The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas

Article (PDF Available)inModern Pathology 25(1):131-9 · August 2011with23 Reads
DOI: 10.1038/modpathol.2011.133 · Source: PubMed
Abstract
AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.

Figures

The expression of phospho-AKT1 and
phospho-MTOR is associated with a favorable
prognosis independent of PTEN expression
in intrahepatic cholangiocarcinomas
Dakeun Lee
1,6
, In-Gu Do
2,6
, Kyusam Choi
2
, Chang Ohk Sung
1
, Kee-Taek Jang
1
,
Dongwook Choi
3
, Jin Seok Heo
3
, Seoung Ho Choi
3
, Jongmin Kim
4
, Jin Young Park
4
,
Hyung Jin Cha
5
, Jae-Won Joh
3
, Kwan Yong Choi
5
and Dae Shick Kim
1
1
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine,
Seoul, Korea;
2
Experimental Pathology Center, Samsung Cancer Research Institute, Samsung Medical Center,
Seoul, Korea;
3
Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of
Medicine, Seoul, Korea;
4
CbsBioscience, Yuseong-gu, Daejeon, Korea and
5
Department of Life Science,
Pohang University of Science and Technology, Pohang, Korea
AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications
in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic
cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome
10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal
protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101
intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the
expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data
and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and
PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P ¼ 0.0137,
0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and
p-AKT1 showed tendency (P ¼ 0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated
with well-to-moderately differentiated tumors (Po0.001) and tumors without metastasis ( P ¼ 0.046). Expression
levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other,
except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were
associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that
dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic
cholangiocarcinoma, but not necessarily in the progression of the disease.
Modern Pathology (2012) 25, 131–139; doi:10.1038/modpathol.2011.133; published online 26 August 2011
Keywords:
AKT1; intrahepatic cholangiocarcinoma; MTOR; prognosis; PTEN
Cholangiocarcinoma is a rare neoplasm in the
developed world, and this tumor arises from the
epithelial cells of the bile ducts. Anatomically
cholangiocarcinomas are classified as intrahepatic
cholangiocarcinomas, hilar cholangiocarcinomas,
and distal extrahepatic bile duct cancers. Intra-
hepatic cholangiocarcinoma usually refers to a
tumor originating within the second bifurcation
of hepatic ducts. The incidence of intrahepatic
cholangiocarcinoma is even lower as it accounts for
less than 10% of all cholangiocarcinomas,
1
although
some recent studies have showed its increasing
incidence in the West.
2,3
Complete surgical resection
Received 15 February 2011; revised 13 July 2011; accepted 18 July
2011; published online 26 August 2011
Correspondence: Dr DS Kim, MD, PhD, Department of Pathology,
Samsung Medical Center, Sungkyunkwan University School of
Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic
of Korea.
E-mail: oncorkim@skku.edu
6
These authors contributed equally to this paper.
Modern Pathology (2012) 25, 131139
& 2012 USCAP, Inc. All rights reserved 0893-3952/12 $32.00 131
www.modernpathology.org
is the only treatment that offers the possibility of
long-term survival in patients with intrahepatic
cholangiocarcinoma. However, the number of can-
didates who can undergo curative surgery is limited,
because a significant proportion of patients present
with advanced disease.
2
Even for patients who
undergo surgical resection, the 5-year survival rate
is still only 22–36%.
4
At present, little is known
about the molecular carcinogenesis of intrahepatic
cholangiocarcinoma. A better knowledge of the
molecular pathways involved in the development
and progression of this disease may help devise new
treatment strategies to improve the clinical outcome
of patients with intrahepatic cholangiocarcinoma.
AKT1 (also known as protein kinase B) is a serine/
threonine protein kinase and has a central role in the
regulation of diverse biological functions. Phospha-
tidylinositol 3-kinase (PIK3CA), an upstream
regulator of AKT1, is activated by receptor tyrosine
kinases such as epidermal growth factor receptor or
insulin-like growth factor 1 recep tor, and it catalyzes
the conversion of phosphatidylinositol-4,5-bispho-
sphate (PIP2) to phosphatidylinositol-3,4,5-tripho-
sphate (PIP3). PIP3 binds to AKT1, leading to
the membrane recruitment of AKT1 and phosphor-
ylation of AKT1.
