Co‑targeting PI3K/Akt and MAPK/ERK pathways leads to an enhanced
antitumor efect on human hypopharyngeal squamous cell carcinoma

Purpose The present study aims to determine whether co-targeting PI3K/Akt and MAPK/ERK pathways in human
hypopharyngeal squamous cell carcinoma (HSCC) is a potential anticancer strategy.
Methods We retrospectively analyzed the clinical data of HSCC patients, and the phosphorylation status of Akt and Erk in
HSCC and tumor adjacent tissues was evaluated by immunohistochemistry. MTT and colony formation assay were performed
to determine the anti-proliferative efect of PI3K/mTOR inhibitor GDC-0980 and MEK inhibitor Refametinib on HSCC
cell line Fadu. Wound-healing and Transwell migration assay were used to analyze the anti-migrative capability of the two
drugs. The involved anti-tumor mechanism was explored by fow cytometry, qRT-PCR and western blot. The combinational
anticancer efect of GDC-0980 and Refametinib was evaluated according to Chou and Talalay’s method.
Results The levels of p-Akt and p-Erk were increased signifcantly with the progression of clinical stage of HSCC, suggest￾ing PI3K/Akt and MAPK/ERK pathways might be associated with HSCC occurrence and progression. Furthermore, both
GDC-0980 and Refametinib showed obvious antitumor efects on FaDu cells. Treatment by the two drugs arrested FaDu
cell cycle progression in G1 phase, with reduction of cyclin D1 and p-Rb, in contrast to enhancement of p27. GDC-0980
inhibited FaDu cell migration and reduced metastasis related proteins including p-PKCζ, p-Integrin β1 and uPA. Combina￾tion use of GDC-0980 and Refametinib exhibited strong synergistic anti-tumor efect.
Conclusion Dual inhibition of PI3K/Akt and MAPK/ERK pathway by GDC-0980 and Refametinib might be a promising
treatment strategy for HSCC patients.
1 Tianjin Key Laboratory on Technologies Enabling
Development of Clinical Therapeutics and Diagnostics,
School of Pharmacy, Tianjin Medical University,
300070 Tianjin, China
2 Department of Otorhinolaryngology Head and Neck Surgery,
Tianjin First Central Hospital, 300192 Tianjin, China
3 School of Medicine, Tianjin Tianshi College, Tianyuan
University, 301700 Tianjin, China
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therapy generally combines surgery, radiotherapy and
chemotherapy. However, even with these treatments, the
overall survival rate of HSCC patients remains less than
50% (Newman et al. 2015). In recent years, personalized
treatment and molecular targeting anti-tumor agents have
gain great achievements in oncotherapy. With the rapid
development of genomics and proteomics, more and more
potential therapeutic targets have been identifed (Wang
et al. 2015; Ren et al. 2016). Therefore, to understand the
pathogenesis and develop efective therapeutic approaches
for HSCC are imperative.
The phosphatidylinositol-3 kinase (PI3K) pathway is a
key signal transduction pathway that mediates multiple cel￾lular functions critical to cancer initiation, progression, and
outcome, including growth, metabolism, migration, inva￾sion, and angiogenesis (Shaw et al. 2006; Mayer and Arteaga
2016). Hyperactivation of PI3K/Akt/mTOR signaling cas￾cades has been identifed in a variety of human cancers,
making the enzymes of this pathway ideal cancer targets
(Thorpe et al. 2015). PI3K signaling pathway has attracted
attention as promising target for HNSCC therapy, since PI3K
overactivation is observed in up to 60% of HNSCC cases
(Kundu and Nestor 2012; Machiels and Schmitz 2011). As
reported, dual inhibition of PI3K and mTOR signifcantly
suppressed the growth of implanted tumor in HNSCC xeno￾graft model, further supporting PI3K as a target in HNSCC.
Currently, two PI3K inhibiters, BYL719 and BKM120 are
under investigation on HNSCC patients in diferent stages
(Massacesi et al. 2016). However, the role of PI3K pathway
in and the efcacy of PI3K inhibitors on HSCC are still
On the other hand, the mitogen-activated protein kinase/
extracellular signal-regulated (MAPK/ERK) cascade is also
important for human cancer cell growth, survival, and difer￾entiation (De Luca et al. 2012). PI3K/Akt and MAPK/ERK
pathways were reported to be co-activated in HNSCC (Ban￾croft et al. 2001). MAPK/ERK pathway consists a series
of proteins including Ras, Raf, MEK and ERK. Therapies
targeting such molecules have shown efciency for therapy
of various solid tumors such as melanoma and colon cancer
(Bancroft et al. 2001; Cargnello and Roux 2011). A cross￾talk was reported between the PI3K/Akt and MAPK/ERK
pathways in regulating cell survival and inhibition of either
pathway could result in compensatory activation of the other
(Aksamitiene et al. 2012). Inhibition of both pathways has
shown synergistic efect in the treatment of some cancers
(Renshaw et al. 2013; Van Dort et al. 2015; Williams et al.
In this study, we frst demonstrated the role of PI3K/
Akt and MAPK/ERK pathways in HSCC by comparing
the phosphorylation levels of Akt and ERK in HSCC tis￾sues with those in tumor adjacent tissues. Then the anti￾cancer efects of PI3K inhibitor GDC-0980 and MEK
inhibitor Refametinib alone or in combination on HSCC
were investigated.
Materials and methods
Reagents and antibodies
GDC-0980 and Refametinib were purchased from Sell￾eck (London, ON, Canada). MTT (3-(4, 5-Dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) rea￾gent was purchased from Amresco (Solon, OH, USA).
FITC-Annexin V/PI Apoptosis Detection Kit was obtained
from BD Biosciences (San Jose, CA, USA). Propidium
iodide (PI) was purchased from Sigma-Aldrich (St.
Louis, MO, USA). TriZol reagent was purchased from
Life Technologies (Carlsbad, CA, USA). PrimeScript™
RT Master Mix Kit and Power SYBR® Premix Ex Taq™
were obtained from Takara (Tokyo, Japan). Antibodies
against PI3K-p110α, phospho-PDK1 (Ser241), phospho￾Akt (Ser473), Akt, phospho-mTOR (Ser2448), phospho￾p70S6 K (Thr389), phospho-Erk (Thr202/Tyr204), Lamin
B and β-actin were purchased from Cell Signaling Tech￾nology (Danvers, MA, USA). Antibodies against phospho￾pRb (pS780), cyclin D1 and p27 were purchased from BD
Biosciences Pharmingen (San Jose, CA, USA). Mouse and
rabbit HRP-conjugated secondary antibodies (Cell Signal￾ing Technology, Danvers, MA, USA) were used at 1: 2000
in non-fat milk (5% in TBST).
