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Combination treatment
with a PI3K/Akt/mTOR
pathway inhibitor overcomes
resistance to anti‑HER2 therapy
in PIK3CA‑mutant HER2‑positive
breast cancer cells
Yumi Fujimoto1,5, Tomoko Yamamori Morita2,5, Akihiro Ohashi2, Hiroshi Haeno2,3,
Yumi Hakozaki2, Masanori Fujii2, Yukie Kashima2, Susumu S. Kobayashi2,4* &
Toru Mukohara1*

Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) are
observed in 15–20% of breast cancers (HER2+ breast cancers), and anti‑HER2 therapies have
significantly improved prognosis of patients with HER2+ breast cancer. One resistance mechanism
to anti‑HER2 therapies is constitutive activation of the phosphoinositide 3‑kinase (PI3K) pathway.
Combination therapy with small‑molecule inhibitors of AKT and HER2 was conducted in HER2+
breast cancer cell lines with or without PIK3CA mutations, which lead to constitutive activation of
the PI3K pathway. PIK3CA mutations played important roles in resistance to single‑agent anti‑HER2
therapy in breast cancer cell lines. Combination therapy of a HER2 inhibitor and an AKT inhibitor, as
well as other PI3K pathway inhibitors, could overcome the therapeutic limitations associated with
single‑agent anti‑HER2 treatment in PIK3CA‑mutant HER2+ breast cancer cell lines. Furthermore,
expression of phosphorylated 4E‑binding protein 1 (p4EBP1) following the treatment correlated with
the antiproliferative activities of the combination, suggesting that p4EBP1 may have potential as a
prognostic and/or efficacy‑linking biomarkers for these combination therapies in patients with HER2+
breast cancer. These findings highlight potential clinical strategies using combination therapy to
overcome the limitations associated with single‑agent anti‑HER2 therapies in patients with HER2+
breast cancer.

Gene amplification, overexpression, and mutation of human epidermal growth factor receptor 2 (HER2), a
transmembrane tyrosine kinase, are observed in approximately 20% of breast cancers and are associated with
poor prognosis1,2. Oncogenic activation of HER2, driven by these gene alternations, constitutively activates down-
stream signaling pathways, such as the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin
(mTOR) pathway (also called the PI3K pathway), which is involved in metabolism, growth, survival, and protein
synthesis3, and the RAS/RAF/mitogen-activated ERK kinase (MEK)/extracellular signal-regulated kinase (ERK)
pathway (also called the mitogen-activated protein kinase [MAPK] pathway), which is involved in gene expres-
sion, mitosis, differentiation, proliferation, and cell survival/apoptosis4. During the last two decades, develop-
ment of anti-HER2 therapies has significantly improved the prognosis of patients with HER2-positive (HER2+)
breast cancer both at early and metastatic disease stages5; however, some patient populations demonstrate clinical

OPEN

1Department of Breast and Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan. 2Division of
Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa,
Japan. 3Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The
University of Tokyo, Kashiwa, Japan. 4Department of Medicine, Beth Israel Deaconess Medical Center/Harvard
Medical School, Boston, MA, USA. 5These authors contributed equally: Yumi Fujimoto and Tomoko Yamamori
Morita. *email: sukobaya@east.ncc.go.jp; tmukohar@east.ncc.go.jp

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resistance to anti-HER2 therapies6. One mechanism of resistance to anti-HER2 therapies is the constitutive
activation of the PI3K pathway, which can occur through mutations/amplification of the PI3K p110α subunit
(PIK3CA), loss of PTEN, and mutations/amplification of AKT7,8. Among these oncogenic alternations, PIK3CA
mutations are observed in approximately 20–30% of patients with breast cancer, and cause resistance to anti-
HER2 therapies in both preclinical and clinical settings9–16.

