Download Akt Kinase-Interacting Protein 1 Signals through

Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Cancer
Research
Therapeutics, Targets, and Chemical Biology
Akt Kinase-Interacting Protein 1 Signals
through CREB to Drive Diffuse Malignant
Mesothelioma
Tadaaki Yamada1,2, Joseph M. Amann1, Koji Fukuda2, Shinji Takeuchi2,
Naoya Fujita3, Hisanori Uehara4, Shotaro Iwakiri5, Kazumi Itoi5,
Konstantin Shilo6, Seiji Yano2, and David P. Carbone1
Abstract
Diffuse malignant mesothelioma (DMM) is a tumor of
serosal membranes with propensity for progressive local disease. Because current treatment options are largely ineffective,
novel therapeutic strategies based on molecular mechanisms
and the disease characteristics are needed to improve the outcomes of patients with this disease. Akt kinase interacting
protein 1 (Aki1; Freud-1/CC2D1A) is a scaffold protein for
the PI3K–PDK1–Akt signaling module that helps determine
receptor signal selectivity for EGFR. Aki1 has been suggested as
a therapeutic target, but its potential has yet to be evaluated in a
tumor setting. Here, we report evidence supporting its definition as a therapeutic target in DMM. In cell-based assays, Aki1
silencing decreased cell viability and caused cell-cycle arrest of
multiple DMM cell lines via effects on the PKA–CREB1 signaling pathway. Blocking CREB activity phenocopied Aki1 silencing. Clinically, Aki1 was expressed in most human DMM
specimens where its expression correlated with phosphorylated
CREB1. Notably, Aki1 siRNA potently blocked tumor growth in
an orthotopic implantation model of DMM when administered
directly into the pleural cavity of tumor-bearing mice. Our
findings suggest an important role for the Aki1–CREB axis in
DMM pathogenesis and provide a preclinical rationale to target
Aki1 by intrathoracic therapy in locally advanced tumors. Cancer
Introduction
directly implicated in the prognosis of DMM patients (4). The
prognosis of DMM patients is extremely poor because conventional treatment regimens, such as chemotherapy and radiotherapy are inefficient in controlling locally advanced disease
and/or metastasis.
Clinical trials using EGFR tyrosine kinase inhibitors (EGFRTKIs) for treatment of DMM have had disappointing results (5, 6).
This mirrors non–small cell lung cancer (NSCLC) where efficacy
of EGFR-TKIs does not correlate with EGFR expression levels and
the activating mutations in EGFR found in NSCLC are extremely
rare in DMM tumors (7, 8). In addition, previous reports have
shown that unlike EGFR-mutated lung cancer cells, multiple
receptor tyrosine kinases (RTK) are simultaneously activated in
DMM cell lines (9). An increased understanding of the molecular
mechanisms of DMM pathology will identify novel molecules
and signaling pathways responsible for DMM local progression
and provide new targets for therapy (4–6).
Akt kinase–interacting protein 1 (Aki1)/Freud-1/CC2D1A has
been shown to be a scaffold protein in the PI3K–PDK1–Akt
pathway (10). This protein selectively forms a complex with
nonmutated EGFR and Akt in response to EGF stimulation,
mediates Akt activation by PDK1, and contributes to cell survival
and proliferation. Recently, we reported that Aki1 constitutively
associates with mutated EGFR, including the T790M gatekeeper
mutation, which is the most prevalent genetic alteration underlying acquired resistance to EGFR inhibitors. Aki1 has an important role as a determinant of receptor-selective signaling for
mutant EGFR, and mediates the survival signal through activation
of Akt (11). Aki1 is expressed in EGFR-mutant lung cancer (53/56
cases) and pancreatic cancer (25/60 cases; refs. 11, 12). Thus, Aki1
may have novel direct therapeutic potential for the treatment of
many refractory malignancies.
Diffuse malignant mesothelioma (DMM) is an aggressive
tumor arising from the mesothelial cells of serosal membranes.
Several etiologic factors, including exposure to asbestos (1, 2),
have been reported to be involved in the development of DMM.
With introduction of strict occupational protective measures,
the incidence of DMM has already reached its peak in the
United States (3); however, it is predicted that there will be
increases in the number of DMM patients in Europe and
Australia until 2020 (3) and in Japan until 2040 (2). DMM
is most likely to advance locally and the extent of the disease is
1
Department of Internal Medicine, The Ohio State University Medical
Center, Columbus, Ohio. 2Division of Medical Oncology, Cancer
Research Institute, Kanazawa University, Kanazawa, Japan. 3Cancer
Chemotherapy Center, Japanese Foundation for Cancer Research,
Tokyo, Japan. 4Department of Molecular and Environmental Pathology, Institute of Health Biosciences, University of Tokushima Graduate
School, Tokushima, Japan. 5Department of Respiratory Surgery,
Hyogo Prefectural Amagasaki Hospital, Amagasaki, Japan. 6Department of Pathology, The Ohio State University Medical Center, Columbus, Ohio.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: David P. Carbone, The Ohio State University Medical
Center, 460 West 12th Avenue, Columbus, OH 43210. Phone: 614-685-4479; Fax:
614-293-4372; E-mail: [email protected]; and Seiji Yano, Cancer
Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa
920-8641, Japan. Phone: 81-76-265-2780; Fax: 81-76-234-4524; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-15-0858
2015 American Association for Cancer Research.
Res; 75(19); 1–10. 2015 AACR.
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Yamada et al.
Because we do not currently have drugs inhibiting this target,
decreasing its activity through RNAi or targeting downstream
pathways remain options. RNAi is a phenomenon of RNA-dependent sequence-specific gene silencing in mammalian cells. Recently, some drug delivery systems were reported for gene therapy
using nonviral vectors for delivery of DNA, siRNA, and miRNA
(13). In particular, in vivo delivery of siRNAs can be highly
efficacious and of very low toxicity and several clinical trials using
siRNA have shown promising results (13), although nonviral
gene-based therapies have yet to be approved by the FDA (14).