5
Activated AKT1 phosphorylates,
activates, or inhibits many downstream targets to
regulate various cellular functions, including cell
metabolism, protein synthesis, cell survival, inhibi-
tion of apoptosis, and cell cycle progression.
5,6
The
aberrant expression or deregulation of AKT1 has
been observed in many different types of tumors,
and it is thought to be an important step
for tumorigenesis or disease progression in these
tumors.
7–9
On the other hand, the phosphatase
and tensin homolog deleted on chromosome 10
(PTEN) was first discovered as a tumor suppressor
on human chromosome 10q23. PTEN has a lipid
phosphatase activity, and it hydrolyzes the
3-phosphate on PIP3 to generate PIP2; therefore,
PTEN negatively regulates AKT1.
10
Specifically, the
loss or mutation of PTEN leads to activation of
AKT1, which in turn promotes the anti-apoptotic
and pro-cell cycle entry pathw ays, which are
believed to be essential in tumorigenesis. A series
of studies have shown that PTEN is frequently
mutated or lost in many human malignancies,
including glioblastomas, breast cancer, lung cancer,
colon cancer, and melanomas.
11–13
In addition,
decreased PTEN expression has been correlated
with progression of solid cancers.
14,15
Mammalian target of rapamycin (MTOR) is a
member of the PIK3CA-related kinase family, and
it is the key downstream signaling molecule of
AKT1. Activated MTOR supports cell growth, cell
survival, and protein synthesis, largely by phos-
phorylating two downstream targets: p70 ribosomal
protein S6 kinase (RPS6KB2) and eukaryotic
initiation factor 4E (eIF4E)-binding protein-1
(EIF4EBP1).
16
Activated p70S6K subsequently
phosphorylates specific ribosomal protein S6,
and phosphorylated S6, in turn, promotes protein
translation and cell growth. Hypophosphorylated
EIF4EBP1 binds to, and thereby inactivates the cap-
binding protein eIF4E, whereas phosphorylation of
EIF4EBP1 results in release of eIF4E, which then
binds to the cap structure of mRNA to initiate
protein translation.
17
As all of these proteins are
regulators of protein synthesis, they can act as proto-
oncogenes, and in fact, they are frequently activated
in human malignancies.
18,19
Accordingly, the sup-
pression of MTOR activity could inhibit cell growth,
eliciting an anti-tumor effect. Inhibitors of proteins
that are involved in MTOR signaling, such as
rapamycin (sirolimus) and its analogs CCI-779
(temsirolimus), have recently been under active
preclinical or clinical investigations for treating
cancers, and they have been suggested as potential
therapeutic agents.
20
Although the AKT1 signaling pathway has been
under active investigation in a variety of cancers,
there has been limited in vivo data regarding the
activity or the prognostic value of the signaling
proteins of AKT1 pathway in intrahepatic cholan-
giocarcinoma. Further, the coexpression of these
proteins in intrahepatic cholangiocarcinoma has not
yet been described. To better understand the role of
AKT1 signaling pathway in the tumorigenes is and
progression of intrahepatic cholangi ocaricnoma, we
investigated the statuses of the proteins in this
pathway, and their correlations with the clinico-
pathological data in patients with intrahepatic
cholangiocarcinoma. Survival analysis was per-
formed to assess the prognostic relevance of these
biomarkers. Furthermore, we sought to explore the
correlations between the expression levels of
the signaling proteins, and we investigated whether
the conventional model of the PTEN–AKT1–MTOR–
RPS6KB2/EIF4EBP1 pathway is valid in this type
of tumor. Thus, this study will have important
implications for further elucidating the molecular
mechanisms, and finding novel therapeutic targets
in this rare subtype of cholangiocarcinomas.