Several chemical inhibitors targeting the PI3K pathway have been developed as candidate anticancer
therapies17. Evidence from preclinical and clinical studies suggests that combination treatment with anti-HER2
therapies and PI3K pathway inhibitors may have potential efficacy in HER2+ breast cancers with PIK3CA
mutations18–20. However, clinical proof of concept for these combination therapies requires confirmation in
further studies. Of these novel agents, AKT inhibitors have attracted attention as next-generation PI3K pathway
inhibitors. Ipatasertib is an ATP-competitive small molecule pan-AKT inhibitor (AKT1, AKT2, and AKT3)21,
and clinical proof of concept has been confirmed in a phase II clinical trial in which ipatasertib significantly
improved progression-free survival (PFS) compared with placebo when combined with paclitaxel in patients
with advanced triple negative breast cancers with PIK3CA mutation or PTEN loss22. Taken together, these data
suggest that AKT inhibitors may have clinical potential in combination with anti-HER2 therapy, and that this
combination may overcome the limitations associated with anti-HER2 therapy in patients with HER2+ breast
cancer carrying PIK3CA mutations, and a highly-activated PI3K pathway.

In this preclinical study, we investigated combination therapy with small-molecule inhibitors of AKT and
HER2 to overcome limitations associated with anti-HER2 monotherapy in HER2+ breast cancer cell lines with
PIK3CA mutations. We also demonstrated that expression of phosphorylated 4E-binding protein 1 (p4EBP1),
a downstream target of the PI3K pathway, may have potential as an efficacy-linking marker of combination
treatment with AKT and HER2 inhibitors in patients with HER2+ breast cancer with PIK3CA mutations. These
preclinical findings support the therapeutic potential of combination treatment with an AKT inhibitor and HER2
therapies in patients with HER2+ breast cancer carrying PIK3CA mutations.

Results
Analysis of overall survival stratified by PI3K pathway status in patients with HER2+ breast
cancer. Although several trials have reported that patients with PIK3CA mutant have poor prognosis as
previously mentioned, some trials have reported that PIK3CA mutations were not significantly associated with
resistance to anti-HER2 antibody therapies, such as TH3RESA trial treated with trastuzumab emtansin23, and
NeoSphere trial with pertuzumab12. The clinical significance of resistance to anti-HER2 therapies associated
with PI3K pathway activation remains unclear. To evaluate the clinical impact of a constitutively-activated PI3K
pathway in patients with PIK3CA, AKT, PIK3R1 mutation, and PTEN homozygous deletion or mutations, we
retrospectively reanalyzed a large and unbiased clinical dataset of anti-HER2 therapies in the HER2+ metastatic
or recurrent breast cancer patients24 (Supplementary Table S1). Of 186 HER2+ patients treated with anti-HER2
therapy, 44.1% possessed mutations of genes in the PI3K pathway; PIK3CA mutations were the most commonly
observed (Table 1). Patients double-positive for HER2 and ER (HER2+ /ER+) also exhibited a similar frequency
of PI3K pathway alterations (44.9%). The distribution of genomic alterations is shown in Oncoprint (Supple-
mentary Fig. 1). Median OS in HER2+ patients with PI3K pathway alterations was significantly shorter than in
those without PI3K pathway alterations (115.3 vs 79.5 months, respectively; hazard ratio, 1.82; 95% CI, 1.0–3.3;
p = 0.036) (Fig. 1a). In the HER2+/ER+ patients, in addition, the PI3K pathway alternations significantly caused
shorter mOS (115.3 vs 79.0 months, respectively; hazard ratio, 2.10; 95% CI, 1.0–4.5; p = 0.04) (Fig. 1b). These
clinical observations suggest that oncogenic alterations in the PI3K pathway are associated with poor prognosis
in patients with HER2+ breast cancer receiving HER2 therapy.