Patients and clinical data collection
The study was performed on 55 patients who were diag￾nosed as hypopharyngeal squamous cell carcinoma
(HSCC) and were treated in Tianjin frst central hospital
of China from 2012 to 2016. The inclusion criteria were
as follows: primary hypopharyngeal squamous cell carci￾noma confrmed through histopathology, well-preserved
specimens, complete clinical records and pathologic data,
and no anti-tumor treatment before operation including
radiotherapy, chemotherapy, biotherapy. The patients
include 52 (94.5%) male and 3 (5.5%) female. The median
age at the time of the frst operation was 61 years (rang￾ing from 41 to 83). We retrospectively analyzed the clini￾cal data regarding age, gender, histological stage, clinical
stage, T stage and node metastasis. Moreover, the phos￾phorylation status of Akt and Erk on HSCC was evaluated
by immunohistochemistry. The procedures were approved
by the institutional review board (IRB) in accordance with
the ethical standards established by Tianjin frst central
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Immunohistochemistry analysis
The phosphorylation status of Akt and Erk was detected and
evaluated as we previously described (Peng et al. 2018). The
specimens were derived from surgical excision of 55 HSCC
tissues and 20 adjacent non-tumor tissues. The tissues were
fxed in 4% formalin for 20–24 h and embedded in paraf￾fn. Tissue blocks were then cut into 4 μm sections, and the
diagnosis of sections was reconfrmed by the pathologists.
Subsequently, the sections were incubated with primary
antibodies against phospho-Akt (dilution 1:50), phospho￾Erk (dilution 1:200,) and anti-mouse secondary antibody
(PV6000 Kit, ZSGB-BIO, CN). Immunohistochemistry
images were taken using Olympus BX51 microscope and
analyzed using MetaMorpheus software. Images were scored
independently by three well-trained pathologists blind to the
clinical data. Staining intensity was scored as four grades:
0 for negative, 1 for weak positive, 2 for moderate posi￾tive and 3 for strong positive. Staining extent (or positive
frequency) was also scored as four grades: 0 for absence of
staining (<1% cells), 1 for focally staining (<10% cells), 2
for variably staining (10% to 50% cells), and 3 for staining of
more than 50% cells. Composite scores were calculated by
multiplying the intensity score by the staining extent score.
Statistically, composite scores≥4 were considered as posi￾tive, and scores<4 were defned as negative.
Cell culture
Human pharyngeal squamous FaDu cell line was purchased
from the cell bank of Chinese Academy of Sciences (Shang￾hai, China). Cells were maintained in DMEM medium
(Biological Industries, Kibbutz Beit-Haemek, Israel) sup￾plemented with 10% fetal bovine serum (FBS), 100 µg/ml
streptomycin and 100 units/ml penicillin. The cells were
cultured in a humidifed atmosphere environment contain￾ing 5% CO2 at 37 °C.
Cell viability assay
Cell viability was determined using MTT assay, as described
by us previously (Wang et al. 2016a, b). Briefy, 200 μl of
FaDu cell suspension (4×104
cells/ml) was seeded in each
well of a 96-well plate and treated with indicated concen￾trations of GDC-0980 and/or Refametinib for 48 h. Subse￾quently, the cells were added with 20 μl of MTT solution
(5 mg/ml). After a further incubation of 4 h, the culture
medium was removed, and the formazan crystals were dis￾solved with 150 μl of DMSO. The resulting absorbance
at 490 nm was measured using a microplate reader iMark
(BIO-RAD, Hercules, CA, USA).
Colony formation assay
The colony formation assay was performed as we previously
described (Wang et al. 2018), with a little modifcation.
After pre-treatment with GDC-0980 and/or Refametinib for
48 h, the assay was carried out in 60-mm dishes in which
there was 4 ml of 1.8% agarose at the bottom and 3 ml of
1.8% agarose containing the drug-treated cells (1.5 ×104
cells/dish) on the top. After cells growing for 14 days, the
colonies were fxed with 4% paraformaldehyde and stained
with 0.5% crystal violet for 30 min. The number of colonies
was counted using Image J software. At least three parallel
dishes were scored for each treatment.
Flow cytometry for cell cycle distribution analysis
Cell cycle distribution was analyzed by fow cytometer as
we previously described (Wang et al. 2016a, b). FaDu cells
cells/ml) were treated with indicated concentrations
of drugs for 48 h at 37 °C. Cells were then collected, washed
with ice-cold PBS and fxed with 75% ethanol overnight at
4 °C. After centrifugation, the fxed cells were resuspended
in PI solution (25 µg/ml) containing 0.5% Triton X-100 and
2% RNase A, incubated in the dark for 60 min, and run on
BD Accuri C6 fow cytometer (BD Biosciences, San Jose,
Flow cytometry for cell apoptosis analysis
Apoptosis analysis was carried out by Annexin V-FITC/
PI double staining as we described previously (Wang et al.
2016a, b). FaDu cells (3×105
cells/ml) were treated with
indicated concentrations of GDC-0980 or Refametinib in
6-well plates for 48 h. After incubation, the cells were then
trypsinized and resuspended in 100 μl of binding bufer,
followed by incubation with Annexin V-FITC/PI solution
for 15 min in the dark at room temperature. The proportion
of apoptotic cells was detected using BD Accuri C6 fow
cytometer (BD Biosciences, San Jose, CA, USA).
Wound‑healing assay
Wound-healing assay was employed to evaluate cell migra￾tion ability as we described previously (Wang et al. 2018).
Briefy, the FaDu cells (3 × 105
cells/ml) were seeded in
24-well plates and grown until confuent state, and then the
cells were scratched by sterile tips. To remove debris, the
cell monolayer was rinsed twice with PBS. Fresh culture
medium was added with indicated concentrations of GDC-
0980 or Refametinib. Twenty four hours later, the mean
width of each scratch was measured using Image Pro Plus.