Patients and methods
Patients and Tumor Samples
The study subjects included 101 patients with
intrahepatic cholangiocarcinoma, who were surgi-
cally treated at Samsung Medical Center (Seoul)
from July 1996 to December 2007. All the tumor
samples were from the primary cholangiocarcinoma
at the time of surgical resection. None of the patients
underwent chemotherapy or radiotherapy before
surgery. Post-operative adjuvant treatments
were performed in a subset of patients as follows:
chemotherapy in 10, radiotherapy in 5, and both
modalities in 12 patients. The cholangiocarcinomas
that had their epicenter only within the hepatic
parenchyma were included, whereas carcinomas of
the hilar or perihilar bile ducts (Klatskin tumors)
AKT1’s role in intrahepatic cholangiocarcinoma
132 D Lee et al
Modern Pathology (2012) 25, 131139
and carcinomas of the mid to distal bile duct were
excluded. Carcinomas arising in the gallbladder or
extrahepatic bile duct, with extension to the
intrahepatic bile duct, were also excluded in this
study. All the tumors were classified according to
the World Heal th Organization classification.
21
The
medical records were reviewed to obtain data that
included the age and gender of the patients, the
survival time, and the survival status. The histo-
pathologic data such as the tumor location, size, and
histological type were obtained from the pathology
reports. In this study, we used pT classification by
AJCC 6
th
edition. This study received appropriate
human protection approval from the Institutional
Review Board of Samsung Medical Center.
Immunohistochemistry
Immunohistochemi cal (IHC) studies were carried
out on formalin-fixed, paraffin-embedded, 4-mm
thick tissue sections. The primary antibodies used
were rabbit monoclonal anti-human PTEN (138G6,
1:200 dilution, Cell Signaling Technology, Beverly,
MA, USA), phosphorylated (p) AKT 1 (p-AKT1)
(D9E, 1:20 dilution, Cell Signaling Technology),
p-MTOR; (49F9, 1:50 dilution, Cell Signaling
Technology), p-RPS6KB2 (1A 5, 1:500 dilution, Cell
Signaling Technology), and p-EIF4EBP1 (236B4,
1:100 dilution, Cell Signaling Technology). The
tissue sections were deparaffinized three times
in xylene for a total of 15 min, and they were
subsequently rehydrated. Immunostaining for
PTEN, p-MTOR, p-RPS6KB2, and p-EIF4EBP1 was
performed using a Bond-max autoimmunostainer
(Leica Biosystem, Melbourne, VIC, Australia) with
ER1 or ER2 retrieval buffers, and a Bond Polymer
refine detection system DS9800 (Vision Biosystems,
Melbourne, VIC, Australia). For the immunostaining
for p-AKT1, the slides were autoclaved for 1 min at
110 1C in citrate buffer (pH 6.0). After the endo-
genous peroxidase activity was blocked with 3%
hydrogen peroxidase for 10 min, the primary
antibody incubation was carried out overnight at
4 1C. The antigen–antibody reaction was detected
using the DAKO REAL Envision Detection system,
Peroxidase/DAB K5007 (DAKO, Glostrup,
Denmark). Counter-staining was performed with
Mayer’s hematoxylin, and then the slides were
examined by light microscopy. Appropriate positive
controls were selected according to the manufac-
turer’s recommendations. The slides processed
without the primary antibodies were used as
negative controls.
Interpretation of IHC Staining
The expressions of PTEN, p-AKT1, p-MTOR,
p-RPS6KB2, and p-EIF4EBP1 were examined by
two patholog ists (DL and COS), who were blinded to
the clinical data of the patients. The pathologists
inspected the complete tumor sections at 40 and
200 magnifications. The discordant cases were
reviewed under a multi-headed microscope to
achieve a consensus. In this study, the tumor cells
showing cytoplasmic staining with or without the
nucleus or cell membrane staining were judged
to be positive for PTEN, p-AKT1, p-MTOR, and
p-EIF4EBP1, whereas nucleus staining was consid-
ered positive for p-RPS6KB2. The stai ning intensity
was determined as 0 (absent ), 1 (weak), 2 (moder-
ate), an d 3 (strong). The photomicrographs of the
biomarkers tested strong positive in tumor tissue are
depicted in Figure 1. On the other hand, PTEN,
p-AKT1, and p-EIF4EBP1 showed weak cytoplasmic
staining in normal bile ducts in some cases.