PIK3CA mutations attenuate the antiproliferative effects of a HER2 inhibitor and an AKT
inhibitor enhances the antiproliferative activity of a HER2 inhibitor in PIK3CA‑mut HER2+
breast cancer cell lines. To evaluate the effects of oncogenic activation in the PI3K pathway on anti-HER2
therapy, we performed in vitro proliferation assays using a HER2 inhibitor, lapatinib, in HER2+ breast cancer
cell lines. Given that the PI3K pathway alterations have a high impact in patients with ER+/HER2+ breast cancer
(Fig. 1), we first evaluated cell viability using ER+/HER2+ breast cancer cell lines with or without PIK3CA muta-

Table 1. Characteristics of the patient cohort selected from Razavi et al.23. *Shown as median (first and third
quartiles). **One patient has both PIK3CA and PIK3R1 mutations. ***One patient has both PIK3CA and
AKT1 mutations.

Characteristics
All patients
(n = 186)

Estrogen receptor positive
(n = 138)

Age at diagnosis*—yr 48 (40–55) 47 (39–54)

PI3K pathway status—no.

 Wild type 104 (55.9%) 76 (55.1%)

 PIK3CA mutation 68 (36.6%) 51 (37.0%)

 PIK3R1 mutation 6 (3.2%)** 3 (2.2%)

 AKT1 mutation 1 (0.5%)*** 1 (0.7%)***

 PTEN homozygous deletion or mutation 9 (4.8%) 8 (5.8%)

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tions. In this study, BT474 and MDA-MB-361 cell lines were used, which represent PIK3CA-wt and PIK3CA-
mut (E545K) ER+/HER2+ breast cancer cell lines, respectively. BT474 cells possess a non-canonical PIK3CA
mutation at K111N, which would preclinically and clinically contributes to less, or exceptionally-mild, activa-
tion of PI3K and its down-stream pathways22,25. (Supplementary Fig.  2). Due to the limited numbers of the
commercially-available ER+/HER2+ breast cancer cell line, we used BT474 cells as a surrogate cell line model
for PIK3CA-wt ER+/HER2+ breast cancer cell. Since a combination of ER and HER2 therapy is clinically-used
for the treatment of patients with ER+/HER2+ breast cancer, we also applied an ER inhibitor fulvestrant and a
HER2 inhibitor lapatinib to the preclinical studies of in vitro cell viability assays. Lapatinib was also selected as
a HER2 inhibitor in the following studies, because the preclinical studies using lapatinib are well established due
to its usability. In addition, although lapatinib is a dual EGFR/HER2 inhibitor, the EGFR signaling is expected to
have little contribution to proliferative activities in HER2+ breast cancer cells26,27.

BT474 and MDA-MB361 cells were treated with fulvestrant alone, lapatinib alone, or a combination of
both for 0, 2, 4, and 8 days. The viability of drug-treated cells was determined by assessing intercellular ATP
levels (Fig. 2a). Protein expression of ER and phosphorylation of HER2 at Tyr1221/1222 (pHER2) were used to
confirm target engagement for fulvestrant and lapatinib, respectively (Fig. 3a). Phosphorylated ERK (pERK) at
Thr202/204 was used as a biomarker of signal modulation within the MAPK pathway. Phosphorylated 4EBP1 at
Ser65 (p4EBP1) and phosphorylated S6 at Ser235/236 (pS6) were used as biomarkers of signal modulation within
the PI3K pathway. Immunoblotting analysis confirmed that fulvestrant and lapatinib treatments potently inhib-
ited their cellular targets in both BT474 and MDA-MB-361 cells (Fig. 3a). Consistent with previous reports28,29,
pHER2 and/or its downstream targets (pERK or p4EBP1) were mildly upregulated in fulvestrant-treated cells
(Fig. 3a).

