Downregulation of ribophorin II suppresses tumor growth, migration, and invasion of nasopharyngeal carcinoma
OncoTargets and Therapy
Downregulation of ribophorin ii suppresses tumor growth, migration, and invasion of nasopharyngeal carcinoma
Feilong hong Yong li haifeng n i Jing li 0
0 Department of Otolaryngology, hangzhou First People's hospital , hangzhou, china
8 1 0 2 - l u J - 3 1 n o 4 1 2 . 6 1 . 4 5 2 . 1 5 y b / m o c . s s rvpee l.yno PowerdbyTCPDF(ww.tcpdf.org) Background: It has been reported that ribophorin II (RPN2) expression is increased in many cancers, but the role of RPN2 in nasopharyngeal carcinoma (NPC) remains unclear. Patients and methods: This study found that the expression of RPN2 is increased dramatically in NPC tissues of patients compared with that in the adjacent normal tissues. This study attempted at understanding the effect of siRNA-RPN2 treatment on the migration and invasion of NPC cell lines CNE2 and HNE1. Results: RT-PCR and Western blotting showed that RPN2 was highly expressed in CNE2 and HNE1 cells. siRNA-RPN2 treatment significantly inhibited cell viability at 24 and 48 h compared with the control group. Results of the transwell assay showed that, compared to the control groups, migration and invasion of the cells treated with siRNA-RPN2 decreased markedly. In addition, compared to the control groups, caspase-3, caspase-9, and E-cadherin expression levels increased and MMP 2 expression decreased significantly in the siRNA-RPN2-treated group. Phosphorylation of AKT and PI3K was also inhibited after siRNA-RPN2 treatment. Conclusion: siRNA-RPN2 can effectively inhibit the invasion and migration of human NPC cells via AKT/PI3K signaling. This can serve as a novel strategy for NPC treatment.
patients, while RPN2 silencing suppresses cell proliferation
and invasiveness.8 The correlation between RPN2 and NPC
has not been reported.
In this study, we first observed that the expression of RPN2
was significantly higher in the NPC tissue than in the
peritumor tissue. Thus, it would be interesting to determine whether
RPN2 plays a role in the development and metastasis of NPC.
In the present study, we attempted to explore the effect(s)
of siRNA-RPN2 silencing on the migration and invasion of
NPC cells which expressed RPN2. Moreover, the underlying
mechanisms involved were also investigated to provide novel
insights into potential NPC therapeutic strategies.
.154 Patients and methods
.512 Patients and tissue samples
/yb Written informed consent was obtained from all participants
.com before the study. Sixty-eight patients with NPC admitted
rvpee l.yno ttohisthsetuHdayn,gaznhdoouuFrisrtsutdPyewopalse’aspHproosvpeidtablywtehree ienndreoplleenddefonrt
After being dewaxed in xylene and rehydrated through
graded alcohol to distilled water, NPC specimen sections
were immersed in 3% hydrogen peroxide for 15 min at room
temperature to prevent endogenous peroxidase activity. Next,
the sections were boiled in the antigen retrieval solution
(citrate, pH=6) for 4 min in a pressure cooker for antigen
retrieval. After being cooled for 2 h to 26°C, the sections
were incubated with diluted rabbit anti-RPN2 antibody
(1:200; Proteintech Group, Wuhan, China) overnight at
4°C. The next day, after rinsing thrice with phosphate
buffered saline with Tween 20 (PBST), the sections were
incubated with the secondary antibody for 30 min at 37°C.
Next, the sections were rinsed with PBST three times, and
3,3-diaminobenzidine staining was performed for 2 min for
targeted protein identification. The sections were
counterstained with hematoxylin to stain the nucleus. After rinsing
for 2 h under flowing water and dehydrating at 37°C, the
specimen sections were mounted using Neutral Balsam for
submit your manuscript | www.dovepress.com
Human NPC cells, namely CNE2, CNE2, HNE1, SUNE-1,
and 5-8F cells, were obtained from the Shanghai Cell Bank
(Chinese Academy of Sciences, Shanghai, China) and
cultured in RPMI 1640 medium (Gibco, Thermo Fisher
Scientific, Waltham, MA, USA) containing 10% fetal bovine
serum and 1% penicillin/streptomycin (Invitrogen, Thermo
Fisher Scientific) at 37°C in 5% CO2.