p-MTOR showed strong cytoplasmic reactivity in
normal bile ducts as previously described,
22
whereas p-RPS6KB2 was not expressed in normal
bile ducts. For comparison with the clinical vari-
ables and survival, the expression levels of these
proteins were semiquantified using an IHC score
calculated by multiplication of the staining intensity
(0B3) with the percent age of positive tumor cells
(0B100%). The ranges of IHC scores for each
biomarker in tumor tissue are as follows: 0B200
for PTEN (median 3), 0B140 for p-AKT1 (median
10), 0B285 for p-MTOR (median 60), 0B297 for
p-EIF4EBP1 (median 10), and 0B120 for p-RPS6KB2
(median 0) (Supplementary Figure S1). The
cut-off values for each biomarker were set to best
Figure 1 The photomicrographs of IHC staining that showed strong reactivity in this study. (a) PTEN, (b) p-AKT1, (c) p-MTOR, and
(d) p-EIF4EBP1 show strong cytoplasmic staining of the tumor cells, and some positive cells stained for p-AKT1 and p-MTOR also
exhibit membranous staining in these examples. On the other hand, (e) p-RPS6KB2 demonstrates strong nuclear reactivity in the
intrahepatic cholangiocarcinoma specimens.
AKT1’s role in intrahepatic cholangiocarcinoma
DLeeet al 133
Modern Pathology
(2012) 25, 131139
discriminate the patients’ survival statistically. The
cut-off IHC scores that were considered positive are
as follows: 415 for PTEN, p-AKT1, and p-RPS6KB2,
and 450 for p-MTOR and p-EIF4EBP1. The cases
with IHC scores below the cut-off values were
considered negative.
Western Blot Analysis
To verify the correlations of IHC scores with
expression levels of p-AKT1 and p-MTOR in the
intrahepatic cholangiocarcinoma, western blot ana-
lysis was performed for the eight cases of intra-
hepatic cholangiocarcinoma for which fresh-frozen
tumor tissues were available. Frozen tissue exam-
ination confirmed that 70–80% of the specimens
were tumor tissue. The frozen tissues were homo-
genized in tissue lysis buffer (20 mM HEPES
(pH 7.4), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM
EDTA, 0.05% (v/v) Triton X-100, 20 mM beta-
glycerophosphate, 1 mM Na3VO4, 10 mM NaF,
0.5 mM DTT, and the protease inhibitor c omplete
Mini (Roche, Penzberg, German y)). The samples
were incubated for 15 min on ice, and then, they
were centrifuged at 14 000 g at 4 1C for 30 min. The
samples containing 15 mg of protein was separated
on 10% SDS-polyacrylamide gel, and then, the
proteins were transferred to nitrocellulose mem-
branes. These membranes were blocked in Tris-
buffered saline containing 5% skim milk and 0.05%
Tween-20 (TBST) for 1 h at room temperature, before
incubation with the primary antibody overnight at
4 1C. Western blotting was conducted using a 1:1500
dilution of antibody against p-AKT1 (Cell Signaling
Technology, Beverly, MA) and a 1:1000 dilution of
antibody against p-MTOR (Cell Signaling Techno-
logy). Peroxidase-conjugated goat antimouse IgG
diluted 1:5000 was used as a secondary antibody.
Peroxidase was visualized via an enhanced
chemiluminescence assay (Amersham, Piscataway,
NJ, USA). The images of the gels were scanned
using a Gel Docum entation System (Gel Doc
1000, Bio-Rad, Hercules, CA, USA), and the
relative densities were analyzed using a Multi-
analyst fingerprinting program (version 1.1, Bio-Rad).
Statistical Analysis
The stat istical analyses were done using SPSS for
Windows (version 12.0, SPSS Inc, Chicago, IL,
USA). The associations between the expression of
each protein and the clinicopathological variables
were exami ned by Pearson’s w
2
-test or Fisher’s exact
test. Spearma n’s rank test was used to determine the
relationships between the expression levels of any
two proteins. The survival rates were estimated
using the Kaplan–Meier method, and the survival
curves between groups stratified by clinicop atholo-
gical parameters and protei n expression levels were
compared by the log-rank test. Multivariate analysis
for survival was done using a Cox proportional
hazards regression model. A P-value of less than
0.05 was considered statistically significant. All the
reported P-values are two-sided.