In this study, we identify a crucial role for Aki1 in the survival of
DMM cells through the CREB signaling pathway and show that
targeting Aki1 or CREB with siRNA is effective in controlling DMM
in vitro and Aki1 siRNA inhibited the growth of pleural disease in
in vivo orthotopic models. To the best of our knowledge, this is the
first experimental report to identify the pivotal roles of Aki1 in
DMM that provides grounds for targeting Aki1 as novel therapeutic strategy.
Materials and Methods
Cell cultures and reagents
We used seven human DMM cell lines, one noncancerous
mesothelial cell line (Met-5A), and the HEK293T cell line. YMESO-8A and Y-MESO-14 were established in Aichi Cancer
Research Center Institute (15). NCI-H290 were provided by Dr.
Adi F. Gazdar (University of Texas Southwestern Medical Center,
Dallas, TX) and Dr. Yoshitaka Sekido (Aichi Cancer Research
Center Institute, Aichi, Japan). Y-MESO-8A was authenticated by
short tandem repeat analysis. Y-MESO-14 and NCI-H290 authentications were not performed. MSTO-211H, NCI-H2052, NCIH2373, NCI-H2452, Met-5A, and HEK293T were obtained directly from the ATCC within 6 months of the experiments reported.
DMM cell lines are divided into three histologic origins, an
epithelioid type (NCI-H290 and NCI-H2452), a sarcomatoid
type (NCI-H2052 and NCI-H2373), and a biphasic type
(Y-MESO-8A, Y-MESO-14, and MSTO-211H; refs. 15–17). DMM
cell lines were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin
(50 g/mL), in a humidified CO2 incubator at 37 C. Met-5A cells
were cultured according to the instructions of the ATCC. HEK293T
cells were cultured in DMEM supplemented with 10% FBS,
penicillin (100 U/mL), and streptomycin (50 g/mL), in a humidified CO2 incubator at 37 C. All cells were passaged for less than 3
months before renewal from frozen, early-passage stocks. Cells
were regularly screened for Mycoplasma, using MycoAlert Mycoplasma Detection Kits (Lonza). PKA inhibitor KT5720 and CBP–
CREB interaction inhibitor were obtained from Sigma-Aldlich
and Calbiochem, respectively.
Antibodies and Western blotting
Protein aliquots of 25 mg each were resolved by SDS polyacrylamide gel (Bio-Rad) electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). After washing three
times, the membranes were incubated with Blocking One (Nacalai Tesque, Inc.) for 1 hour at room temperature, and then
incubated overnight at 4 C with primary antibodies to Akt,
phospho-Akt (Thr308), p-CREB1, t-CREB1, p-PKAC, t-PKACa,
HA, DYKDDDDK (FLAG), anti–beta-actin (13E5; 1:1,000 dilution; Cell Signaling Technology), Aki1 (1:1,000 dilution; Bethyl
Laboratories), cyclin A, or EGFR antibodies (1:1,000 dilution;
OF2 Cancer Res; 75(19) October 1, 2015
Santa Cruz Biotechnology). After washing three times, the membranes were incubated for 1 hour at room temperature with
secondary Ab [horseradish peroxidase (HRP)–conjugated species-specific Ab]. Immunoreactive bands were visualized with
SuperSignal West Dura Extended Duration Substrate Enhanced
Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was performed at least three times independently.
RNAi transfection
Stealth RNAi siRNA for Aki1 (HSS123402, HSS123400; Invitrogen), EGFR (HSS103114, HSS103116; Invitrogen), and Silencer Select siRNA for CREB1 (s3489, s3490; Invitrogen) were
transfected with Lipofectamine RNAiMAX (Invitrogen) in accordance with the manufacturer's instructions. Stealth RNAi siRNA
Negative Control Low GC Duplex no.3 (Invitrogen) was used as
scramble control throughout the experiment. Aki1, EGFR, and
CREB1 knockdowns were confirmed by Western blotting analysis.
Each experiment was performed at least in triplicate, and three
times independently.
Proliferation assay
The cells were reseeded at 2 103 per well in 96-well plates, and
incubated in antibiotic-containing RPMI-1640 with 10% FBS.
After 24 hours of incubation, KT5720 (0.1 and 1 mmol/L), or
CBP–CREB interaction inhibitor (0.1 and 0.3 mmol/L) was added
to each well, and incubation was continued for a further 48 hours.
These cells were then used for proliferation assay, which was
measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium) dye reduction method. An aliquot of MTT
solution (2 mg/mL; Sigma) was added to each well followed by
incubation for 2 hours at 37 C as previously described (18). The
media were removed and the dark blue crystals in each well were
dissolved in 100 mL of DMSO. Absorbance was measured with an
MTP-120 microplate reader (Corona Electric) at test and reference
wavelengths of 550 and 630 nm, respectively. The percentage of
growth is shown relative to untreated controls. Each sample was
assayed in triplicate, with each experiment repeated at least three
times independently.
Cell-cycle assay
Cells treated by Aki1 siRNA or scramble control siRNA were
collected by centrifugation and resuspended at 1 106 cells/mL in
propidium iodide (PI) staining buffer (0.1% sodium citrate, 0.1%
Triton X-100, and 50 mg/mL PI) and were treated with 1 mg/mL
RNase at room temperature for 30 minutes. Cell-cycle histograms
were generated after analysis of PI-stained cells by FACS with a BD
Biosciences FACScan. For each culture, at least 1 104 events were
recorded. Histograms generated by FACS were analyzed by ModFit cell-cycle analysis software (Verity) to determine the percentage of cells in each phase (G0–G1, S, and G2–M). Each sample was
assayed in triplicate, with each experiment repeated at least two
times independently.