The cell viability assay revealed that treatment with fulvestrant alone exerted on no antiproliferative effects in
BT474 cells (Fig. 2a, left panel, green line,), while treatment with lapatinib alone or in combination of lapatinib
and fulvestrant demonstrated potent antiproliferative effects in a time-dependent manner, leading to growth
regression (Fig. 2a, left panel, blue and red lines,). In those cells, treatment with lapatinib alone or in combina-
tion with fulvestrant effectively inhibited pERK and p4EBP1, both of which are downstream targets of HER2.
In contrast, lapatinib alone or in combination with fulvestrant showed limited antiproliferative effects and failed
to induce tumor regression in MDA-MB-361 cells (Fig. 2a, right panel, blue and red lines). Comparison of the
effect in each drug treatment between BT474 and MDA-MB-361 cell lines (Fig. 2b) also revealed that lapatinib
treatment in either a single-agent (Fig. 2b, lower-left panel) or a fulvestrant-combination (Fig. 2b, lower-right
panel) significantly induced a highly-potent anti-proliferative effects in BT474 cells, while the growth rates in
DMSO treatment were equivalently (Fig. 2b, upper-left panel). Immunoblotting analysis revealed that, although

a

b

0 50 100 150 200
0

50

100

patients with HER2 positive breast cancer

Months
P

ro
po

rti
on

of
O

ve
ra

ll
S

ur
vi

va
l(

%
)

PI3K pathway wild type (n=104)

PI3K pathway mutation (n=82)
Hazard ratio, 1.82 (95% CI: 1.0 to 3.3)
P=0.036

0 50 100 150 200
0

50

100

patients with HER2 positive / ER positive breast cancer

Months

P
ro

po
rti

on
of

O
ve

ra
ll

S
ur

vi
va

l(
%

)

PI3K pathway wild type (n=76)

PI3K pathway mutation (n=62)

Hazard ratio, 2.10 (95% CI: 1.0 to 4.5)
P=0.04

Figure 1. Mutations in the PI3K pathway are associated with poor overall survival for patients with HER2-
positive (HER2+) and HER2 and ER double-positive (HER2+/ER+) breast cancer. (a) Overall survival (OS)
was analyzed in patients with HER2+ breast cancer with (red) or without (blue) mutations in the PI3K
pathway. Median OS was 115.3 (blue) vs 79.5 months (red), respectively (hazard ratio: 1.82; 95% CI 1.0–3.3,
p value = 0.036). (b) OS was analyzed in patients with HER2+/ER+ breast cancer with (red) or without (blue)
mutations in PI3K pathway. Median OS was 115.3 (blue) vs 79.0 months (red), respectively (hazard ratio: 2.10;
95% CI: 1.0–4.5, p value = 0.04).

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Figure 2. The antiproliferative effect of treatment with fulvestrant and lapatinib is limited in MDA-MB-361
cell line. (a) Antiproliferative activity of fulvestrant and lapatinib, alone and in combination, in ER+/HER2+
breast cancer cell lines with and without PIK3CA mutations. BT474 (left) and MDA-MB-361 (right) cells were
used as representative PIK3CA-wild-type and PIK3CA-mutant ER+/HER2 breast cancer cell lines, respectively.
Cells were treated with DMSO (black), fulvestrant (100 nM, green), or lapatinib (100 nM, blue), alone and in
combination (red), for 0, 2, 4, and 8 days (mean ± standard deviation [SD; n = 3]). Relative levels of ATP were
calculated by chemiluminescence assay and compared with the chemiluminescence of DMSO on day 0. (b)
Comparison of the effect in each drug treatment between BT474 and MDA-MB-361 cell lines. Differences on
day 8 were analyzed using Student’s t-test. The data represent the mean ± SD (n = 3). F, Fulvestrant; L, Lapatinib.