Results
Clinicopathological Characteristics
The ages of the patients ranged from 13–76 years
(median age: 59 years). Sixty patients were men
and 41 were wome n. The tumor sizes ranged from
1–15.8 cm (median: 4.5 cm). Most of the cases
(92 out of 101) were histologically diagnosed as
adenocarcinoma, whereas others included muci-
nous carcinoma (three cases), adenosquamous
carcinoma (one case), sarcomatous carcinoma (three
cases), and bile duct cystadenocarcinoma (two
cases). Only for 92 cases of adenocarcinomas, tumor
differentiation was assessed. Of these, 64 cases were
well-to-moderately differentiated, whereas 28 cases
were poorly differentiated. The patients’ follow-up
time ranged from 0.6–158.2 months, and the median
survival at the last follow-up was 21.8 months.
Relationships between the Clinicopathological
Variables and the Protein Expressions
The surviva l rates according to each clinicopatho-
logical parameter asses sed by Kaplan–Meier method
are summarized in Table 1. The significant prog-
nostic factors for poor survival were the presence of
satellite nodule(s) (P ¼ 0.0013), poorly differentiated
tumor (P ¼ 0.025 7), presence of vascular invasion
(P ¼ 0.0029), perineural invasion (P ¼ 0.0034), and
lymph node metastasis (Po0.0001). The pT classi-
fication also had an impact on the prognosis
(P ¼ 0.0181). Gender, age, and the resection margin
status were not signi ficantly correlated with the
patients’ survival. The size of tumor only showed a
trend (P ¼ 0.05 24). Pearson’s w
2
-test or Fisher’s exact
test was used to investigate the relationships
between the clinical variables and the expressions
of the analyzed pathway proteins (Supplementary
Table S1). The expression of p-MTOR was more
frequently observed in the well-to-moderately dif-
ferentiated tumors (Po0.001) and in the tumors
without lymph node metastasis (P ¼ 0.046).
Status of the Expressions of Signaling Proteins and
their Relationships with Survival
Positive reactivity for PTEN, p-AKT1, p-MTOR,
p-RPS6KB2, and p-EIF4EBP1 was found in 33.7%.
(34 of 101 cases), 33.7% (34 cases), 53.5% (54 cases),
22.8% (23 cases), and 23.8% (24 cases), respectively,
of all the intrahepatic cholangiocarcinoma cases.
The expression of p-AKT1 and p-MTOR was further
confirmed by western blot analysis in eight repre-
sentative patients whose fresh-frozen tissues were
AKT1’s role in intrahepatic cholangiocarcinoma
134 D Lee et al
Modern Pathology (2012) 25, 131139
available, and there was a tight correlation between
the results of the immunohistochemistry and the
western blot analysis (Figure 2). The 5-year overall
survival rate was significantly higher for the patients
whose tumors overexpressed PTEN (P ¼ 0.0337),
p-AKT1 (P ¼ 0.0137), and p-MTOR (P ¼ 0.0194;
Figure 3). Patients with tumors that were positive
for p-RPS6KB2 and p-EIF4EBP1 showed better
Table 1 Overall survival rates of the patients with intrahepatic
cholangiocarcinoma according to the clinicopathologic variables
No. 1-year
(%)
3-year
(%)
5-year
(%)
P-value
Gender
Male 60 58.33 39.36 31.53 0.62
Female 41 75.61 43.62 34.98
Age
o65 53 66.04 40.82 31.02 0.92
X65 48 64.58 41.16 35.84
Satellite nodule(s)
Present 15 33.33 13.33 13.33 0.0013*
Absent 86 70.93 45.77 36.32
Size (cm)
p5 63 73.02 44.89 39.01 0.0524
45 38 52.63 31.58 21.65
Differentiation
Well/moderate 64 67.19 45.12 35.71 0.0257*
Poor 28 53.57 21.43 16.07
Vascular invasion
Present 42 47.62 25.54 19.15 0.0029*
Absent 59 77.97 51.83 40.9
Perineural invasion
Present 31 51.61 21.77 12.1 0.0034*
Absent 70 71.43 49.32 41.75
LN metastasis
Present 27 33.33 18.52 12.35 o0.0001*
Absent 74 77.03 49.23 40.04
Resection margin
Involved 7 85.71 28.57 28.57 0.76
Clear 94 63.83 42 33.33
pT classification
1 52 76.92 54.95 42.28 0.0181*
2 32 62.5 26.52 19.89
3 15 33.33 26.67 26.67
425000
*Significant at the level of Po0.05.