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Transwell migration assay
Transwell chambers with 8 μm pores (Costar; Corning, New
York, NY, USA) were used to perform the migration assays
as reported by us previously (Wang et al. 2018). The cells
cells/ml) treated with GDC-0980 were seeded into
the upper chamber which contained 200 μl of serum-free
medium, and the lower chamber was flled with 650 μl of
medium containing 10% FBS. The chambers were main￾tained at 37 °C, 5% CO2 for 48 h. Afterward, the unmigrated
cells were removed by cotton swabs and the inserts were
then fxed in 95% ethyl for 1 h and stained with 1% eosin for
30 min. Photographs were captured and the migrated cells
were counted in at least 3 random felds.
Quantitative real‑time PCR (qRT‑PCR)
Total RNA was extracted from FaDu cells using TriZol rea￾gent according to the manufacturer’s instructions and quanti￾fed by a Nanodrop spectrophotometer (Thermo Fisher Sci￾entifc, Waltham, MA, USA). Quantitative RT-PCR was then
performed as we previously described (Zhou et al. 2016).
Briefy, using PrimeScriptTM RT master mix kit, 500 ng
of total RNA was reverse-transcribed into cDNA. The PCR
reaction was then carried out in a system of 20 µl volume
containing 1 µl of cDNA, 0.4 µl of former primer and reverse
primer, 8.2 µl RNase Free Water and 10 µl of 2 × Power
SYBR® Premix Ex Taq™, using a CFX96™ Real-Time
PCR Detection System (BIO-RAD, Hercules, CA, USA).
The relative gene expression levels were quantifed using
the comparative Ct (ΔΔCt) method.
Western blot analysis
Western blot analysis was performed as described in our
previous report (Wang et al. 2016a, b). After incubation
with indicated drugs for 24 h, the cells were collected and
lysed using RIPA lysis bufer (Roche Diagnostics, Basel,
Switzerland). The nuclear lysates were prepared using NE￾PER Nuclear and Cytoplasmic Extraction Kit (Thermo
Fisher Scientifc, Waltham, MA, USA). Cell lysates with
equal amount of protein were subjected to 10% SDS- PAGE
and transferred onto PVDF membranes. Membranes were
then hybridized with primary antibodies, followed by incu￾bation with HRP-conjugated secondary antibodies at room
temperature for 1 h. The resulting protein bands were visual￾ized with ECL system and digitalized by scanning.
Synergism assay
The combinational anticancer effect of GDC-0980 and
Refametinib on FaDu cells was evaluated as reported
by the isobologram and Fa-CI plot based on Chou
and Talalay’s method (Zhou et  al. 2017). Briefly,
FaDu cells seeded in a 96-well plate were exposed to
GDC-0980 and Refametinib at three constant ratios
(IC50 GDC-0980:IC50 Refametinib, 0.5×IC50 GDC-0980:IC50 Refametinib,
2×IC50 GDC-0980:IC50 Refametinib) for 48 h. The cell viability
was determined using MTT assay. The combination index
(CI) was calculated according to Chou and Talalay’s method
using the CalcuSyn software. CI<1, CI=1 and CI>1 indi￾cates synergism, additivity and antagonism, respectively.
Statistical analysis
Quantitative results were analyzed by two-tailed unpaired
Student’s t test, representative of at least three independ￾ent experiments. p<0.05 was considered statistically sig￾nifcant. All statistical analyses were performed using the
GraphPad Prism 5.0 (San Diego, CA, USA). The difer￾ences were analyzed by χ2
or Fisher exact test, when appli￾cable. We explored the correlation between expression of p-Akt and p-Erk through Pearson R test.
The phosphorylation of Akt and Erk is up‑regulated
in HSCC tissues and positively correlated with tumor
It has been reported that over-activation of PI3K/Akt and/
or MAPK/ERK signaling are frequent events in human can￾cers (Martini et al. 2013; Cargnello and Roux 2011). To
investigate the role of PI3K/Akt and MAPK/ERK pathways
in the occurrence and progression of HSCC, we examined
the phosphorylation status of Akt and Erk in 55 HSCC tis￾sues and 20 normal adjacent hypopharyngeal tissues. The
phosphorylation level of Akt was signifcantly higher in
tumor tissues (54.5%) compared to normal tissues (10.0%)
(p≤0.05) (Table 1, Fig. 1a–c). Similar result was found in
Table 1 Expression patterns
of p-Akt in hypopharyngeal
squamous cell carcinoma
(HSCC) and adjacent non-tumor
Group No. p-Akt expression Positive rate (%) χ2 p value
Positive Negative
Normal tissues 20 2 18 10.0 10.146 0.001
HSCC tissues 55 30 25 54.5
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the phosphorylation status of Erk, which was higher in tumor
tissues (69.1%) than in normal tissues (25.0%) (p≤0.05)
(Table 2, Fig. 1d–f). The correlation coefcients between
the two factors were analyzed using Pearson R tests, and
no statistical correlation was observed between p-Akt and
p-Erk (Table 3). Additionally, we analyzed p-Akt and p-Erk
in patients with diferent stages and tumor pathologic fea￾tures (Tables 4 and 5). Among 55 tumor tissues, 75% (24/32)
of T3-T4 stage tumors showed membrane staining of p-Akt,
but interestingly, in 23 patients of T1-T2 stage, detection of
p-Akt was restricted to only 6 (26.1%) patients. The p-Akt
was increased signifcantly with the progression of clini￾cal stage (p=0.004). However, no association was shown
between p-Akt expression and the patients’ age, gender,
histological grades and node metastasis. In contrast, p-Erk
was positively associated with the progression of clini￾cal stage and cervical lymph node metastasis (p≤0.05).
Whereas, p-Erk expression showed no correlation with
age, gender, histological grades and T stages (p>0.05).
These results suggested PI3K/Akt and MAPK/ERK path￾ways might be critical in regulating HSCC initiation and
Inhibitory efect of GDC‑0980 and Refametinib
on cell viability of FaDu cells
To investigate whether the suppression of PI3K/Akt and
MAPK/ERK pathways can inhibit HSCC growth, we
explored the respective efect of PI3K/mTOR inhibitor￾GDC-0980 and MEK inhibitor- Refametinib on human
hypopharyngeal cancer FaDu cells. As shown in Fig. 2a,
after 48 h treatment, both GDC-0980 and Refametinib efec￾tively reduced FaDu cells viability in a dose-dependent man￾ner, with IC50 value as 3.387 μM and 14.54 μM, respec￾tively. Also, the phosphorylation level of Akt and Erk in
FaDu cells was dramatically decreased after GDC-0980 and
Refametinib treatment (Fig. 2b).