Human phospho-kinase antibody array
We used the Human Phospho-Kinase Array Kit (R&D Systems)
to measure the relative level of phosphorylation for 43 kinases
and two related total proteins. The assay was performed according
to the manufacturer's instructions with modifications. Briefly,
cells were cultured in RPMI-1640 containing 10% FBS, and then
were lysed with array buffer before they reached confluence. The
arrays were blocked with blocking buffer and incubated with 450
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Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Aki1 Is a Therapeutic Target in DMM
mg of cell lysate overnight at 4 C. Arrays were washed, incubated
with an HRP-conjugated phospho-kinase antibody and treated
with ECL solution (GE Healthcare), before being exposed to film.
Each experiment was performed at least two times independently.
Luciferase reporter assay
We designed a dual-luciferase reporter driven by a 3x CRE
consensus–binding sequence in the promoter region in addition
to a TATA box, which was inserted into a lentiviral construct
upstream of luciferase as previously described (19). Cells were
stably transduced to express CRE wild-type reporters and ratios
between the two were compared after RNAi transfection of Aki1 or
scramble control. Each sample was assayed in triplicate, with each
experiment repeated at least two times independently.
Plasmid construction
pCF plasmid–expressing empty Vector (Vector), FLAG-tagged
human CREB (CREB-WT), or FLAG-tagged human CREB-S133A,
which was CREB-kinase dead–mutated (CREB-KD) clones were
purchased from Addgene, Inc. Phosphorylation of CREB on
Serine133 is essential for the transcriptional function of CREB
(20). The X-tremeGene HP (Roche) transfection reagent was used
to transfect 211H cells with Vector, CREB-WT, or CREB-KD
plasmid following the manufacturer's instructions. 211H and
HEK293T cells were stably transfected with pHM6 plasmid–
expressing HA-tagged recombinant human WT-Aki1 proteins
(Aki1-WT) or empty vector as a control protein by using XtremeGene Transfection Reagent. The plasmid was constructed
by Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research (Tokyo, Japan) as previously described (10). The successfully transduced clones were identified by G418 (SigmaAldrich) selection. Whole-cell lysates (25 mg) were subjected to
Western blot analysis.
EGFP-Eluc gene transfection
The cDNA of EGFP from pIRES-EGFP Vector (Invitrogen) and
Emerald Luc (Eluc) from Emerald Luc Vector (ELV-101,
TOYOBO) were cloned into MaRXIVf Puro retrovirus vector as
described previously (21). All cDNA were sequenced. Transfection was performed using Lipofectamine LTX and PLUS Reagent
(Invitrogen), and retroviral infection into 211H cells was performed using the Retrovirus Packaging Kit (Ampho) according to
the manufacturer's instructions. Then, the cells were selected in
Puromycin (Sigma-Aldrich).
Orthotopic model in SCID mice and in vivo RNAi
We used 5-week-old female SCID mice (Clea) for this study. All
animal experiments were maintained under specific pathogenfree conditions throughout the study and complied with the
Guidelines for the Institute for Experimental Animals, Kanazawa
University Advanced Science Research Center (approval no. AP081088). An orthotopic implantation model of human DMM was
established as described previously (22). Briefly, 1 106 211H
transfected the Eluc/EGFP gene cells (211H/Eluc) in 100 mL of PBS
were injected into the right pleural cavity of mice. On day 7 after
cell inoculation, 100 mg of either scramble or Aki1 siRNA complexed with invivofectamine (Invitrogen) was injected into the
right pleural cavity. siRNA and invivofectamine complex was
prepared in accordance with the manufacturer's instructions
(Invitrogen). Aki1 knockdown in tumor tissue was confirmed by
www.aacrjournals.org
Western blotting analysis on day 7 after injection of siRNA
complex. We evaluated tumor progression using in vivo imaging
by the luminescence as described previously (21). The mice were
sacrificed 19 days after tumor inoculation. Luminescence was
evaluated twice per week from day 2 to day 19 after cell inoculation. All surgeries were performed under sodium pentobarbital
anesthesia, and efforts were made to minimize the suffering of the
animals.
Patients
A total of 68 tumor specimens were obtained from 67 DMM
patients with written informed consent at the Ohio State University (Columbus, Ohio) and the Hyogo Prefectural Amagasaki
Hospital (Amagasaki, Japan) in studies with Institutional Review
Board approval. Of the 67 patients, 36 were epithelioid, 12 were
biphasic, 10 were sarcomatoid, seven were desmoplastic, and two
patients showed other types. Specimens from the 34 patients (35
tumors) were obtained in tissue microarray (TMA) at the Ohio
State University, and the 33 patients (33 tumors) were obtained in
the tissue at the Hyogo Prefectural Amagasaki Hospital.
Immunohistochemical studies for Aki1 and p-CREB
Paraffin-embedded tissue was cut at 4 mm and sections were
placed on positively charged slides. Slides were then placed in a
60 C oven for 1 hour, cooled, deparaffinized, and rehydrated
through xylene, and graded ethanol solutions to water. All slides
were quenched for 5 minutes in a 3% hydrogen peroxide aqueous
solution to block for endogenous peroxidase. Antigen retrieval
was performed by Heat-Induced Epitope Retrieval, in which the
slides were placed in a 1 x solution of Target Retrieval Solution, pH
6.0 (Dako, S1699) for 25 minutes at 96 C using a vegetable
steamer (Black & Decker) and cooled for 15 minutes in solution.
Slides were stained with the Intellipath Autostainer Immunostaining System. Detection system included Mach 3 Rabbit HRP
(Biocare Medical; M3R531L) and Liquid DABþ Chromogen
(Dako; K346811). All incubations on the Autostainer were performed at room temperature. Slides were counterstained in
Richard Allen hematoxylin, dehydrated through graded ethanol
solutions, cleared with xylene, and coverslipped. Dilution experiments for immunohistochemical studies were performed in
resection specimens according to the manufacturer's instructions.