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Figure 3. Lapatinib suppresses 4EBP1 phosphorylation insufficiently in MDA-MB-361 cells and the antiproliferative effect of
lapatinib is limited in HER2+ breast cancer cell lines with PIK3CA mutations. (a) Inhibitory effects of fulvestrant (100 nM) and
lapatinib (100 nM), alone and in combination treated for 24 h, on 4EBP1 phosphorylation in ER+/HER2+ breast cancer cell lines with
and without PIK3CA mutation. Full-length blots are presented in Supplementary Fig. 3. (b) Newly synthesized protein in DMSO-,
fulvestrant-, lapatinib-, and combination-treated cells. BT474 (upper) and MDA-MB-361 (lower) cells were treated with the indicated
inhibitors for 24 h; Click-iT HPG was incorporated into the drug-treated cells for 15 h. Green indicates the newly synthesized proteins
detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. (c) PIK3CA mutation, ER expression, HER2
amplification, PTEN Loss and PIK3R1 status of the breast cancer cell lines used in this study. Information on gene modification and
expression was obtained from the COSMIC database and ATCC information. (d) Antiproliferative activity of combined fulvestrant
and lapatinib in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3,
and ZR-75-30; red bars) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; blue bars) HER2+ breast cancer cell
lines were used. The cells were treated with fulvestrant and lapatinib in combination for 8 days (mean ± SD [n = 3]). Y-axis indicates
relative amounts of ATP (%), which were calculated with a chemiluminescence assay and compared with the chemiluminescence value
of DMSO treatment on day 8. The data represent the mean ± SD (n = 3). (e) Inhibitory effects of fulvestrant (100 nM) and lapatinib
(100 nM) in combination treatment for 24 h, on 4EBP1 phosphorylation in HER2+ breast cancer cell lines with and without PIK3CA
mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30) (left panel) and three PIK3CA-mutant (UACC893, HCC1954,
and MDA-MB-361) (right panel) HER2+ breast cancer cell lines were used. Full-length blots are presented in Supplementary Fig. 4.

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pHER2 was completely inhibited, substantial amounts of p4EBP1 and pS6 were present in MDA-MB-361 cells
treated with lapatinib alone or in combination with fulvestrant. In MDA-MB-361 cells, furthermore, expression
level of pERK was relatively low and pERK modification was also less affected in treatment with lapatinib, imply-
ing that proliferation in MDA-MB-361 cells may be more addicted to the PI3K pathways than the ERK pathway.
These results suggest that lapatinib failed to completely inhibit PI3K signaling, as the PIK3CA mutation leads
to constitutive activation of the PI3K pathways in MDA-MB-361 cells, thus resulting in less inhibition, or only
partial inhibition, of p4EBP1 and pS6.

Since p4EBP1 positively regulates protein synthesis though Eukaryotic translation initiation factor 4E
(eIF4E)7,21, we next determined how newly-synthesized proteins are modulated by lapatinib to identify differ-
ence between BT474 and MDA-MB-361 cells. The results of the Click-iT assay revealed the presence of newly-
synthesized proteins in MDA-MB-361 cells following treatment with lapatinib; conversely lapatinib alone or in
combination with fulvestrant potently inhibited protein synthesis in BT474 cells (Fig. 3b, upper). These data
suggest that inactivation of HER2 kinase by lapatinib fails to fully inhibit PI3K signaling, and partial activation
of PI3K attenuates antiproliferative activity of lapatinib in PIK3CA-mut cells. Furthermore, to confirm that
constitutive activation of PI3K signaling caused by PIK3CA-mutations attenuates the antiproliferative effects of
HER2 inhibition regardless of ER status, we tested four additional ER−/HER2+ breast cancer cell lines: SK-BR-3
cells and ZR-75-30 cells (PIK3CA-wt/ER−/HER2+), and UACC893 cells and HCC1954 cells (both PIK3CA-
mut [H1047R]/ER−/HER2+) (Fig. 3c). The results of a cell viability assay revealed that antiproliferative activity
measured by ATP was more potent in PIK3CA-wt/HER2+ breast cancer cell lines (Fig. 3d, red bars) than in
PIK3CA–mut HER2+ breast cancer cell lines (Fig. 3d, blue bars). Additionally, inhibitory effect of lapatinib on
p4EBP1 was evaluated in these cell lines (Fig. 3e). In accordance with the results in BT-474 and MDA-MB-361
cells (Fig. 3a), lapatinib treatment also exhibited less inhibition of p4EBP1 in the PIK3CA–mut HER2+ cells
compared to the PIK3CA–wild HER2+ cells (Fig. 3e). Taken together, these results demonstrate that the residual
expression of p4EBP1 in the post-treatment, which is mediated by PIK3CA mutations, would attenuate the
antiproliferative effects of lapatinib in both ER+/HER2+ and ER−/HER2+ breast cancer cell lines, supporting
the clinical results demonstrating relatively poor prognosis in patients with HER2+ breast cancer patients with
active mutations in the PI3K pathway regardless of the status of ER expression (Fig. 1).