Fig. 1 Immunohistochemical analysis of p-Akt and p-Erk in HSCC
tissues and adjacent non-tumor tissues. Level of p-Akt in HSCC tis￾sues (a×100; b×200); Level of p-Akt in non-tumor tissues (c×200);
Level of p-Erk in HSCC tissues (d×100; e×200); Level of p-Erk in
non-tumor tissues (f×200)
Table 2 Expression patterns
of p-Erk in hypopharyngeal
squamous cell carcinoma
(HSCC) and adjacent non-tumor
Group No. p-Erk expression Positive rate (%) χ2 p value
Positive Negative
Normal tissues 20 5 15 25.0 9.923 0.002
HSCC tissues 55 38 17 69.1
Table 3 Correlation analysis between p-Akt and p-Erk expression
R=0.259, p=0.057
p-Erk expression
Positive Negative
p-Akt expression Positive 24 6
Negative 14 11
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GDC‑0980 and Refametinib induced cell cycle arrest
at G1 phase
To understand the anticancer mechanism of GDC-0980 and
Refametinib on FaDu cells, we examined whether cell cycle
arrest and/or apoptosis was involved in their growth inhibi￾tory efect. The results showed that both GDC-0980 and
Refametinib signifcantly induced G1 phase arrest in FaDu
cells, compared with control group (Fig. 3a and b). On the
other hand, there was no sub-G1 population was observed
Table 4 P-Akt expression
and patient clinicopathologic
characteristics in both drugs-treated groups; and the Annexin V/PI staining

assay also showed no apoptotic cell population increased,
suggesting that the two drugs did not induce apoptosis in
FaDu cells (Fig. 3c and d). Cell cycle progression is known
to be promoted by CDK (cyclin dependent kinase)-cyclin
complexes and blocked by CDK inhibitors, such as p27
(Malumbres and Barbacid 2009). We next investigated the
efect of GDC-0980 and Refametinib on cell cycle-related
proteins involved in G1/S checkpoint to explore the potential
mechanism by which the two drugs-induced G1 phase arrest.
As shown in Fig. 4a, the expression of p27 increased, and
the levels of cyclin D1 and phosphorylated pRb decreased
dose-dependently. In addition, the RNA expression levels of
p27 increased obviously after GDC-0980 and Refametinib
treatment (Fig. 4b). These data indicated that the downreg￾ulation of cyclin D1 and p-pRb, and upregulation of p27
might be involved in G1 arrest induced by GDC-0980 and
The efect of GDC‑0980 and Refametinib on the key
proteins of PI3K/Akt and MAPK/ERK pathways
Since GDC-0980 is a dual PI3K/mTOR inhibitor, and PI3K/
Akt/mTOR signaling plays a vital role in regulation of cell
cycle progression (Powles et al. 2016; Zi et al. 2015). To
further elucidate the mechanism involved in GDC-0980-in￾duced G1 phase arrest, we investigated the efect of GDC-
0980 on the key regulating proteins of PI3K/Akt pathway.
As shown in Figure S1A, a dose-dependent reduction of
p-PDK1, p-Akt, p-mTOR and p-p70S6 K was observed after
GDC-0980 treatment. The expression of the upstream of
PI3K, the catalytic subunit PI3K-p110α, was inhibited by
GDC-0980 dose-dependently as well. Also, the MAPK/ERK
cascades are essential in cell cycle regulation (Zhang and
Liu 2002). We then determined the efect of MEK inhibitor
Refametinib on the phosphorylation of Erk, the key efecter
of MAPK/ERK pathway. Figure S1B showed p-Erk was
remarkably reduced by Refametinib treatment.
GDC‑0980 inhibited FaDu cell migration
Tumor cell detachment from the primary tumor and migra￾tion into the surrounding tissue is the initiation step in the
process of tumor metastasis (Chafer and Weinberg 2011).
To test whether GDC-0980 and Refametinib can inhibit
FaDu cell migration, we frst performed wound-healing
assay with both drugs at non-cytototic concentrations. As
presented in Fig. 5a and b, migration distance of FaDu cells
was decreased with the increasing concentrations of GDC-
0980. The inhibition rate of 0.5 and 1 μM of GDC-0980
Fig. 2 Antiproliferative activities of GDC-0980 and Refametinib on
FaDu cells. a FaDu cells were treated with indicated concentrations
of GDC-0980 (a) and Refametinib for 48  h, respectively, and the
cell viabilities were determined by MTT assay. The results are presented as mean±SD, representative of three independent experiments
(n=3), compared with respective control. b The level of p-Akt, Akt and p-Erk was determined by
Western blot
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was approximately 34% and 53%, respectively. However,
the cell migration distance was not signifcantly changed
by Refametinib treatment (Fig. 5c and d). In addition, Tran￾swell migration assay was carried out to further confrm the
anti-migratory capacity of GDC-0980. As shown in Fig. 5e
and f, GDC-0980 markedly inhibited cell migration from
the upper to the lower chamber, with the inhibition rate as
61% and 84% for 0.5 and 1 μM of GDC-0980, respectively.
Furthermore, to investigate the antimigratory mechanism
of GDC-0980, we examined the efect of GDC-0980 on
several proteins that mediate cell migration as downstream
molecules of Akt. It was found that GDC-0980 inhibited
the phosphorylation of PKCζ and Integrin β1, decreased the
expression of uPA in a dose-dependent manner (Fig. 5g).
Such results indicated inhibition of PI3K/Akt pathway and
the downstream PKCζ, Integrin β1 and uPA might be related
to the antimigraory efect of GDC-0980.