siRNA-RPN2 was designed and synthesized by Genepharma
(Shanghai, China). The interference sequence is 5′-GCA
GAGCAGAGCAGTAGATTGGCAA-3′. The NPC cells,
CNE2 and HNE1, were selected for transfection. After
transfection, RPN2 expression was confirmed using reverse
transcription polymerase chain reaction (RT-PCR) and
Western blotting. For siRNA transfection, the cells were seeded
onto 12-well tissue culture plates at a density of 6×104 cells/
well. When the cells were 70% confluent, they were
transfected with the RPN2 siRNA (siRPN2) or control siRNA
(siNC), according to the manufacturer’s instructions. After
48 h, the transfected cells were collected and processed for
the subsequent experiments.
cell viability assay
The effect of siRNA-RPN2 treatment on CNE2 and
HNE1 cell viability was evaluated using the cell counting
kit-8 (CCK-8) assay. Briefly, 0, 12-, 24-, 48-, and 72-h
post-transfection, the cells were seeded at a density of
4×103 cells/well in 96-cell plates and incubated for the
indicated time periods. Next, 20 μL CCK-8 was added to each
well and the cells were incubated for 1 h. The OD values
were recorded using a microplate reader (Thermo Fisher
Scientific) at 450 nm.
cell cycle assay
After transfection for 48 h, the cells were harvested, washed
with PBS, and fixed with 70% ethanol at 4°C overnight.
Next, the cells were washed with PBS and resuspended in the
RNase A–propidium iodide solution (100 mg/mL RNase A
and 5 μg/mL propidium iodide). The cells were incubated at
room temperature for 1 h. Stained cells were analyzed using
the FACScan flow cytometer (BD Biosciences, Mountain
View, CA, USA).
cell apoptosis assay
Cell apoptosis was detected using the annexin V/PI
fluorescence-activated cell sorting (FACS) assay, as described
previously. Briefly, the cells (3×105 cells/well) were harvested
and washed in cold PBS. After fixing with 70% ethanol, the
cells were treated with RNase (5 mmol/L) and incubated
for 10–15 min in the dark at 37°C. Subsequently, they were
stained with 195 μL Annexin V and 5 μL PI. The fluorescence
intensities were determined using fluorescence-activated cell
sorting (FACS) to analyze the percentage of apoptotic cells.
cell migration and invasion assays
The cell migration assay was performed using a 24-well
transwell chamber with a pore size of 8 μm (Sigma-Aldrich,
Munich, Germany). Then, 5×104 siRNA-RPN2-transfected
cells, mock cells, and non-transfected cells were resuspended
in a serum-free medium and transferred to the upper chamber.
The lower chamber was filled with a medium containing 10%
fetal bovine serum. After 24-h cultivation, the number of cells
stained by 0.1% crystal violet was counted visually under a
microscope (OLYMPUS, Hamburg, Germany). The cell
invasion assay was performed using the same procedure, except
that the upper chamber was coated with matrigel. Results from
three independent experiments were averaged and reported.
Quantitative reverse transcription
Total RNA was extracted from transfected cells, mock cells,
and non-transfected cells and reverse transcribed to cDNA
using the First Strand cDNA Synthesis kit (Sigma-Aldrich),
according to the manufacturer’s protocol. The PCR cycling
conditions were as follows: 95°C for 10 min, followed by 40
cycles of denaturation at 95°C for 15 s and
annealing/extension at 60°C for 45 s. The ABI 7300 thermocycler (Applied
Biosystems, Foster City, CA, USA) and SYBR Premix Ex
Taq kit (Takara, Beijing, China) were used.
Western blot analysis
The concentration of proteins extracted from cell samples
was determined using the BCA assay (Beyotime). Next, the
proteins were subjected to SDS-polyacrylamide gel
electrophoresis and electroblotted onto polyvinylidene fluoride
membranes. Following blocking with 5% non-fat dry milk in
PBS for 1 h, the blotting membranes were probed overnight
at 4°C with the following antibodies individually:
anticaspase-3, anti-caspase-9, anti-E-cadherin, anti-MMP2,
antiMMP9, anti-PI3K, anti-AKT, anti-p-PI3K, and anti-p-AKT
antibodies. The polyvinylidene fluoride membrane was
exposed to an X-ray film, and immunoreactive bands were
detected by reaction with the ECL detection system reagents
(Amersham, Arlington Heights, IL, USA).