On the basis of the observed expression patterns, the tumor cell
staining in tissue and TMA was evaluated separately for p-CREB
(1:700, clone 87G3; Cell Signaling Technology) and Aki1 (1:200;
Aki1 aka CC2D1A, rabbit polyclonal; Sigma). Because p-CREB
showed only nuclear staining that varied in intensity and distribution within a tumor, its expression was assessed in regard to the
staining intensity as negative (0), low (1þ), intermediate (2þ),
and high (3þ). Because immunohistochemical studies for Aki1
showed only cytoplasmic staining evenly distributed within a
tumor but varied in intensity, its expression was assessed as
negative (0), low (1þ), intermediate (2þ), and high (3þ).
Statistical analysis
Data from MTT assay, luciferase reporter assay, and tumor
progression using in vivo imaging are expressed as means of SD. The statistical significance of differences was analyzed by oneway ANOVA and Spearman rank correlations performed with
GraphPad Prism Ver. 6.0 (GraphPad Software, Inc.). For all
analyses, a two-sided P value less than 0.05 was considered
statistically significant.
Cancer Res; 75(19) October 1, 2015
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Yamada et al.
Results
Met-5A, were only marginal. These results suggest that Aki1
knockdown inhibits viability of at least a subset of DMM cells
and is independent of the EGFR signaling.
Western blotting analyses showed that Aki1 knockdown in the
five most sensitive DMM cell lines, reduced cyclin A protein
expression, consistent with the decrease in cell viability
(Fig. 1C). Cyclin A is known to moderate the initiation and
completion of DNA replication during S phase and reduction of
cyclin A with Aki1 knockdown suggests that Aki1 contributes to
the regulation of cell-cycle progression. Indeed, cell-cycle analysis
confirmed that knockdown of Aki1 decreased the number of cells
in S phase in 211H and H2052 (Fig. 1D).
Aki1 maintains the viability of DMM cells through CREB1
activation
To elucidate the downstream activities regulated by Aki1 that
are involved in cell viability of DMM cells, we performed a human
phosphokinase array to compare Aki1 containing with Aki1
knockdown cells (Fig. 2A). Several kinase phosphorylation sites
were changed with Aki1 knockdown. Interestingly, the CREB1
phosphorylation level at serine 133 (pS133) was decreased when
cells were transfected with Aki1 siRNA. Examination of DMM cell
lines by Western blot analysis for CREB1 pS133 indicates that all
of the DMM cell lines appear to have higher levels of pS133 than
C
Met-5A
Y-meso8A
H2452
H2373
H2052
H290
211H
A
Y-meso14
Specific downregulation of Aki1 inhibits cell viability and
induces cell arrest in DMM cells
To assess the involvement of Aki1 in EGFR-mediated signaling
of DMM cells, we first examined the expression of Aki1 protein
and EGFR-related proteins (Akt and EGFR) in seven human DMM
cell lines and compared them with an immortalized human
mesothelial cell line, Met-5A, by Western blotting analysis
(Fig. 1A). All of the cell lines examined expressed Aki1, Akt, and
EGFR protein at various levels. However, the levels of Aki1 were
generally higher in DMM cell lines than Met-5A. EGFR and
phosphorylated Akt were also detected in all DMM and mesothelial cell lines at various levels. These results indicate that Aki1
expression is not correlated with EGFR-related proteins in DMM
cells.
To determine the role of Aki1 in DMM cell lines, we used
specific siRNA (siRNA) to knockdown Aki1 or EGFR. Treatment
with Aki1-specific siRNA decreased the viability of DMM cells by
40% or more in 5 of the 7 DMM cell lines examined (Fig. 1B and
Supplementary Fig. S1). Treatment with an EGFR-specific siRNA
decreased the cell viability in H2373 dramatically and Y-meso8A
to a lesser degree; however, most DMM cell lines and the Met-5A
line were left unaffected with EGFR knockdown. The effects
of EGFR or Aki1 siRNAs in a noncancerous mesothelial cells,
Aki1 siRNA
Aki1
211H
H290
H2052
H2373
Y-meso14
− +
− +
− +
− +
− +
Aki1
p-Akt
EGFR
Akt
CyclinA
EGFR
β-Acn
β-Acn
B
D
Medium
Scramble siRNA
Aki1 siRNA
211H
EGFR siRNA
120
100
100
80
80
60
60
40
40
20
20
20
0
0
0
siAki1
40
G0−G1
Scramble
60
% Cell populaon
80
siAki1
G2−M
S
Scramble
120
100
% Cell viability
H2052
120
Figure 1.
Role of Aki1 on cell viability and cell cycle in malignant pleural mesothelioma cells. A, the expression of Aki1- and EGFR-related proteins in malignant pleural
mesothelioma cell lines (211H, H290, H2052, H2373, H2452, Y-meso8A, and Y-meso14) and human mesothelial cell lines (Met-5A). They were lysed and the indicated
proteins were detected by Western blotting analysis. B, cells were treated with Aki1, EGFR, or scramble control siRNA. After 72 hours incubation, cell viability
was determined by MTT assay. C, cells were treated with scramble control or Aki1 siRNA. After 24 hours incubation, cells were lysed and the indicated proteins
were detected by Western blotting. D, cells were treated with scramble control or Aki1 siRNA. After 48 hours incubation, cell cycle was determined with an
Annexin V–FITC Apoptosis Detection Kit I. The figure shows percentages of cell population (G2–M, S, and G0–G1).