Synergistic efect of GDC‑0980 and Refametinib
on FaDu cells
To examine whether co-targeting PI3K and MEK by com￾bination of GDC-0980 and Refametinib could show better
therapeutic efcacy, we carried out a combination study
using Chou and Talalay’s method. Firstly, MTT assay was
conducted to determine the inhibitory activities using a
series of drug combinations (20%, 40%, 60%, 80%, 100%
of the IC50 values of each drug). Three constant ratios of
0.5×IC50 GDC-0980:IC50 Refametinib, IC50 GDC-0980:IC50 Refametinib,
and 2 × IC50 GDC-0980:IC50 Refametinib were used. All of the
three drug combinations indicated greater growth inhibi￾tory efect than either drug alone, as shown in Fig. 6a.
Then combination index (CI) profles were prepared, and
the values at ED50, ED75 and ED90 were calculated by
CalcuSyn software and presented in Fig. 6b and Table 6.
Both combinations of 2× IC50 GDC-0980:IC50 Refametinib and
IC50 GDC-0980:IC50 Refametinib exhibited synergistic effects
(CI<1), and the former combination showed stronger syn￾ergistic efcacy. Subsequently, to confrm the anti-prolifer￾ative efect of 2×IC50 GDC-0980:IC50 Refametinib combination,
soft agar assay was carried out. Combination of GDC-0980
Fig. 3 GDC-0980 and Refametinib induced cell cycle arrest at G1
phase in FaDu cells. a FaDu cells were incubated with indicated
concentrations of GDC-0980 (0, 0.5, 1, and 5 μM) and Refametinib
(0, 5, 10, and 20 μM) for 48 h, respectively. After PI staining, fow
cytometry analysis was performed to determine cell cycle distribu￾tion. b The histograms show the percentages of cell population in G1,
S, and G2/M phases. c The apoptosis inducing efect of GDC-0980
and Refametinib on FaDu cells. Cells were treated with the indicated
concentrations of GDC-0980 and Refametinib for 48  h. The cells
were harvested, double stained with Annexin V and PI, and analyzed
with fow cytometry. d Quantifcation of the apoptotic cells. Data are
mean±SD, representative of three independent experiments (n=3)
Fig. 4 GDC-0980 and Refametinib inhibited cell cycle regulatory
proteins. a Western blot analysis of cell cycle-related proteins fol￾lowing GDC-0980 and Refametinib treatment. FaDu cells were
exposed to indicated concentrations of GDC-0980 or Refametinib for
24 h. The expression levels of cyclin D1, p27, and the phosphoryla￾tion level of pRb in the nuclei were determined. Lamin B was used
for normalization. b Quantitative RT-PCR analysis of p27 mRNA
expression. The relative gene expression levels were quantifed using
the comparative Ct (ΔΔCt) method. Data are mean±SD, representa￾tive of three independent experiments (n=3). *p<0.05, **p<0.01,
compared with respective control
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(1.356 μM) and Refametinib (2.908 μM) (the concentra￾tion of 20% of 2×IC50 GDC-0980:IC50 Refametinib) signifcantly
decreased the number of colonies, compared with either
drug alone (Fig. 6c and d). Over all, these data demonstrated
synergistic efect of combination treatment of GDC-0980
and Refametinib on FaDu cells.
Combination of GDC‑0980 with Refametinib
induced an enhanced G1 phase arrest compared
with either drug alone
To further explore the synergistic mechanism of GDC-0980
and Refametinib, we performed cell cycle analysis with PI
staining after GDC-0980/Refametinib co-treatment. The
result showed the combination treatment led to a great
increase of G1 phase cells, compared with GDC-0980 or
Refametinib alone (Fig. 7a and b). Meanwhile, the levels of
cyclin D1 and phosphorylated Rb decreased, and the expres￾sion of p27 increased (Fig. 7c). At mRNA levels, we found
GDC-0980/Refametinib co-treatment signifcantly enhanced
the expression of p27, compared with either drug alone
(Fig. 7d). Interestingly, the phosphorylation status of Akt
also reduced when treated with Refametinib alone (Fig. 7e).
Since the Ras/ERK and PI3K/AKT pathways were reported
to cross-activate each other (Mendoza et al. 2011), the inhi￾bition of ERK phosphorylation by Refametinib might also
afect PI3K/AKT pathway, therefore leading to the reduc￾tion of p-Akt. Above all, these results indicated GDC-0980/
Refametinib co-treatment induced an enhanced G1 phase
arrest, which might be attributed to co-targeting of both
PI3K/Akt and MAPK/ERK pathways.
It remains to be a challenge for oncologists to manage HSCC
in diagnosis and treatment (Kwon and Miles 2019; Newman
et al. 2015). Therefore, the identifcation of specifc markers
associated with the pathogenesis and development of HSCC
and exploration of new treatment options are critical. In the
present study, we examined the phosphorylation status of
2 key molecules known to be involved in carcinogesis, Akt
and Erk, in 55 HPC tissue samples and 20 normal adjacent
hypopharyngeal tissues. The results indicated the levels of
p-Akt and p-Erk were signifcantly higher in HSCC tissues
compared with those in non-cancerous tissues, suggesting
PI3K/Akt and MAPK/ERK pathways may play an important
role in the development of HSCC.
Both PI3K/Akt and MAPK/ERK pathways regulate cell
survival, proliferation and motility. Besides their independ￾ent signaling that provides compensatory adjustment action,
the two pathways have extensive cross-talk to regulate each
other’s activity positively or negatively (Mendoza et al.
2011; Steelman et al. 2011). Since the interaction between
the two pathways in HSCC has not been reported yet, we
analyzed the relevance of p-Akt and p-Erk in HSCC tissues.
The results showed a p value of 0.057, which is very close
to that (0.05) with statistical diference. Hence, we think we
can not completely exclude the association between the two
signaling pathways in HSCC. To support this hypothesis,
further investigation with a larger sample size is needed.
Furthermore, we found the amounts of p-Akt and p-Erk
were correlated with clinical stage. Elevated levels of p-Akt
and p-Erk were found in late stage of HSCC, suggesting the
activitation of the two pathways might be associated with
poor prognosis in cancer patients. Dan et al. (2010) demon￾strated that the levels of phosphorylated Akt at S473 corre￾late to sensitivities of cancer cells to the PI3K inhibitors by
use of a panel of 39 human cancer cell lines (JFCR39), they
also used immunohistochemical method to confrm that the
phosphorylated Akt levels correlate to the in vivo efcacy
of PI3K inhibiter ZSTK474 (Isoyama et al. 2012). On the
other hand, it was reported that relapsed neuroblastomas har￾boring hyperactivated ERK signaling was sensitive to MEK
inhibition therapy (Eleveld et al. 2015). As the enhanced
expression of signaling pathways could be associated with
altered sensitivity to targeted therapy compared to patients
that do not exhibit increased expression (McCubrey et al.