Xenograft model experiments
All experimental protocols involving animals were approved
by the institutional animal care and use committee of the
Hangzhou First People’s Hospital and performed
following the Guide for the Care and Use of Laboratory Animals
issued by Institute of Laboratory Animal Resources.
Fourweek-old male severe combined immunodeficiency (SCID)
mice were purchased from Beijing HFK Bioscience Co.
Ltd. (Beijing, China). Cells transfected with siRNA-RPN2
(Genecopeia, Rockville, MD, USA) were injected into
the mice subcutaneously (1×106 cells per mouse). Tumor
growth in the mice was monitored every 7 days. All mice
were euthanized after 40 days, following which, the tumor
nodules in the mice were removed. Tumor sizes were
measured using a caliper, and tumor volume was calculated
using the following equation: tumor volume (mm3) = length
(mm) × width (mm)2/2.
All results represent the mean±SD of three independent
experiments. Statistical analysis was performed using the
SPSS 13.0 statistical package, and data were subjected
to one-way analysis of variance (ANOVA), followed by
Dunnett’s test. A P-value0.05 was considered to be
high expression of rPn2 in tumor tissue
To verify the biological role of RPN2 in NPC, the expression
levels of RPN2 in tissues of NPC patients were evaluated
using RT-PCR and IHC. As shown in Figure 1A, the mRNA
expression of RPN2 was higher in the NPC tissues than in
the adjacent normal tissues (P0.01). IHC analysis also
showed that the expression of RPN2 was higher in the NPC
tissues than in the adjacent normal tissues (Figure 1B). These
results indicated that RPN2 overexpression may result in the
initiation and/or progression of NPC.
sirPn2 inhibits cell proliferation of nPc
RPN2 mRNA was silenced in CNE2 and HNE1 cell lines,
as described previously. The interference efficient was then
identified using RT-PCR and Western blotting. Transfection
with siRNA-RPN2 resulted in a marked decrease in RPN2
mRNA and protein levels in the siRNA-RPN2 group,
compared to the control group and mock group, which confirmed
that siRNA-RPN2 was effective in silencing RPN2
expression (Figure 1C and D).
The effect of siRNA-RPN2 treatment on cell viability
measured using the CCK-8 assay is shown in Figure 1E
and F. Compared to the control and mock cell groups, cell
viability significantly (P0.05) decreased in the
siRNARPN2 group 24-, 48-, and 72-h post-transfection.
Decrease in rPn2 expression induces
cell cycle arrest and apoptosis
To explore the potential mechanism(s) by which RPN2
suppresses NPC cell growth, we evaluated the cell cycle
distribution of siRNA-RPN2 transfected cells and siRNA-NC cells
using flow cytometry. It was observed that knockdown of
RPN2 in the CNE2 and HNE1 cells resulted in an increase in
the number of cells in the G0–G1 phase (CNE2, 61.1%±1.1%;
HNE1, 59.4%±2.34%) and a decrease in those in the
S phase (CNE2, 28.9%±2.32%; HNE1, 29.65%±1.32%),
as compared to the siRNA-NC-transfected cells (CNE2:
G0–G1, 33.25%±2.09%; S, 55.14%±1.72%; HNE1: G0–G1,
31.18%±1.57%; S, 57.22%±1.67%) (Figure 2A). Additionally,
results from the annexin V/PI assay showed that, compared to
siRNA-NC-transfected cells (CNE2, 9.77%±1.24%; HNE1,
8.74%±1.14%), the CNE2 and HNE1 cells transfected with
A) and cell apoptosis (B) of cne2
submit your manuscript | www.dovepress.com
siRNA-RPN2 (CNE2, 33.5%±2.76%; HNE1, 28.7%±1.89%)
showed increased apoptosis (Figure 2B). Taken together,
these data suggested that RPN2 promotes cell proliferation
and suppresses apoptosis in NPC cells in vitro.