Survival rates were estimated using Kaplan–Meier method and the
P-value was drawn by log-rank test.
Figure 2 Representative results of the western blot analysis of the
intrahepatic cholangiocarcinoma specimens. Eight patients with
positive ( þ ) or negative () p-AKT1 or p-MTOR expression as
assessed by immunohistochemistry were analyzed by western
blot analysis to validate these antibodies.
Figure 3 Kaplan–Meier survival analysis of the intrahepatic
cholangiocarcinomas for comparing the overall survival for
(a) PTEN, (b) p-AKT1, and (c) p-MTOR.
AKT1’s role in intrahepatic cholangiocarcinoma
DLeeet al 135
Modern Pathology
(2012) 25, 131139
overall survival rates, but the differences were
not statistically significant (P ¼ 0.3164 and 0.097,
respectively; Supplementary Table S2).
Correlations between the Expressions of the
Signaling Proteins
The correlations between the expression levels of
the semi-quantitatively assessed biomarkers were
explored using Spearman’s rank test (Table 2).
On this analysis, significant correlations were found
in many pairs of proteins analyzed, and the Spear-
man’s coefficients (r) were all positive values.
The expression of p-AKT1 was correlated with the
expression of p-MTOR (P ¼ 0.042). The expression
of p-EIF4EBP1 was strongly correlated with the
expressions of PTEN (P ¼ 0.015), p-AKT1
(Po0.001), p-MTOR (P ¼ 0.015), and p-RPS6KB2
(Po0.001). The expression of p-RPS6KB2 was
also positively correlated with the expressions of
p-AKT1 and p-MTOR (P ¼ 0.033 and 0.025, respec-
tively). On the other hand, no correlation was
observed between the expression of PTEN and
the expressions of the other proteins, except for
p-EIF4EBP1.
Multivariate Analysis of the Clinicopathological
Factors for Survival
Multivariate analysis was done with a Cox regres-
sion model, and we included only the clinical
variables and biomarkers that showed significance
on the univariate analysis (Table 3). We found
expression of PTEN (P ¼ 0.032) was an independent
favorable prognostic fact or, whereas the presence of
vascular invasion (P ¼ 0.017), perineural invasion
(P ¼ 0.020), and LN metastasis (Po0.001) were
independent prognostic factors that predicted poor
survival. The expression of p-AKT1 showed a
tendency, but did not attain statistical significance
in the multivariate analysis (P ¼ 0.051).
Discussion
By evaluating expression of multiple components of
the AKT1 pathway in a large number of intrahepatic
cholangiocarcinomas using immunohistochemistry,
this study was able to show, for the first time,
the interactions between various members of the
AKT1 pathway including its downstream targets,
p-RPS6KB2 and p-EIF4EBP1, and their prognostic
significance in patients with intrahepatic cholan-
giocarcinoma. We found that the overexpression of
PTEN was an independent favorable progno stic
factor for intrahepati c cholangiocarcinoma, and that
the overexpression of p-AKT1 and p-MTOR could
also predict a favorable prognosis for pa tients with
intrahepatic cholangiocarcinoma in the univariate
analysis. In addition, AKT1 and its downstream
pathway did not appear to be strongly influenced by
PTEN in the intrahepatic cholangiocarcinoma.
AKT1 and its downstream proteins have been
reported to be frequently activated in human
cancers, and these activated forms were shown to
be poor prognostic factors in several malignant
tumors, including non-small cell lung cancer
23
and
breast cancer.
24
Studies suggest that this may be the
case for malignant tumors of the biliary tract as well.
Herberger et al
22
found that overall survival was
significantly shorter for patients with p-MTOR-
positive tumors of the biliary tract, as compared
with that of patients with p-MTOR-negative tumors.