2008; Fedorov et al. 2007), inhibition of PI3K, Akt, mTOR,
Raf or MEK, may be useful in HSCC treatment.
To verify our hypothesis, we determined the therapeutic
efcacy of a dual PI3K/mTOR GDC-0980, and a MEK1/
MEK2 inhibitor Refametinib against hypopharyngeal can￾cer FaDu cells in vitro. As expected, either GDC-0980 or
Refametinib alone signifcantly decreased FaDu cell via￾bility, with IC50 of 3.39 μM and 14.54 μM, respectively.
The underlying mechanism involved G1 phase cell cycle
arrest, but no apoptosis. To explore the mechanism for
causing G1 phase arrest, we examined several cell cycle
regulators that control G1-S transition, including cyclin
D1, pRb and p27. In G1/S cell cycle checkpoint, cyclin D1
binds and activates CDK4, and then the resulting cyclin
Fig. 5 GDC-0980 inhibited migration of FaDu cells. a–d FaDu
cells were cultured with various concentrations of GDC-0980 and
Refametinib for 48  h, respectively. a, c Images of the wound heal￾ing assay were taken at 0  h and 48  h after scratching. b, d Migra￾tion distances to the wound area following treatment with various
concentrations of GDC-0980 and Refametinib. e Cells were treated
with GDC-0980 for 48 h, and the representative pictures of migrated
cells were taken. Images showed cells that migrated through the Tran￾swell chamber membrane. f Percentage of cells migrated after GDC-
0980 treatment compared to those without treatment. g The metas￾tasis related proteins p-PKCζ, p-Integrin β1 and uPA were reduced
in FaDu cells after treatment with GDC-0980 for 24  h. β-actin was
used for normalization. Data are mean±SD, representative of three
independent experiments compared with control
Journal of Cancer Research and Clinical Oncology
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Journal of Cancer Research and Clinical Oncology
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D1-CDK4 complex targets pRb via phosphorylation, so
that E2F is released and activated to promote cell cycle
progression through G1 phase (Fedorov et al. 2007). On
the other hand, the activity of cyclin D1-CDK4 complex
is negatively regulated by CDK inhibitor p27 (Malumbres
and Barbacid 2009). Treatment with either GDC-0980 or
Refametinib led to the upregulation of p27, downregu￾lation of cyclin D1, and the decreased phosphorylation
of pRb. Consistant with the western blot result, the RNA
level of p27 was also enhanced after drug treatment.
Therefore, the downregulation of CDK4-cyclin D1 com￾plex and upregulation of p27 might lead to blockade of
cell cycle, which could contribute to the anticancer efect
of the two drugs.
As reported, both PI3K/Akt and MAPK/ERK pathways
are responsible for the regulation of cell cycle progres￾sion (Zhang and Liu 2002; Liang and Slingerland 2003
and TSC2, leading to the reduction of p27, inactivation of
GSK3-β, leading to increased cyclin D1/Rb/E2F (Liang and
Slingerland 2003). In MAPK/ERK pathway, the phosphoryl￾ation of ERK results in the activation of cyclin D1-CDK4/6
complex, which phosphorylates pRb and further promotes
G1-S switch (Zhang and Liu 2002). Here, we found GDC-
0980 remarkably suppressed the key regulators in PI3K/Akt
pathway, such as the phosphorylation of PDK1, Akt, mTOR,
p70S6 K, as well as the expression of PI3K-p110α. Mean￾while, Refametinib exhibited strong inhibition of ERK phos￾phorylation. Therefore, the efect of inducing G1 arrest by
GDC-0980 and Refametinib could be attributed to the inhi￾bition of PI3K/Akt and MAPK/ERK pathway, respectively.
As poor survival rate of HSCC is partly due to the devel￾opment of distant metastasis (Wyclife et al. 2007), dis￾covery of innovative pharmacotherapies to control HSCC
metastasis is of great importance. We next explored the
efect of GDC-0980 and Refametinib on FaDu cell migra￾tion. A strong inhibition against FaDu migration was
observed in GDC-0980 treated cells, but not in those treated
with Refametinib. To further investigate the anti-migration
mechanism of GDC-0980, we detected efect of GDC-0980
on p-PKCζ, p-Integrinβ1 as well as uPA, which are all
downstream efectors of PI3K/Akt pathway. Protein kinase C
(PKC) family plays pleiotropic roles in cell polarity, migra￾tion, and adhesion (Hirai and Chida 2003). It was reported
that PKCζ was regulated by PI3K/Akt in human non-small
cell lung cancer and breast cancer (Sun et al. 2005; Liu et al.
2009). Integrinβ1 and uPA also mediate cell migration and
angiogenesis (Wang et al. 2013; Dutta et al. 2014). Our
results suggested that GDC-0980 might attenuate migration
of FaDu cells via inhibiting p-PKCζ, p-Integrin β1 and uPA.
Studies have suggested that a link between PI3K/Akt and
MAPK/ERK pathways might give rise to redundancy in reg￾ulating cell survival. Thus, the use of combination therapy
by co-targeting the molecules of both pathways may be more
sufcient to treat tumor (Van Dort et al. 2015). Therefore,
we analyzed the anti-tumor efency with the joint use GDC-
0980 and Refametinib. The two drugs showed a strong syn￾ergistic suppressive efect on FaDu cell viability, with an
enhanced G1 phase arrest.
In conclusion, we verifed PI3K/Akt and MAPK/ERK
signaling pathways were activated in most HSCC tissues.
PI3K/mTOR inhibitor GDC-0980 and MEK inhibitor
Refametinib, exhibited antitumor activity on HSCC FaDu
cells through inhibiting cell proliferation and arresting cell
cycle at G1 phase. GDC-0980 could attenuate the migration
of FaDu cells via inhibiting the activity of PKCζ, Integrin
β1 and uPA. In addition, a signifcant synergy was observed
when GDC-0980 combined with Refametinib in suppress￾ing FaDu cells viability. Therefore, our research suggests
that co-targeting multiple signaling nodes of the PI3K path￾way and MAPK pathway might ofer potential therapeutic
benefts and provide a promising therapeutic approach for
HSCC therapy.
