary Figure 3). The phospho-/total ERK1/2 ratio in the untreated control of each breast cancer cell line was set to 1 (dark
grey bars), and the effect of everolimus (light grey bars) and PP242 (white bars) treatments on the phospho-/total ERK1/2 ratio in each
cell line is presented relative to its control. Relative levels of ERK1/2 activation were significantly higher in resistant cell lines than in
sensitive cell lines after everolimus (P ¼ 0.0103) or PP242 treatment (P ¼ 0.0261; two-tailed heteroscedastic t-test). Ctrl, control; MDA,
MDA-MB; null, loss of function; wt, wild type; *HER2 amplification.
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mechanisms in a large panel of breast cancer cell lines.
We have shown that breast cancer cells harbouring
activating PIK3CA mutations but not lacking PTEN
function were selectively sensitive to the allosteric
mTORC1 inhibitor everolimus and to the active-site
mTORC1/2 inhibitor PP242, confirming the observa-
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tions that mutations in multiple components of the
PI3K pathway are not necessarily equivalent in their
biological impact (Stemke-Hale et al., 2008; Vasudevan
et al., 2009; Dan et al., 2010). Our results corroborate
and expand on the previous observations that PTEN
loss of function and PIK3CA mutations have different
functional effects on PI3K pathway activation in human
breast cancers and in breast cancer cell lines (StemkeHale et al., 2008; Dan et al., 2010). Here, we demonstrate that the distinct functional effects of lack of
PTEN function versus activating PIK3CA mutations
seem to have crucial implications for the use of therapies
targeting the PI3K–AKT–mTOR pathway in breast
cancer.
It has recently been reported that the efficacy of 25
PI3K pathway inhibitors did not correlate with either
gain-of-function mutations of PIK3CA or PTEN loss in
a panel of 39 human cancer cell lines derived from
tumours from various anatomical sites (Dan et al.,
2010). On the other hand, PIK3CA mutations were
recently reported to predict sensitivity to everolimus in
glioblastoma, breast, ovarian, prostate, endometrial and
colorectal cancer cells (Di Nicolantonio et al., 2010), to
predict response to temsirolimus alone or in combination in patients with advanced cervical, endometrial,
ovarian and breast cancer in a phase I clinical trial
(Janku et al., 2011), and the PI3K/mTOR dual inhibitor
NVP-BEZ235 has been shown to selectively induce cell
death in breast cancer cells harbouring PIK3CA
mutations (Serra et al., 2008; Brachmann et al., 2009)
but not in cells lacking PTEN function (Brachmann
et al., 2009). Taken together, these findings and the
results presented herein suggest that the predictive
nature of PIK3CA mutations for response to everolimus
and PP242 may be tissue specific and of particular
relevance to breast cancer.
A feedback loop depending on an S6K-PI3K-RAS
pathway has been shown to act as a potential mechanism of resistance to mTOR inhibition in metastatic
cancer patients and in in vitro and in vivo models, which
leads to MAPK pathway activation and cell survival
(Carracedo et al., 2008; Brachmann et al., 2009). We
detected increased ERK1/2 activation in breast cancer
cell lines that lack PTEN function or are wild-type for
PIK3CA/PTEN when treated with the active-site
inhibitor PP242, and combined inhibition of
mTORC1/2 and MEK resulted in decreased viability
of these cells. As a group, mTOR inhibitor sensitive
PIK3CA-mutated breast cancer cell lines did not display
increased MAPK pathway activation upon treatment
with everolimus or PP242. There is burgeoning evidence
to demonstrate that breast cancer cells harbouring
PIK3CA mutations are physiologically dependent on
this kinase for the activation of RPS6 and survival,
whereas PIK3CA wild-type cell lines do not necessarily
require PI3K for the activation of the mTOR pathway
(Crowder et al., 2009; Dan et al., 2010). These
observations suggest a greater dependency of PIK3CA
mutant cell lines to the canonical PI3K–AKT–mTOR
pathway, which is consistent with our observations.