Next, we determined whether pharmacological inhibition of the PI3K pathway would enhance the antipro-
liferative effects of lapatinib in PIK3CA-mut ER+/HER2+ breast cancer cell lines. For this, BT474 and MDA-
MB-361 cells were treated with fulvestrant, lapatinib, and ipatasertib alone or in various combinations for 0,
2, 4, and 8 days (Supplementary Fig. 5). Combination treatment with fulvestrant and lapatinib inhibited the
proliferation of BT474 cells in a time-dependent manner (Fig. 4a, left panel, blue line); however, addition of
ipatasertib had little impact when a triple combination was used (Fig. 4a, left panel, red line). In contrast, addition
of ipatasertib to the triple combination markedly decreased cell viability and led to cellular regression in MDA-
MB-361 cells compared with those treated with the double combination of fulvestrant and lapatinib (Fig. 4a right
panel, blue vs. red lines). Comparison of the effect in each drug treatment between BT474 and MDA-MB-361
cell lines (Fig. 4b) also revealed that the triple combination induced a highly-potent anti-proliferative effects
in MDA-MB-361 cells, equivalently to, or even more than, the anti-proliferative effects in the double- or the
triple-combination in BT474 cells (Fig. 4b). The results of the crystal violet assay also demonstrated that the
triple combination inhibited the proliferation of MDA-MB-361 cells, overcoming the limited antiproliferative
effects observed following treatment with the double combination (Fig. 4c,d, and supplementary Fig. 6a,b).

Figure 4. Ipatasertib enhances the antiproliferative activity of fulvestrant and lapatinib combination in a
PIK3CA-mutant HER2+/ER+ breast cancer cell line. (a) Antiproliferative activity of BT474 (PIK3CA-wild-type,
left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black), combination of fulvestrant
(100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant, lapatinib, and ipatasertib
(1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were compared with the
chemiluminescence value of DMSO treatment on day 0. (b) Comparison of the inhibitory effect in each drug
treatment between BT474 and MDA-MB-361 cell lines. Differences on day8 were analyzed using Student’s
t-test. The data represent the mean ± SD (n = 3). (c) Representative images of crystal violet staining in BT474 or
MDA-MB-361 cells treated with DMSO, the combination of fulvestrant (100 nM) and lapatinib (100 nM), and
the triple-combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) for 8 days. (d) Quantified data from
Fig. 4c. Crystal violet absorbance indicating the amount of normalized protein was measured with a microplate
reader and compared with a DMSO control. The data represent mean ± SD (n = 3). Crystal violet absorbance
was statistically analyzed using Student’s t-test to compare the double-combination and the triple-combination
treatments. Differences were considered significant at p ≤ 0.05 (*). (e) Effect of combined fulvestrant, lapatinib,
and ipatasertib treatment on the apoptosis of BT474 (left) and MDA-MB-361 (right) cells. The cells were treated
with DMSO (black), the combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-
combination of fulvestrant, lapatinib, and ipatasertib (1000 nM) (red) for 24 h (mean ± SD [n = 3]). Relative
caspase-3/7 activities were calculated based on luminescence compared with the DMSO control. Caspase-3/7
activities were analyzed using Student’s t-test to compare the DMSO treatment and the double-combination
treatment, and the double-combination and the triple-combination treatments. Differences were considered
significant at p ≤ 0.05 (*). F, Fulvestrant; L, Lapatinib; I, Ipatasertib; n.s., not significant. (f) Representative
images of 3D culture in BT474 (left) or MDA-MB-361 (right) cells. (g) Antiproliferative activity of 3D-cultured
BT474 (PIK3CA-wild-type, left) and MDA-MB-361 (PIK3CA-mutant, right) cells treated with DMSO (black),
combination of fulvestrant (100 nM) and lapatinib (100 nM) (blue), and the triple-combination of fulvestrant,
lapatinib, and ipatasertib (1000 nM) (red) for 0, 2, 4, and 8 days (mean ± SD [n = 3]). Relative ATP amounts were
compared with the chemiluminescence value of DMSO treatment on day 0.