Fig. 6 GDC-0980 and Refametinib synergized to inhibit FaDu cell
proliferation. a FaDu cells were incubated with GDC-0980 and/
or Refametinib for 48  h. Three fxed ratios of IC50 GDC:IC50 Refa,
2×IC50 GDC:IC50 Refa, and 0.5×IC50 GDC:IC50 Refa were used to assess
the combinational efect. Cell viability after diferent treatments
was determined by MTT assay. Data are mean±SD, representa￾tive of three independent experiments. b Combinational efect was
analyzed using CalcuSyn software and the resulting CI-Fa plots are
shown (right). The horizontal line of CI=1, representing additivity,
is indicated. Values of drug combinations below the horizontal line
indicate synergism. CI combination index, Fa fraction afected. c The
cells were treated with GDC (1.356 μM) and/or Refa (2.908 μM) for
48 h, then grown in soft agar at 37 °C for 14 days. The colonies were
observed and counted under a microscope. d Quantifcation results
from the data of colony formation assay. The results are mean±SD,
representative of three independent experiments (n=3). **p<0.01,
compared with control. GDC GDC-0980, Refa Refametinib
Acknowledgements The work was supported by grant from National
Natural Science Foundation of China (81673464, 81373441), the Grant
for Major Project of Tianjin for New Drug Development (17ZXX￾YSY00050), and the Grant from Natural Science Foundation of Tian￾jin-Science and Technology (15JCYBJC27500).
Author contributions XP, YL and SZ performed the experiments. XP,
HL and WJ analyzed the data. ZZ, YQ and MJ prepared the fgures.
YL, XP and RW wrote the main manuscript. DK revised the manu￾script. PL, RW and DK designed the experiments. All authors reviewed
the manuscript.
Fig. 7 Combination treatment with GDC-0980 and Refametinib
induced an enhanced cell cycle arrest in FaDu cells. a FaDu cells
exposed to GDC-0980 (1.356 μM) and/or Refametinib (2.908 μM) for
48 h, were stained with PI solution and analyzed using fow cytom￾eter. b The histograms show the percentages of cell population in G1,
S, and G2/M phases. c–e After treatment with GDC-0980 (1.356 μM)
and/or Refametinib (2.908  μM) for 24  h, c the indicated cell cycle￾related proteins were detected by Western blot. Lamin B was used
for normalization. d p27 mRNA expression was examined by qRT￾PCR. e the indicated proteins in PI3K/Akt and MAPK/ERK pathways
were determined by Western blot. β-actin was used for normalization.
Data are mean±SD, representative of three independent experiments
(n=3). *p<0.05, compared with control. GDC GDC-0980, Refa
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Compliance with ethical standards
Conflict of interest The authors declare that there are no conficts of
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti￾tutional research committee and with the 1964 Helsinki declaration and
its later amendments or comparable ethical standards.
Aksamitiene E, Kiyatkin A, Kholodenko BN (2012) Cross-talk
between mitogenic Ras/MAPK and survival PI3K/Akt pathways:
a fne balance. Biochem Soc Trans 40:139–146
Bancroft CC, Chen Z, Dong G et al (2001) Coexpression of proangio￾genic factors IL-8 and VEGF by human head and neck squamous
cell carcinoma involves coactivation by MEK-MAPK and IKK￾NF-kappaB signal pathways. Clin Cancer Res 7:435–442
Cargnello M, Roux PP (2011) Activation and function of the MAPKs
and their substrates, the MAPK-activated protein kinases. Micro￾biol Mol Biol Rev 75:50–83
Chafer CL, Weinberg RA (2011) A perspective on cancer cell metas￾tasis. Science 331:1559–1564
Dan S, Okamura M, Seki M (2010) Correlating phosphatidylinositol
3-kinase inhibitor efcacy with signaling pathway status: in silico
and biological evaluations. Cancer Res 70:4982–4994
Davies L, Welch HG (2006) Epidemiology of head and neck cancer
in the United States. Otolaryngol Head Neck Surg 135:451–457
De Luca A, Maiello MR, D’Alessio A et al (2012) The RAS/RAF/
MEK/ERK and the PI3K/AKT signalling pathways: role in cancer
pathogenesis and implications for therapeutic approaches. Expert
Opin Ther Targets 16:S17–S27
Dutta S, Bandyopadhyay C, Bottero V et al (2014) Angiogenin inter￾acts with the plasminogen activation system at the cell surface of
breast cancer cells to regulate plasmin formation and cell migra￾tion. Mol Oncology 8:483–507
Eleveld TF, Oldridge DA, Bernard V et al (2015) Relapsed neuroblas￾tomas show frequent RAS-MAPK pathway mutations. Nat Genet
Fedorov SN, Shubina LK, Bode AM et al (2007) Dactylone inhibits
epidermal growth factor-induced transformation and phenotype
expression of human cancer cells and induces G1-S arrest and
apoptosis. Cancer Res 67:5914–5920
Hirai T, Chida K (2003) Protein kinase Czeta (PKCzeta): activation
mechanisms and cellular functions. J Biochem 133:1–7
Isoyama S, Yoshimi H, Dan S et al (2012) Development of an immu￾nohistochemical protein quantifcation system in conjunction
with tissue microarray technology for identifying predictive
biomarkers for phosphatidylinositol 3-kinase inhibitors. Biol
Pharm Bull 35:1607–1613
Kundu SK, Nestor M (2012) Targeted therapy in head and neck cancer.