Although our findings should be interpreted as hypothOncogene
esis generating and the underlying mechanism for our
observations is yet to be fully established, one could
posit that these distinct effects on the MAPK pathway
according to PIK3CA and PTEN status may be due to
(1) the distinct levels of PI3K pathway activation in cells
with different patterns of mutations in this pathway
(Stemke-Hale et al., 2008; Dan et al., 2010), (2) AKTindependent signal operant within PIK3CA mutant cell
lines (Vasudevan et al., 2009), (3) the increased
dependency of PTEN null cancers on the PI3K isoform
p110b (Torbett et al., 2008; Wee et al., 2008), (4) the
complex cross-talk between PTEN and other pathways,
including the MAPK pathway (Zhang and Yu, 2010) or
(5) importantly, the redundancy and complex feedback
regulation in the PI3K–AKT–mTOR pathway (Efeyan
and Sabatini, 2010; Zhang and Yu, 2010). Further
mechanistic studies to delineate the genotype-dependent
impact of mTOR inhibitors on the signalling networks
in breast cancer cells are warranted.
It should be noted that the prevalence of PIK3CA
mutations in human breast cancers (that is 18–32.5%;
Levine et al., 2005; Saal et al., 2005; Stemke-Hale et al.,
2008; Kalinsky et al., 2009) is somewhat higher than the
proportion of pre-treated metastatic breast patients who
showed clinical benefit with everolimus monotherapy
(12%) (Ellard et al., 2009). This may be reconciled by
the fact that (1) patients with PIK3CA mutant cancers
have a better outcome (Kalinsky et al., 2009) and may
be under-represented in the group of patients with
metastatic disease, (2) B30% of breast cancers show
PTEN protein loss (Hennessy et al., 2005; Perez-Tenorio
et al., 2007; Stemke-Hale et al., 2008), which in contrast
to PTEN mutations is not mutually exclusive
with PIK3CA mutations (Perez-Tenorio et al., 2007;
Stemke-Hale et al., 2008) and that (3) MAPK pathway
activation due to other genomic/molecular aberrations
may not be uncommon (Adelaide et al., 2007; Roidl
et al., 2010), which would result in mTOR inhibitor
resistance in a subset of PIK3CA-mutated cancers
according to our findings. In addition, it has been
suggested that the strength of PI3K–AKT pathway
activation may differ in vitro compared with human
PIK3CA-mutated breast cancer (Loi et al., 2010).
Response rates to everolimus could potentially be
further compounded by the reported anti-angiogenic
effects of rapalogues (Guba et al., 2002; Del Bufalo
et al., 2006); however, this is unlikely given that only
6.7% of pre-treated metastatic breast cancer patients
showed response to the bona fide VEGF inhibitor
bevacizumab as a single agent (Cobleigh et al., 2003).
In the phase II clinical trial reported by Ellard et al.
(2009), PTEN status was evaluated by immunohistochemistry for a possible correlation with everolimus
response in patients with recurrent/metastatic breast
cancer; however, no statistical association was found.
This may be due to the limited number of patients
assessed (Ellard et al., 2009) and to the lack of validated
methodology and scoring system for immunohistochemical evaluation of loss of functional PTEN. In fact,
different antibodies used for immunohistochemical
analysis of PTEN provided divergent results, which
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often did not correlate with PTEN mutations and/or
loss of function (Pallares et al., 2005). Thus, methodologies and scoring systems for immunohistochemical
analysis of PTEN need to be improved and standardised
before its use in the context of clinical trials. Furthermore, recent massively parallel sequencing studies have
provided direct evidence to demonstrate that the
repertoire of gene mutations in a primary breast tumour
and its metastasis differ (Ding et al., 2010). One could
speculate that the PTEN status of a primary tumour
may therefore not necessarily be identical to that of the
metastasis. Our results suggest PIK3CA mutation status
rather than PTEN loss of function would constitute a
positive predictive marker for response to mTOR
inhibitors in breast cancer patients. Given that B80%
of PIK3CA mutations are located in exons 9 and 20
(Cosmic database; Bachman et al., 2004; Samuels et al.,
2004; Forbes et al., 2008) and that sequencing of
PIK3CA mutation hot-spots can be reliably performed
in archival material (Kalinsky et al., 2009), analysis of
Figure 5 PIK3CA mutations determine response to mTOR inhibitors independent of the cell culture environment. (a) PIK3CA and
PTEN wild-type, (b) PIK3CA mutant, and (c) PTEN null cells were grown in 2D (grey) or 3D (black) cell culture models and treated
with serial dilutions of the rapalogue everolimus (left) or the active-site inhibitor PP242 (right). Response was assessed 72 h after
treatment relative to untreated cells using the CellTiter-Blue assay. PIK3CA/PTEN wild-type and PTEN null cells were resistant,
whereas PIK3CA mutant cells were sensitive to everolimus and PP242 treatment in both 2D and 3D culture environments. Error bars
depict standard error of median. MDA, MDA-MB; mut, mutant; null, loss of function; wt, wild type.