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Consistent with these results, the effects of ipatasertib on apoptosis as part of triple therapy were also observed
in these cell lines. The caspase-3/7 assay revealed that the double combination with fulvestrant and lapatinib
significantly elevated caspase-3/7 activity in BT474 cells (Fig. 4e, left, black vs. blue bars), while not significantly
in MDA-MB-361 cells (Fig. 4e, right panel, black vs. blue bars). In contrast, addition of ipatasertib to the triple
combination markedly significantly elevated caspase-3/7 activity in MDA-MB-361 cells (Fig. 4e, right panel, blue
vs. red bars, supplementary Fig. 6c, right panel), while exhibited no significant change in BT474 cells (Fig. 4e,
left panel, blue vs. red bars, supplementary Fig. 6c, left panel). Additionally, we conducted antiproliferation
studies in a 3D culture system (Fig. 4f ), which also demonstrated the potent antiproliferation in treatment with
the triple combination in MDA-MB-361 cells, overcoming the limited antiproliferative effects with the double
combination (Fig. 4g, supplementary Fig. 5c,d). These results suggest that combination treatment with lapatinib
and ipatasertib could be effective against PIK3CA-mutant HER2+ breast cancers, overcoming the therapeutic
difficulty in the currently-used cancer drugs against these cancers.

Next, we examined the signaling pathways downstream of HER2 in the PIK3CA-wild-type or the PIK3CA-
mutant HER2+ breast cancer cell lines treated with each inhibitor alone or in combination. As reported
previously30, our studies also revealed that ipatasertib notably elevated phosphorylated AKT at Ser-473 (pAKT)
in a dose-dependent manner in both BT474 and MDA-MB-361 cells (Fig. 5a and Supplementary Fig. 7). This
paradoxical observation may be explained by ipatasertib binding to the active site in AKT, subsequently pro-
tecting these sites from phosphatases and increasing AKT phosphorylation21,31, while the downstream of Akt
pathway is expected to be suppressed in treatment with ipatasertib30. Therefore, AKT phosphorylation does not
reflect its activation. In BT474 cells, lapatinib alone or in combination with fulvestrant potently inhibited both
pERK and p4EBP1, and no additional effect of ipatasertib was detected (Fig. 5a, left panel, lane 3, 7 and lane
8). In contrast, in MDA-MB-361 cells, lapatinib treatment as a single-agent or in combination with fulvestrant
potently inhibited pERK; however, a substantial amount of p4EBP1 was still detected (Fig. 5a, right panel, lane
3 and lane 7). Furthermore, addition of ipatasertib to lapatinib and fulvestrant markedly decreased p4EBP1
expression (Fig. 5a, right panel, lane 7 and lane 8). Results of the Click-iT assay also revealed that, although sig-
nals for newly-synthesized proteins were detected in MDA-MB-361 cells treated with the double combination,
ipatasertib treatment as part of triple therapy reduced these signals to the detection limit (Fig. 5b,c). In BT474
cells, combination treatment with fulvestrant and lapatinib effectively inhibited the synthesis of new proteins
(Fig. 5b,c). Dephosphorylated 4EBP1 binds to eIF4E to inhibit the complex formation of eIF4E–eIF4G–eIF4A
subunits, and suppresses cap-dependent protein translation32. Next, to determine the effects of the triple combi-
nation treatment on the complex formation of these translation subunits, we performed a pulldown assay with
a γ-aminophenyl-7-methyl guanosine (m7 GTP) …

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