sirna-rPn2 inhibits tumor growth
8 We investigated the effects of siRNA-RPN2 treatment on
-012 repressing tumor growth in vivo. NPC cells transfected
l-Ju with siRNA-RPN2 or a negative control were
subcutanen13 ously injected into SCID mice. After 40 days, the mice were
1o4 euthanized, and RPN2 expression levels in their tissues
.612 were measured. The expression of RPN2 was observed to
.54 be lower in the group treated with siRNA-RPN2 than in the
.125 siNC group. Tumors with lower RPN2 expression showed
/yb slower growth and were smaller in size than control tumors
.com (Figure 3). The average tumor weight was approximately
ss 2.3-fold lower in the miR-148a-overexpressing tumors than
rvpee l.yno in the negative controls. These results suggested that
siRNARPN2 treatment may inhibit NPC cell growth in vivo.
effects of sirna-rPn2 treatment on
nPc cell migration and invasion
Cell invasion and migration are crucial factors for cancer
metastasis.10 The transwell assay was employed to
investigate the effect of siRNA-RPN2 treatment on the
migration and invasion of CNE2 and HNE1 cells. As shown in
Figure 4A and B, transfection with siRNA-RPN2 resulted
in significant lowering of the migration ability of CNE2 and
HNE1 cells. Similarly, the transwell invasion assay
demonstrated that the invasion abilities of CNE2 and HNE1 cells
transfected with siRNA-RPN2 were notably lower than those
of the control and mock cells (Figure 4C and D). These results
indicated that siRNA-RPN2 treatment significantly inhibited
the migration and invasion of CNE2 and HNE1 cells.
sirna-rPn2 regulates the expression
of caspase-3, caspase-9, e-cadherin, and
Caspase-3, caspase-9, E-cadherin, and MMP2 expressions were
monitored by Western blot analysis. Results showed that,
compared to the siNC group, the siRNA-RPN2-treated group
showed a significant increase in the expression of caspase-3,
caspase-9, and E-cadherin in both the CNE2 and HNE1 cells
(P0.05, Figure 5A and B). MMP2 expression decreased
significantly in NPC cells following siRNA-RPN2 treatment
(P0.05, Figure 5C and D). Therefore, inhibition of cell
invasion and migration mediated by E-cadherin and MMP2
may play an important role in inhibiting NPC metastasis.
sirna-rPn2 suppressed the
phosphorylation of Pi3K/aKT signaling
The phosphorylation of PI3K/AKT signaling plays a crucial
role in NPC occurrence and pathogenesis. Therefore, we
sciD mice. representative
submit your manuscript | www.dovepress.com
analyzed the effect of siRNA-RPN2 treatment on
phosphorylation of PI3K and AKT. We demonstrated that, compared
to the siNC and control groups, the siRNA-RPN2-treated
group showed significantly higher inhibition of AKT and
PI3K phosphorylation in the CNE2 and HNE1 cells (P0.05,
Abnormal expression of RPN2 has been reported in breast
cancer, non-small cell lung cancer, gastric cancer, colorectal
cancer, and prostatic carcinoma.11–13 In the present study,
we observed a marked increase in RPN2 expression in
NPC tissues. The subsequent experiments were designed to
explore the effect of siRNA-RPN2 treatment on tumor
migration and invasion in NPC. CNE2 and HNE1 were selected
for further investigation and validating the high expression
of RPN2. siRNA-RPN2 treatment effectively inhibited cell
proliferation (in vivo and in vitro), and suppressed the
invasion and migration of CNE2 and HNE1 cells. siRNA-RPN2
treatment also effectively induced cell apoptosis, and cell
cycle arrest in the G1 phase. The critical role of siRNA-RPN2
in NPC cell growth, invasion, and migration encouraged
us to explore the potential mechanism(s) responsible for
the aforementioned observations by measuring the changes
in expression of relevant genes and proteins. Zhang et al10
reported that RPN2 regulated colorectal cell
proliferation through mediating the glycosylation of EGFR. Fujita
et al8 demonstrated that RPN2 promoted cell proliferation
and inhibited cell apoptosis by regulation of Bax/Bcl-2 in
non-small cell lung cancer. In this study, we identified the
submit your manuscript | www.dovepress.com
expression of caspase-3/-9. Caspase-3 and caspase-9 are
known to participate in mitochondrial apoptosis, which
mediates the apoptotic cascade reaction.14–16 Caspase-3 is a
downstream target in the mitochondrial apoptosis pathway.