OF4 Cancer Res; 75(19) October 1, 2015
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Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Aki1 Is a Therapeutic Target in DMM
Fig. S2). Notably, the reduction in cell viability is very similar to
cells treated with Aki1 siRNA. The immortalized mesothelial cell
line, Met-5A, does not appear to be as affected by CREB1 and Aki1
knockdown as the DMM cell lines (Supplementary Fig. S3). These
findings suggest that the Aki1–CREB1 axis has an important role
in maintaining the viability of DMM cells.
To further prove that CREB1 activity is important in DMM cell
viability and is downstream of Aki1, we surmised that enforced
expression of CREB1 might overcome Aki1 knockdown and
maintain cell viability. To do this, we transfected 211H cells with
empty Vector, CREB-WT, and a mutated CREB that lacks transcriptional activity, CREB-S133A. Western blot analysis of transfected 211H cells clearly shows an increase in both the total
CREB1 and phosphorylated CREB1 (Fig. 2F). Treatment of the
transfected cells with Aki1 siRNA reduced the phosphorylation
of CREB1 in the vector control cells as expected. Although
CyclinA
β-Acn
D
Scramble
Luciferase acvity (relave to scramble)
siAki1
t-CREB1
β-Acn
siCREB1
Scramble
Y-meso14
siCREB1
Scramble
siAki1
siCREB1
Scramble
siAki1
siCREB1
Scramble
H2373
p-CREB1
t-CREB1
b
H2052
Aki1
p-CREB1
p-CREB1 (S133)
Scramble
211H
H290
H2052
Y-meso14
Met5A
a
H290
211H
siAki1
C
B
siCREB1
A
siAki1
the immortalized mesothelial cell line, Met-5A (Fig. 2B). Western
blotting analyses confirmed the phosphokinase array result showing that Aki1 downregulation decreases phospho-CREB1 levels in
multiple DMM cell lines (Fig. 2C). The Western blot analysis also
showed that the specific downregulation of CREB1 by siRNA
reduced cyclin A protein expression in five DMM cell lines, which
is also seen with Aki1 knockdown. To evaluate how Aki1 loss
affects CREB activity, we transfected DMM cell lines with a CRE
reporter. The CRE-Luciferase reporter assay demonstrated that the
treatment with Aki1 siRNA significantly inhibits CREB1 activity in
211H and H2052 cells compared with scramble control (P < 0.05
by one-way ANOVA; Fig. 2D).
We next evaluated the role of CREB1 activity in maintaining
DMM cell viability. DMM cells treated with a CREB1 siRNA show
remarkably reduced cell viability when compared with cells
treated with the scrambled control (Fig. 2E and Supplementary
siAki1
1.5
1
*
0.5
0
500
Scramble
siAki1
siCREB1#1
siCREB1#2
300
200
Day 1 Day 2 Day 3 Day 4
% Cell viability
% Cell viability
800
600
400
Y-meso14
Scramble
siAki1
siCREB#1
siCREB#2
G
Aki1 siRNA
Aki1
Vector
CREB-WT
− +
− +
100
80
siAki1
*
60
40
20
0
p-CREB1
200
t-CREB1
0
Day 1 Day 2 Day 3 Day 4
120
β-Acn
H290
Scramble
siAki1
siCREB#1
siCREB#2
Scramble
t-CREB1
Day 1 Day 2 Day 3 Day 4
500
400
300
200
100
0
FLAG
0
H2052
H
p-CREB1
100
0
CREB-KD
400
% Cell viability
1,000
Scramble
siAki1
siCREB1#1
siCREB1#2
% Cell viability
% Cell viability
1,500
F
Vector
H2052
211H
CREB-WT
211H
E
*
Day 1 Day 2 Day 3 Day 4
β-Acn
Figure 2.
Aki1 maintains the viability of DMM cells through CREB1 activation. A, human kinase phosphorylation array analysis in 211H cells with scramble control siRNA (a) or Aki1
siRNA (b) after 48 hours incubation. B, total and phosphorylation of CREB expressions in four DMM (211H, H290, H2052, and Y-meso14) and mesothelial
(Met-5A) cells. Cells were lysed and the indicated proteins were detected by Western blotting. C, luciferase activity assay against CREB1. Cells were stably transduced
to express wild-type CRE reporters, which binds to CREB1 as a certain DNA sequences, and ratios between the two were compared after RNAi transfection
of Aki1 or scramble control; , P < 0.05 versus scramble control by one-way ANOVA. D, cells were treated with scramble control, Aki1, or CREB1 siRNA. After
24 hours incubation, cells were lysed and the indicated proteins were detected by Western blotting analysis. E, cells were treated with Aki1, CREB1 (#1, #2), or
scramble control siRNA. After 24, 48, 72, and 96 hours incubation, cell viability was determined by MTT assay. F, pCF plasmid–expressing empty vector
(Vector), FLAG-tagged human CREB (CREB-WT), or FLAG-tagged human CREB-kinase dead–mutated (CREB-KD) clones were transfected into 211H cells.
They were lysed and the indicated proteins were detected by Western blotting analysis. G, total and phosphorylation of CREB1 expressions in 211H cells with
transfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 or scramble control siRNA. After 24 hours incubation, cells were lysed and the
indicated proteins were detected by Western blotting. H, 211H cells with transfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 or
scramble control siRNA. After 72 hours incubation, cell viability was determined by MTT assay; , P < 0.05 versus cells with vector or CREB-KD clones treated
with Aki1 siRNA by one-way ANOVA.
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Yamada et al.
phosphorylated CREB1 in CREB-WT transfected cells was reduced
in the Aki1 knockdown cells, it was still expressed at a relatively
high level compared with vector control (Fig. 2G). More importantly, cells expressing wild-type CREB had a significantly higher
viability than vector control cells or cells expressing the mutated
form of CREB (P < 0.05 by one-way ANOVA; Fig. 2H). These
findings confirm that there is indeed an Aki1–CREB axis and that
increased CREB1 activity downstream of Aki1 is important in
maintaining cell viability of DMM cells.