Oncogene
PIK3CA mutation predicts mTOR inhibitor response
B Weigelt et al
3230
the PIK3CA gene status in relation to clinical response
to everolimus is warranted.
There is evidence to suggest that HER2-amplified
breast cancers may be ‘addicted’ to (that is physiologically dependent on) continued PI3K–AKT pathway
activation (She et al., 2008). We observed here that a
subset of HER2-amplified breast cancer cells showed
increased sensitivity to the mTOR kinase inhibitor
PP242, but not to the rapalogue everolimus, independent of the PIK3CA mutation status. PP242, unlike
everolimus, also inhibits mTORC2 and its main
substrate AKT, which leads to a more effective decrease
in PI3K–AKT signalling. Taken together, our findings
suggest that at least a subgroup of HER2-amplified
breast cancers may harbour ‘PI3K–AKT pathway
addiction’ and are consistent with the observation that
the PI3K/mTOR dual inhibitor NVP-BEZ235 induces
cell death in breast cancer cells with HER2 gene
amplifications (Brachmann et al., 2009). The mechanisms leading to PI3K–AKT independence in a subgroup
of HER2-amplified breast cancers merits further
investigation.
Both cell–ECM interactions and 3D morphology of
breast cancer cells have been reported to modulate
signalling pathway activation and drug response (Weaver et al., 2002; Serebriiskii et al., 2008; Weigelt and
Bissell, 2008). Interestingly, breast cancer cell lines
grown in 3D ECM culture models or as floating
aggregates without the supply of exogenous ECM (that
is poly-HEMA cultures) showed differential response to
the HER2 targeting antibodies trastuzumab and/or
pertuzumab compared with cells grown in conventional
2D models (Pickl and Ries, 2009; Weigelt et al., 2010).
In this study, we assessed the response of a panel of
breast cancer cells to mTOR inhibition when grown in
3D ECM models. Similarly to in 2D cultures, cell lines
harbouring PIK3CA mutations were selectively sensitive
to everolimus and PP242 in 3D growth conditions.
These data suggest that the ECM signals upstream of
PIK3CA gene mutations tested in this study have a
negligible effect on the dependence of breast cancer cells
on the PI3K–AKT–mTOR signalling pathway. Importantly, our results suggest that 2D models may be
adequate to investigate the genotypic–phenotypic correlations between mutations in components of this
signalling pathway and response to mTOR inhibitors.
In conclusion, in this study we demonstrate that
breast cancer cell lines with PIK3CA mutations but not
lack of PTEN function are selectively sensitive to
allosteric mTORC1 and active-site mTORC1/2 inhibitors independent of the cell culture environment. In
addition, we showed that a subset of HER2-amplified
breast cancer cell lines have increased sensitivity to the
mTOR kinase inhibitor PP242 but not to the rapalogue
everolimus. Our results provide circumstantial evidence
to suggest that PIK3CA mutations rather than PTEN
loss of protein expression by immunohistochemical
analysis should be investigated as a predictive marker
to guide the selection of breast cancer patients who
would benefit from mTOR inhibitor therapy in future
clinical trials.