Activated caspase-8 can lead to the release of caspase-9 and
then give rise to the activation of caspase-3, and ultimately
to induce apoptosis.16 Therefore, we detected the
caspase3/-9 expressions after cells transfected with RPN2 siRNA.
E-cadherin expression is known to decrease in several
carcinoma cells. Further, the extracellular matrix also gets
destroyed, which results in tumor migration and invasion.17,18
Tumor cell invasion and metastasis are characteristic features
of malignant phenotypes and require regulated expression
of MMPs. Among all MMPs, MMP2 has been suggested
to have well-characterized roles in cancer cell invasion and
metastasis.19,20 We also provided evidence suggesting that
the mechanism underlying the aforementioned effects was
related to the inhibition of expression of MMP2, which is
known to play a major role in tumor invasion and metastasis
by proteolyzing the extracellular matrix.21,22 These results
indicated that the inhibition of cell proliferation, invasion,
and migration in response to siRNA-RPN2 treatment may
be mediated via the regulation of caspase-3, caspase-9,
E-cadherin, and MMP2. PI3K/AKT signaling plays crucial
roles in cell proliferation, migration, and invasion of human
NPC.23,24 Our study showed that the phosphorylation of PI3K
and AKT was blocked by siRNA-RPN2.
Our study showed that RPN2 was highly expressed in human
NPC tissues. Additionally, the mRNA and protein levels
of RPN2 were upregulated in the NPC cell lines, namely
CNE2 and HNE1. Furthermore, siRNA-RPN2 can markedly
inhibit the invasion and migration of NPC cells by regulating
caspase-3, caspase-9, E-cadherin, and MMP2 expression,
and PI3K/AKT signaling. RPN2, therefore, may serve as a
biomarker for the diagnosis and prognosis of NPC. Finally,
targeting the PI3K/AKT signaling pathway may be an
effective therapeutic strategy for treating NPC.
The authors report no conflicts of interest in this work.
submit your manuscript | www.dovepress.com
Publish your work in this journal
OncoTargets and Therapy is an international, peer-reviewed, open
access journal focusing on the pathological basis of all cancers, potential
targets for therapy and treatment protocols employed to improve the
management of cancer patients. The journal also focuses on the impact
of management programs and new therapeutic agents and protocols on
1. Fountzilas G , Psyrri A , Giannoulatou E , et al. Prevalent somatic BRCA1 mutations shape clinically relevant genomic patterns of nasopharyngeal carcinoma in Southeast Europe . Int J Cancer . 2018 ; 142 ( 1 ): 66 - 80 .
2. Chee J , Loh KS , Tham I , et al. Prognostic stratification of patients with metastatic nasopharyngeal carcinoma using a clinical and biochemical scoring system . J Cancer Res Clin Oncol . 2017 ; 143 ( 12 ): 2563 - 2570 .
3. Hsu C , Lee SH , Ejadi S , et al. Safety and antitumor activity of pembrolizumab in patients with programmed death-ligand 1-positive nasopharyngeal carcinoma: results of the KEYNOTE-028 study . J Clin Oncol . 2017 ; 35 ( 36 ): 4050 - 4056 .
4. Wang M , Liu G , Shan GP , Wang BB . In vivo and in vitro effects of ATM/ATR signaling pathway on proliferation, apoptosis, and radiosensitivity of nasopharyngeal carcinoma cells . Cancer Biother Radiopharm . 2017 ; 32 : 193 - 203 .
5. Ou X , Miao Y , Wang X , Ding J , He X , Hu C. The feasibility analysis of omission of elective irradiation to level IB lymph nodes in lowrisk nasopharyngeal carcinoma based on the 2013 updated consensus guideline for neck nodal levels . Radiat Oncol . 2017 ; 12 : 137 .
6. Meng H , Zhu X , Li L , et al. Identification of CALM as the potential serum biomarker for predicting the recurrence of nasopharyngeal carcinoma using a mass spectrometry-based comparative proteomic approach . Int J Mol Med . 2017 ; 40 : 1152 - 1164 .
7. Chen WB , Zhang B , Liang L , et al. To predict the radiosensitivity of nasopharyngeal carcinoma using intravoxel incoherent motion MRI at 3 .0 T. Oncotarget . 2017 ; 8 : 53740 - 53750 .