Aki1 regulates CREB by modulating protein kinase A
phosphorylation and activity.
To gain a better understanding of how Aki1 regulates CREB1
activity, Aki1 was overexpressed and PKA, a major regulator of
CREB phosphorylation at S133, was examined. To do this, we
made an Aki1 expression construct that was transfected into both
HEK293T and 211H cells. The exogenously expressed Aki1 protein induced the phosphorylation of CREB1 and the catalytic
subunit of Protein Kinase A (PKAC) in both cell lines (Fig. 3A).
Alternatively, treatment of two DMM cell lines with Aki1 siRNA
reduced PKAC phosphorylation (Fig. 3B). We also determined the
effect of the PKA inhibitor KT5720 on the cell viability of DMM
and Met-5A cells. Treatment of DMM cells with KT5720 reduces
211H
Aki1-WT
Aki1-WT
Vector
293T
Vector
A
CREB1 phosphorylation after 4 and 8 hours incubations (Fig. 3C).
KT5720 also inhibits the growth of DMM cells in a dose-dependent manner with little effect on the Met-5A cells (Fig. 3D).
In addition, we investigated the efficacy of the CBP–CREB interaction inhibitor (CBP–CREB-I) on the cell growth of DMM and
Met-5A cells. CBP–CREB-I inhibits the CREB1 phosphorylation in
DMM cells (Fig. 3E) and strongly suppresses the viability of DMM
cells, with marginal effects on the Met-5A cells (Fig. 3F). Thus, our
findings clearly show that Aki1 mediates the activation of the
PKA–CREB axis in DMM cells and may provide an attractive
therapeutic option in DMM (Supplementary Fig. S4).
Direct injection of Aki1 siRNA into the pleural cavity of a DMM
orthotopic mouse model dramatically decreases tumor
formation
We next examined the antitumor potential of Aki1 siRNA by
directly injecting the siRNA into pleural cavity of a DMM orthotopic mouse model. The DMM 211H cell line expressing luciferase
was implanted into the pleural cavity of immunocompromised
mice. Seven days after implantation of the DMM cells Aki1 siRNA
or a control siRNA complexed with invivofectamine was injected
as a single dose. Bioimaging was used to monitor disease progression. The single injection of Aki1 siRNA into the pleural cavity
C
E
Y-meso14
211H
KT5720
−
4h 8h
−
CBP-CREB-I
4h 8h
HA
p-CREB1
p-CREB1
Aki1
t-CREB1
t-CREB1
β-Acn
β-Acn
p-CREB1
211H
H2052
Y-meso14
− +
− +
− +
t-CREB1
D
F
t-PKACα
Medium
B
211H
Aki1 siRNA
− +
H290
− +
p-PKAC
PKACα
p-CREB1
t-CREB1
Medium
1 μmol/L
120
100
100
% Cell viability
β-Acn
0.1 μmol/L
120
80
60
40
% Cell viability
p-PKAC
0.1 μmol/L
0.3 μmol/L
80
60
40
20
20
0
0
β-Acn
Figure 3.
Aki1 regulates CREB by modulating protein kinase A phosphorylation and activity. A, HEK293T and 211H cells were stably transfected with pHM6 plasmid–expressing
HA-tagged recombinant human WT-Aki1 proteins (Aki1-WT) or empty vector (Vector). They were lysed and the indicated proteins were detected by
Western blotting analysis. B, cells were treated with scramble control or Aki1 siRNA. After 24 hours incubation, cells were lysed and the indicated proteins
were detected by Western blotting. C, cells were treated with KT5720 (0.1 mmol/L). After 4 and 8 hours incubation, cells were lysed and the indicated proteins were
detected by Western blotting. D, cells were treated with KT5720 (0.1 and 1.0 mmol/L). After 72 hours incubation, cell viability was determined by MTT assay.
E, cells were treated with CBP–CREB interaction inhibitor (CBP–CREB-I; 0.1 mmol/L). After 4 hours incubation, cells were lysed and the indicated proteins were
detected by Western blotting. F, cells were treated with CBP-CREB-I (0.1 and 0.3 mmol/L). After 72 hours incubation, cell viability was determined by MTT assay.
OF6 Cancer Res; 75(19) October 1, 2015
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Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Aki1 Is a Therapeutic Target in DMM
significantly inhibited tumor growth in comparison with that of
scramble control 12 days after Aki1 treatment (P < 0.05 by oneway ANOVA; Fig. 4A and B). We confirmed the knockdown of
Aki1 and the inhibition of CREB1 phosphorylation in tumors by
Western blotting analysis (Fig. 4C). These results clearly indicate
the therapeutic potential of Aki1 siRNA against DMM localized to
the pleural cavity.
Aki1 and phosphorylated CREB are frequently expressed in
human DMM tumors
We examined Aki1 expression in 68 clinical specimens
obtained from 67 DMM patients (Fig. 5A and B and Supplementary Fig. S5). Of 68 DMM tumors, Aki1 was expressed at some level
in 65 (95.6%). High cytoplasmic Aki1 protein expression was
detected in 15 (22.1%), intermediate in 22 (32.4%), low in 28
(41.2%), and negative expression in 3 (4.4%) of 68 DMM tumors.