Tumour Biol 33:707–721
Kuo P, Sosa JA, Burtness BA et al (2016) Treatment trends and sur￾vival efects of chemotherapy for hypopharyngeal cancer: analy￾sis of the national cancer data base. Cancer 122:1853–1860
Kwon DI, Miles BA (2019) Education Committee of the American
Head and Neck Society (AHNS). Hypopharyngeal carcinoma: do
you know your guidelines? Head Neck 41:569–576
Liang J, Slingerland JM (2003) Multiple roles of the PI3K/PKB (Akt)
pathway in cell cycle progression. Cell Cycle 2:339–345
Liu Y, Wang B, Wang J et al (2009) Down-regulation of PKCzeta
expression inhibits chemotaxis signal transduction in human lung
cancer cells. Lung Cancer 63:210–218
Machiels JP, Schmitz S (2011) New advances in targeted therapies
for squamous cell carcinoma of the head and neck. Anticancer
Drugs 22:626–633
Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a
changing paradigm. Nat Rev Cancer 9:153–166
Martini M, Ciraolo E, Gulluni F et al (2013) Targeting PI3K in cancer:
any good news? Front Oncol 3:108
Massacesi C, Di Tomaso E, Urban P et  al (2016) PI3K  inhibi￾tors  as  new  cancer  therapeutics:  implications  for  clinical
trial design. Onco Targets Ther 9:203–210
Mayer IA, Arteaga CL (2016) The PI3K/AKT pathway as a target for
cancer treatment. Ann Rev Med 67:11–28
McCubrey JA, Steelman LS, Abrams SL et al (2008) Targeting survival
cascades induced by activation of Raf/Raf/MEK/ERK, PI3K/
PTEN/Akt/mTOR and Jak/STAT pathways for efective leukemia
therapy. Leukemia 22:708–722
Mendoza MC, Er EE, Blenis J (2011) The Ras-ERK and PI3K-mTOR
pathways: crosstalk and compensation. Trends Biochem Sci
Newman JR, Connolly TM, Illing EA et al (2015) Survival trends in
Hypopharyngeal cancer: a population based review. Laryngoscope
Parfenov M, Pedamallu CS, Gehlenborg N et al (2014) Characteriza￾tion of HPV and host genome interactions in primary head and
neck cancers. Proc Natl Acad Sci USA 111:15544–15549
Peng X, Liu Y, Peng X et al (2018) Clinical features and the molecu￾lar biomarkers of olfactory neuroblastoma. Pathol Res Pract
Powles T, Lackner MR, Oudard S et al (2016) Randomized open-label
phase II trial of apitolisib (GDC-0980), a novel inhibitor of the
PI3K/mammalian target of rapamycin pathway, versus everoli￾mus in patients with metastatic renal cell carcinoma. J Clin Oncol
Ren H, Guo H, Thakur A et al (2016) Blockade efcacy of MEK/
ERK-dependent autophagy enhances PI3K/Akt inhibitor NVP￾BKM120′s therapeutic efectiveness in lung cancer cells. Onco￾target 7:67277–67287
Renshaw J, Taylor KR, Bishop R et al (2013) Dual blockade of the
PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244)
pathways synergistically inhibits rhabdomyosarcoma cell growth
in vitro and in vivo. Clin Cancer Res 19:5940–5951
Shaw RJ, Cantley LC (2006) Ras, PI(3)K and mTOR signalling con￾trols tumour cell growth. Nature 441:424–430
Siegel R, Naishadham D, Jemal A (2013) Cancer statistics. CA Cancer
J Clin 63:11–30
Steelman LS, Chappell WH, Abrams SL et al (2011) Roles ofthe Raf/
MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in control￾ling  growth  and  sensitivity  to  therapy-implications  for  can￾cer and aging. Aging (Albany NY) 3:192–222
Sun R, Gao P, Chen L et al (2005) Protein kinase C zeta is required
for epidermal growth factor-induced chemotaxis of human breast
cancer cells. Cancer Res 65:1433–1441
Thorpe LM, Yuzugullu H, Zhao JJ (2015) PI3K in cancer: diver￾gent roles of isoforms, modes of activation and therapeutic tar￾geting. Nat Rev Cancer 15:7–24
Van Dort ME, Galbán S, Wang H et al (2015) Dual inhibition of allos￾teric mitogen-activated protein kinase (MEK) and phosphati￾dylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional
inhibitor. Bioorg Med Chem 23:1386–1394
Wang H, Wu C, Wan S et al (2013) Shikonin attenuates lung cancer
cell adhesion to extracellular matrix and metastasis by inhibiting
integrin β1 expression and the ERK1/2 signaling pathway. Toxi￾cology 308:104–112
Journal of Cancer Research and Clinical Oncology
1 3
Wang Y, Qu X, Shen HC et al (2015) Predictive and prognostic bio￾markers for patients treated with anti-EGFR agents in lung cancer:
a systemic review and meta-analysis. Asian Pac J Cancer Prev
Wang R, Zhang Q, Peng X et al (2016a) Stellettin B induces G1 arrest,
apoptosis and autophagy in human non-small cell lung cancer
A549 cells via blocking PI3K/Akt/mTOR pathway. Sci Rep
Wang Y, Liu J, Qiu Y et  al (2016b) ZSTK474, a specific class
Iphosphatidylinositol 3-kinase inhibitor, induces G1 arrest and
autophagy in human breast cancer MCF-7 cells. Oncotarget
Wang Z, Wang Y, Zhu S et al (2018) DT-13 inhibits proliferation and
metastasis of human prostate cancer cells through blocking PI3K/
Akt pathway. Front Pharmacol 9:1450
Williams TM, Flecha AR, Keller P et al (2012) Cotargeting MAPK and
PI3K signaling with concurrent radiotherapy as a strategy for the
treatment of pancreatic cancer. Mol Cancer Ther 11:1193–1202
Wyclife ND, Grover RS, Kim PD et al (2007) Hypopharyngeal cancer.
Top Magn Reson Imaging 18:243–258
Zhang W, Liu HT (2002) MAPK signal pathways in the regulation of
cell proliferation in mammalian cells. Cell Res 12:9–18
Zhou Q, Chen Y, Chen X et al (2016) In vitro antileukemia activity of
ZSTK474 on K562 and multidrug resistant K562/A02 cells. Int
J Biol Sci 12:631–638
Zhou Q, Chen Y, Zhang L et al (2017) Antiproliferative efect of
ZSTK474 alone or in combination with chemotherapeutic drugs
on HL60 and HL60/ADR cells. Oncotarget 8:39064–39076
Zi D, Zhou ZW, Yang YJ et al (2015) Danusertib induces apoptosis,
cell cycle arrest, and autophagy GDC-0980 but inhibits epithelial to mesen￾chymal transition involving PI3K/Akt/mTOR signaling pathway
in human ovarian cancer cells. Int J Mol Sci 16:27228–27251
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