8. Fujita Y , Yagishita S , Takeshita F , Yamamoto Y , Kuwano K , Ochiya T. Prognostic and therapeutic impact of RPN2-mediated tumor malignancy in non-small-cell lung cancer . Oncotarget . 2015 ; 6 : 3335 - 3345 .
9. Lu X , Nowicka U , Sridharan V , et al. Structure of the Rpn13-Rpn2 complex provides insights for Rpn13 and Uch37 as anticancer targets . Nat Commun . 2017 ; 8 : 15540 .
10. Zhang J , Yan B , Spath SS , et al. Integrated transcriptional profiling and genomic analyses reveal RPN2 and HMGB1 as promising biomarkers in colorectal cancer . Cell Biosci . 2015 ; 5 : 53 .
11. Chen X , Yue B , Zhang C , et al. MiR -130a-3p inhibits the viability, proliferation, invasion, and cell cycle, and promotes apoptosis of nasopharyngeal carcinoma cells by suppressing BACH2 expression . Biosci Rep . 2017 ; 37 :pii.
12. Hu H , Wang G , Li C. miR-124 suppresses proliferation and invasion of nasopharyngeal carcinoma cells through the Wnt/beta-catenin signaling pathway by targeting Capn4 . Onco Targets Ther . 2017 ; 10 : 2711 - 2720 .
13. Wang JY , Jin X , Li XF . Knockdown of TMPRSS3, a transmembrane serine protease, inhibits the proliferation, migration, and invasion in human nasopharyngeal carcinoma cells . Oncol Res . 2018 ; 26 ( 1 ): 95 - 101 .
14. Jiang C , Zhou L , Wang H , Zhang Q , Xu Y. Axl Is a Potential Cancer Prognostic Marker for the Migration and Invasion of Nasopharyngeal Carcinoma . Adv Clin Exp Med . 2016 ; 25 : 531 - 537 .
15. Hsu CC , Huang SF , Wang JS , et al. Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion . BMC Cancer . 2016 ; 16 : 800 .
16. Oudejans JJ , Harijadi A , Cillessen SA , et al. Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome . Mod Pathol . 2005 ; 18 : 877 - 885 .
17. Zhang Z , Bu X , Chen H , Wang Q , Sha W. BMI -1 promotes the invasion and migration of colon cancer stem cells through the downregulation of E-cadherin . Int J Mol Med . 2016 ; 38 : 1199 - 1207 .
18. Cheng JC , Chang HM , Xiong S , So WK , Leung PC . Sprouty2 inhibits amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells . Oncotarget . 2016 ; 7 : 81645 - 81660 .
19. Li Y , Huang Y , Qi Z , Sun T , Zhou Y . MiR-338 -5p promotes glioma cell invasion by regulating TSHZ3 and MMP2 . Cell Mol Neurobiol . 2018 ; 38 : 669 - 677 .
20. Xu W , Xu H , Fang M , Wu X , Xu Y. MKL1 links epigenetic activation of MMP2 to ovarian cancer cell migration and invasion . Biochem Biophys Res Commun . 2017 ; 487 : 500 - 508 .
21. Yang H , Liang J , Zhou J , et al. Knockdown of RHOC by shRNA suppresses invasion and migration of cholangiocellular carcinoma cells via inhibition of MMP2, MMP3, MMP9 and epithelial-mesenchymal transition . Mol Med Rep . 2016 ; 13 : 5255 - 5261 .
22. Ghosh A , Dasgupta D , Ghosh A , et al. MiRNA199a -3p suppresses tumor growth, migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA, VEGFR1, VEGFR2, HGF and MMP2 . Cell Death Dis . 2017 ; 8 : e2706 .
23. Xie M , Yi X , Wang R , et al. 14 -Thienyl methylene matrine (YYJ18), the derivative from matrine, induces apoptosis of human nasopharyngeal carcinoma cells by targeting MAPK and PI3K/Akt pathways in vitro . Cell Physiol Biochem . 2014 ; 33 : 1475 - 1483 .
24. Wong CH , Ma BB , Cheong HT , Hui CW , Hui EP , Chan AT . Preclinical evaluation of PI3K inhibitor BYL719 as a single agent and its synergism in combination with cisplatin or MEK inhibitor in nasopharyngeal carcinoma (NPC) . Am J Cancer Res . 2015 ; 5 : 1496 - 1506 .