Aki1 protein expression was detected in 37 of 37 (100%) of
epithelioid DMM, 8 of 10 (80%) sarcomatoid DMM, and 12 of
12 (100%) biphasic DMM (Supplementary Fig. S6A). We next
examined phosphorylated CREB expression in 35 clinical specimens obtained from 34 DMM patients (Fig. 5A and B and
Scramble siRNA
A
Aki1 siRNA
6 x 109
4 x 109
*
siRNA treatment
2 x 109
Da
y
Da 2
y
Da 5
y7
Da
Da y 9
y
Da 12
y
Da 14
y
Da 16
y1
9
Total flux (p/sec/cm2)
8 x 109
9
19
Scramble
siRNA
5
Luminescence
Scramble
siRNA
Day
C
2.5
2.0
Aki1 siRNA
1.5 ×107
Aki1 siRNA
B
Aki1
p-CREB1
1.0
0.5
Radiance
(p/sec/cm2/sr)
t-CREB1
β-Acn
Figure 4.
Direct injection of Aki1 siRNA into the pleural cavity of a DMM orthotopic
mouse model dramatically decreases tumor formation. 211H transfected the
6
Eluc/EGFP gene cells (211H/Eluc; 1 10 cells per 100 mL of PBS) and were
injected into the right pleural cavity of 5-week-old male SCID mice. On day 7
after cell inoculation, 100 mg of either scramble or Aki1 siRNA complexed with
invivofectamine was injected into the right pleural cavity. A, luminescence
was evaluated twice per week from day 2 to day 19 after cell inoculation. Bars,
SE is shown for groups of 8 mice; , P < 0.05 by one-way ANOVA. B, the
appearance of representative mice is shown. C, the harvested tumors, which
are on day 7 after injection of siRNA complex, were examined for Aki1 and
phosphorylated CREB1 by Western blotting analysis.
www.aacrjournals.org
Supplementary Fig. S5). Of 35 DMM tumors, phosphorylated
CREB was expressed at some level in 30 tumors (85.7%). High
nuclear expression of phosphorylated CREB protein was detected
in 16 (45.7%), intermediate in 12 (34.3%), low in 2 (5.7%), and
negative expression in 5 (14.3%) of 35 DMM tumors. Phosphorylated CREB was detected in 20 of 21 (95%) epithelioid, in 1 of 5
(20%) sarcomatoid, and in 6 of 6 (100%) biphasic DMM (Supplementary Fig. S6A). There was a statistically significant correlation between expression of Aki1 and phosphorylated CREB
(Spearman rank correlations ¼ 0.521; P ¼ 0.002, N ¼ 34; Fig.
5C). The epithelioid DMM demonstrated the strongest correlation between expression levels of Aki1 and phosphorylated CREB
(Spearman rank correlations ¼ 0.561; P ¼ 0.009, N ¼ 21;
Supplementary Fig. S6B). These findings suggested involvement
of phosphorylated CREB in Aki1-mediating signaling in DMM
tumors, especially epithelioid types.
Discussion
Prior observations where the knockdown of EGFR by siRNA
hardly affected cell viability of DMM (23), suggested that targeting
molecules downstream of the multiple RTKs is a more effective
therapeutic option.
Previously, we identified Aki1 as a scaffold protein critical for
transmitting the signal through the EGFR–PI3K–PDK1–Akt pathway. Downregulation of Aki1 dramatically decreased cell viability
in EGFR-mutant lung cancer cell lines (11). These data suggested
that Aki1 had a role in aberrant signaling through the EGFR–
PI3K–Akt pathway. Our screening approach through monitoring
altered kinase phosphorylation in Aki1 knockdown DMM cells
led to the discovery that Aki1 was signaling through a different
pathway. On the basis of in vitro studies, we showed for the first
time, that Aki1 is critical in proliferation and cell viability of
DMM. This process is independent of EGFR signaling and involves
CREB1 activation by modulating PKA phosphorylation.
The observation that phosphorylation of CREB1 was decreased
by knockdown of Aki1 agrees with previous studies in nonsyndromic mental retardation where Aki1 is a regulator of the cAMP–
PKA pathway in neuronal cell differentiation (24, 25). Mutant
mice with a truncated Aki1 showed defective PKA activation and
CREB phosphorylation in the neuronal system (25). Hence, Aki1
has important roles in regulating the activity of the PKA–CREB
axis in neuronal differentiation and in brain development as well
as in malignant tumors.
CREB1 is a 43 kDa basic/leucine zipper transcription factor that
mediates signaling from extracellular stimulation and can be
phosphorylated and activated by many kinases, including PKA,
protein kinase C, Ca2þ/calmodulin-dependent kinase, extracellular
signal–regulated kinases, Akt, and p90 ribosomal S6 kinase (26).
Phosphorylation of CREB1 on Serine 133 has an important role in
the transcriptional activity of CREB1 by promoting the interaction
with the coactivator CREB-binding protein (CBP)/p300. CREB
promotes cell proliferation, cell-cycle progression, and survival of
myeloid cells through induction of specific target genes (27). In
addition, the overexpression and activation of CREB1 has been
associated with poor prognosis in nonsmokers with NSCLC (28),
melanoma metastasis (29), and acute myelogenous leukemia (30).
With regard to the roles of CREB1 in DMM, CREB-induced inflammation is associated with cell growth and asbestosis-related DMM
(31). Recent studies in DMM cells, both in vitro and in vivo, as well as
our study, demonstrate that CREB inhibition by RNAi or small
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Yamada et al.
A
Aki1
(n = 68)
Score
p-CREB
(n = 35)
Case
%
Case
%
0 (negative)
1 (low)
2 (intermediate)
3 (high)
3
28
22
15
4.4
41.2
32.4
22.1
5
2
12
16
14.3
5.7
34.3
45.7
positive
65
95.6
30
85.7
B
C
r = 0.521 (Spearman rank)
P = 0.0016
Calrenin
p-CREB
Aki1
3
Aki1 score (0–3)
H&E
Figure 5.