Oncogene
Materials and methods
Cell culture and drug treatment
Human breast cancer cell lines were obtained as NCI-ICBP45
kit procured through American Type Culture Collection
(ATCC) (ATCC Breast Cancer Cell Panel, Manassas, VA,
USA). Cell lines were authenticated by ATCC using short
tandem repeat DNA profiling, and each cell culture was
examined by light microscopy and compared with images
published by ATCC and the Integrative Cancer Biology
Program (ICBP; http://icbp.lbl.gov/breastcancer/celllines.php)
to verify identity. For a detailed list of the 31 breast cancer cell
lines and growth conditions used, see Supplementary Table 2.
For drug treatment in 2D monolayers, cells were inoculated
into 96-well microtiter plates in 100 ml at plating densities
ranging from 2500 to 25 000 cells/well depending on the
doubling time and cell size of individual cell lines. After 24 h,
cells were treated with serial dilutions (100 pM to 10 mM) of
everolimus (RAD001; LC Laboratories, Woburn, MA, USA),
PP242 (gift from K Shokat, UCSF; Sigma-Aldrich, Dorset,
UK), U0126 (Cell Signaling Technology, New England
Biolabs, Hitchin, UK) or AZD6244 (Axon Medchem,
Groningen, The Netherlands). For combination mTOR and
MEK inhibition, 10 mM everolimus or 1 mM PP242 were added
to serial dilutions (100 pM to 10 mM) of U0126 or AZD6244,
respectively. Cell viability was assessed after 72 h of treatment
by incubation with CellTiter-Blue (Promega, Southampton,
UK) for 1.5 h; fluorescence was read on an EnVision 2102
plate-reader (Perkin-Elmer, Waltham, MA, USA). The drug
dose required for survival of 50% of cells relative to untreated
cells (surviving fraction 50, SF50 (Turner et al., 2010)) was
determined using Graphpad Prism version 5.0c. For drug
treatment in 3D cell cultures, cells were seeded in 96-well plates
on top of growth factor reduced phenol red-free Engelbreth–
Holm–Swarm tumour matrix (Matrigel, BD Biosciences,
Bedford, MA, USA) in 100 ml of their respective medium with
5% Matrigel at densities ranging from 3000 to 5000 single cells/
well depending on the doubling time of individual cell lines (Lee
et al., 2007; Weigelt et al., 2010). Serial dilutions of everolimus or
PP242 (100 pM to 10 mM) were added 4 days after plating, and cell
viability was assessed after 72 h of treatment using CellTiter-Blue.
For combined mTOR and MEK inhibition, cells were treated
with serial dilutions (100 pM to 10 mM) of U0126 or AZD6244
alone, or in combination with 10 mM everolimus or 1 mM PP242,
respectively, for 120 h given the decreased level of proliferation of
cells grown on top of Matrigel.
Western blot analysis and protein quantification
Cells were rinsed with phosphate-buffered saline and lysed in
NuPAGE LDS Sample Buffer (Invitrogen, Paisley, UK).
Samples were resolved on 4–12% gradient NuPAGE Novex
Bis-Tris gels (Invitrogen) and proteins were transferred onto
a nitrocellulose membrane (Whatman, Dassel, Germany).
Membranes were blocked for 1 h in 5% BSA at room
temperature and probed overnight at 4 1C with primary antibody
in 1% BSA. Antibodies against poly (ADP-ribose) polymerase
(#9532), b-actin (#4967), PTEN (#9188), AKT (#9272), phospho-AKT (Ser473; #9271), S6 Ribosomal Protein (#2217),
phospho-S6 Ribosomal Protein (#2211), 4E-BP1 (#9452),
phospho-4E-BP1 (Thr37/46; #9459), ERK1/2 (#9102) and
phospho-ERK1/2 (Thr202/Tyr204; #9101) were obtained at Cell
Signaling Technology, anti-a-tubulin (clone B-5-1-2) was obtained at Sigma-Aldrich (St Louis, MO, USA). After incubation
with horseradish peroxidase-conjugated secondary antibody in
5% skimmed milk, proteins were detected using chemiluminescence (GE Healthcare, Little Chalfont, UK).