Aki1 and phosphorylated CREB are
frequently expressed in DMM. Clinical
specimens from DMM patients
evaluated for Aki1 and phosphorylated
CREB by immunohistochemistry. A,
high cytoplasmic Aki1 protein
expression was detected in 15 (22.1%),
intermediate in 22 (32.4%), low in 28
(41.2%), and negative expression
in 3 (4.4%) of 68 DMM tumors. High
nuclear expression of phosphorylated
CREB protein was detected in 16
(45.7%), intermediate in 12 (34.3%),
low in 2 (5.7%), and negative
expression in 5 (14.3%) of 35 DMM
tumors. B, representative images of
epithelioid DMM, hematoxylin and
eosin stain (H&E), showing diffuse
expression of calretinin, p-CREB, and
Aki1. C, the correlation of expression
scores between Aki1 and
phosphorylated CREB in tumors of
DMM patients (Spearman rank
correlations ¼ 0.521; P ¼ 0.002,
N ¼ 34).
2
1
0
0
molecules, results in significant attenuation of proliferation, migration, and drug resistance to cytotoxic chemotherapy, such as
doxorubicin (31, 32). In addition, our study demonstrates that
treatment with small-molecule inhibitors targeting PKA or CREB
reduces proliferation of DMM cells in vitro.
Our data would suggest that inhibition of Aki1, or a downstream pathway component such as PKA, would be an effective
strategy to treat DMM. However, Aki1 is a scaffold protein without
a known small-molecule inhibitor. An inhibitor of PKA would be
an option, and finding a PKA inhibitor is currently an area of active
research (33, 34). Phase I trials of an antisense RNA against the
PKA regulatory subunit, RIa, were performed in multiple solid
tumors (35). This was a systemic approach with i.v. infusion of the
antisense RNA, and although it appeared that PKA activity
decreased as measured by serum extracellular PKA (ECPKA)
activity, there were no objective responses to treatment. A previous study showed that tumor growth of several cancer types was
blocked by multiple i.p. injections of siRNAs for Zcchc11, Lin28A,
or Lin28B using a xenograft model (36). A recent article reports the
pleural and peritoneal injection of a recombinant human p53containing adenovirus to control malignant effusions (37). This
treatment provided excellent local control of the effusion, was
safe, and could be an option for patients unable to undergo
standard chemotherapeutic treatments. As for a local therapy
using pleural injection, we have demonstrated the efficacy of a
single direct injection into the pleural cavity of a siRNA to Aki1 in
OF8 Cancer Res; 75(19) October 1, 2015
1
2
p-CREB score (0–3)
3
an orthotopic mouse model of DMM. Given the unique aspects of
mesothelioma biology, and particularly its localization to the
pleural cavity in most cases, direct injection of an antisense RNA
could be practical, and provide local control and reduce side
effects associated with standard systemic cytotoxic chemotherapy.
Phosphorylated CREB1 has been shown to be increased in mesothelioma compared with reactive mesothelial hyperplasia and
normal lung tissue (31). The results of human tissue analysis in
our study are in keeping with that observation, but also for the first
time document that Aki1 is also frequently overexpressed and
correlates with expression of phosphorylated CREB in DMM.
Among DMM subtypes, the strongest correlation between the
expression levels of these molecules was seen in epithelioid DMM,
although the cell-based analysis showed the high expression of
both proteins even in sarcomatoid type. We cannot explain why
these correlations were not apparent in our TMA samples, but is
likely related to the small number of sarcomatoid samples in our
TMA. Further experiments are needed to confirm these histologic
differentiations in DMM.
In summary, we have demonstrated that Aki1 has roles in the
cell growth, cell cycle, and transcription though the activation of
PKA–CREB1 signaling for DMM cells. Direct application of a
single dose of Aki1 siRNA into the pleural cavity of mice significantly inhibited the growth of 211H cells within the pleural cavity
in an orthotropic implantation model. Furthermore, Aki1 is
frequently expressed in DMM tumors and correlated with
Cancer Research
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Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Aki1 Is a Therapeutic Target in DMM
phosphorylation of CREB1, especially epithelioid types. Our data
provide a rationale for targeting Aki1 by intrathoracic therapy in
patients with local invasive DMM tumors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: T. Yamada, J.M. Amann, D.P. Carbone
Development of methodology: T. Yamadai
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): K. Fukuda, K. Shilo
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): T. Yamada, J.M. Amann, K. Fukuda, S. Takeuchi,
H. Uehara, K. Shilo, D.P. Carbone
Writing, review, and/or revision of the manuscript: T. Yamada, J.M. Amanni,
K. Shilo, D.P. Carbone
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): K. Fukuda, N. Fujita, S. Iwakiri, K. Itoi, S. Yano,
D.P. Carbone
Study supervision: J.M. Amann, D.P. Carbone
Acknowledgments
The authors appreciate that NCI-H290 was provided by Dr. Adi F. Gazdar
(University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Yoshitaka Sekido (Aichi Cancer Research Center Institute, Aichi, Japan). The authors
thank Dr. Takayuki Nakagawa and Mr. Kenji Kita (Cancer Research Institute,
Kanazawa University, Kanazawa, Japan) for technical advice and for technical
support.
Grant Support
This study was supported by Grants-in-Aid for Cancer Research (25860638 to
T. Yamada), Scientific Research on Innovative Areas "Integrative Research on
Cancer Microenvironment Network" (22112010A01 to S. Yano) from the
Ministry of Education, Culture, Sports, Science, and Technology of Japan, and
the Dellapezze Family Foundation (D.P. Carbone).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received April 1, 2015; revised June 6, 2015; accepted June 26, 2015;
published OnlineFirst August 20, 2015.
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Research.
Published OnlineFirst August 20, 2015; DOI: 10.1158/0008-5472.CAN-15-0858
Akt Kinase-Interacting Protein 1 Signals through CREB to
Drive Diffuse Malignant Mesothelioma
Tadaaki Yamada, Joseph M. Amann, Koji Fukuda, et al.
Cancer Res Published OnlineFirst August 20, 2015.
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