PIK3CA mutation predicts mTOR inhibitor response
B Weigelt et al
3231
For quantification of phospho-ERK1/2 and ERK1/2
protein bands, samples were resolved on 4–12% gradient
NuPAGE Novex Bis-Tris gels and proteins were transferred
onto an immobilon polyvinylidene difluoride fluorescence
membrane (Millipore, Billerica, MA, USA). Membranes
were dried and blocked for 1 h in Odyssey Blocking Buffer
(LI-COR, Cambridge, UK) at room temperature and probed
overnight at 4 1C simultaneously with anti-ERK1/2 (#9107)
and anti-phospho-ERK1/2 (Thr202/Tyr204; #9101; Cell Signaling Technology) in Odyssey Blocking Buffer. After incubation with IRDye 800CW Goat anti-Mouse IgG (926-32210)
and IRDye 680LT Goat anti-Rabbit IgG (926-68021) secondary antibodies in Odyssey Blocking Buffer, membranes were
scanned using the Odyssey Infrared Imaging System and
analysed and quantified using the 500 Odyssey Software.
Apoptosis and cell-cycle analysis
For cell-cycle analysis, cells were treated after 24 h of seeding
in 10 cm dishes with everolimus or PP242 at indicated
concentrations or vehicle (dimethyl sulfoxide). After 48 h, cells
were harvested, fixed in cold 70% ethanol, stained using
propodium iodide (Sigma-Aldrich, Dorset, UK), and DNA
content measured on a LSRII flow cytometer (BD Biosciences). Cell-cycle distribution was assessed using FlowJo
9.0.1 software. For analysis of apoptosis induction, cells were
treated after 24 h of seeding with everolimus or PP242 at
indicated concentrations or vehicle (dimethyl sulfoxide) for
48 h or Staurosporin (positive control; Sigma-Aldrich) for 3 h
and analysed by western blotting for poly (ADP-ribose)
polymerase cleavage. Alternatively, cells were treated after
24 h of plating in 96-well microtiter plates with everolimus,
PP242, vehicle (dimethyl sulfoxide) or Etoposide (positive
control; Sigma-Aldrich) at indicated concentrations. After
48 h, cell viability was determined using CellTiter-Blue by
incubation with cells for 1.5 h, and apoptosis induction
using the Apo-ONE Caspase-3/7 assay (Promega) by incubation of cells for 5 h. Fluorescence was read on an EnVision
2102 plate-reader.
PIK3CA and PTEN mutation analysis
Mutation analysis was performed of the complete coding
sequence of PTEN (NM_000314), and of exons 2, 4–11 and
19–21 of PIK3CA (NM_006218). Cells were rinsed with
phosphate-buffered saline and total RNA was extracted using
RNA-Bee (Ams Biotechnology, Milton, UK) following the
manufacturer’s instructions. cDNA synthesis of 2 mg total
RNA was performed using the SuperScript VILO cDNA
Synthesis Kit (Invitrogen). Three and four PCRs were
performed for amplification of the PTEN and PIK3CA
transcripts, respectively (see Supplementary Table 1 for primer
sequences) using the Platinum Taq DNA Polymerase High
Fidelity (Invitrogen) according to the manufacturer’s instructions. PCR specificity was assessed by agarose gel electrophoresis. For sequence analysis, amplified products were
sequenced in both directions with the BigDye Terminator
v3.1 using an ABI3730 DNA Analyser (Applied Biosystems,
Foster City, CA, USA) (Supplementary Table 1). Sequences
were analysed using Mutation Surveyor software (Softgenetics,
State College, PA, USA).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Miriam Molina Arcas, Ralph Fritsch, Elza de Bruin,
Charles Swanton (CRUK London Research Institute) and
Maryou Lambros and Jorge Reis-Filho (Breakthrough Breast
Cancer Centre, London, UK) for helpful discussions, technical
advice or critical reading of the manuscript, members of the
LRI Equipment Park for sequencing and of the LRI FACS
facility for cell-cycle analysis. We thank Morri Feldman and
Kevan Shokat (UCSF) for providing PP242. This work was
funded by Cancer Research UK.
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Oncogene