A recent study demonstrated that a low PTEN
expression and decreased PTEN/p-AKT1 and
PTEN/p-MTOR expression ratios in extrahepatic
cholangiocarcinoma were poor prognostic factors,
Table 2 Correlations between the expressions of PTEN, p-AKT1, p-MTOR, p-RPS6KB2, and p-EIF4EBP1
PTEN
(P-value)
p-AKT1
(P-value)
p-MTOR
(P-value)
p-RPS6KB2
(P-value)
p-EIF4EBP1
(P-value)
PTEN
p-AKT1 0.116 (r ¼ 0.158)
p-MTOR 0.238 (r ¼ 0.119) 0.042* (r ¼ 0.203)
p-RPS6KB2 0.533 (r ¼ 0.063) 0.033* (r ¼ 0.213) 0.025* (r ¼ 0.223)
p-EIF4EBP1 0.015* (r ¼ 0.242) o0.001* (r ¼ 0.390) 0.015* (r ¼ 0.241) o0.001* (r ¼ 0.362)
Abbreviations: PTEN, phosphatase and tensin homolog deleted on chromosome 10; p-AKT1, phosphorylated AKT1; p-MTOR, phosphorylated
mammalian target of rapamycin; p-RPS6KB2, phosphorylated p70 ribosomal protein S6 kinase; p-EIF4EBP1, phosphorylated eukaryotic initiation
factor 4E-binding protein-1; r, Spearman’s coefficient.
*Significant at the level of Po0.05.
Table 3 Multivariate analysis by the Cox proportional hazard
regression model
Hazard
ratio
95% CI P-value
Poorly differentiated tumor 1.598 0.880–2.901 0.124
Presence of vascular invasion 1.963 1.127–3.417 0.017*
Presence of perineural invasion 1.867 1.105–3.154 0.020*
Presence of satellite nodule(s) 1.626 0.771–3.426 0.201
Presence of LN metastasis 4.135 2.240–7.636 o0.001*
Expression of PTEN 0.523 0.290–0.946 0.032*
Expression of p-AKT1 0.549 0.301–1.003 0.051
Expression of p-MTOR 1.064 0.609–1.859 0.827
Abbreviations: PTEN, phosphatase and tensin homolog deleted on
chromosome 10; p-AKT1, phosphorylated AKT1; p-MTOR, phos-
phorylated mammalian target of rapamycin; CI, confidence interval.
*Significant at the level of Po0.05.
AKT1’s role in intrahepatic cholangiocarcinoma
136 D Lee et al
Modern Pathology (2012) 25, 131139
although there were no significant differences in
survival rates based on the single protein expression
status of p-AKT1 and p-MTOR.
25
Moreover,
an earlier study demonstrated that PIK3CA is an
important media tor of Ras-induced radiation resis-
tance in T24 (human bladder carcinoma) cells,
26
and
Tanno et al
27
drew a similar conclusion that
constitutively active AKT1 markedly decreased
radiosensitivity in human bile duct cancer cells.
In contrast, in the present study, we found
the overexpression of PTEN, p-AKT1, and
p-MTOR could predict better survival in patients
with intrahepatic cholangiocarcinoma. Similarly,
patients wit h p-RPS6KB2- or p-EIF4EBP1-positive
tumors exhibited better 5-year overall survival rates
than patients with p-RPS6KB2- or p-EIF4EBP1-
negative tumors, although the differences were not
statistically significant. These unexpected results
are intriguing, as most of the previous studies,
whether they dealt with cholangiocarcinoma or
other cancers, considered overexpression of these
biomarkers to be poor prognostic factors. To our
knowledge, there are only two previous reports on
human cholangiocarcinomas that are in line with
our results. Javle et al
28
studied 24 cases of
cholangiocarcinoma using immunohistochemistry,
and they revealed that AKT1 and p-AKT1 expres-
sions were associated with an improved prognosis.
However, that study did not provide the specific
location of the tumor in the bile duct, and the number
of cases was too small to draw meaningful conclu-
sions. In another study, Schmitz et al
29
showed that
patients with strong positive p-AKT1 staining had
higher 5-year survival rates among 59 patients with
intrahepatic cholangiocarcinoma. However, they con-
cluded that p-AKT1 expression was not a significant
prognostic factor, because the differences did not
reach statistical significance.
An important and well-know n function of
AKT1 signaling pathway is the maintenance of
cell survival and inhibition of apoptosis. There is
growing evidence, on the other hand, that this
pathway is also involved in the promotion of cell
death. Perez-Tenorio and Stal
24
showed that p-AKT1
was strongly associated with a lower S-phase
fraction, and La wlor and Rotwein
30
observed induc-
tion of the cell cycle inhibitor p21 by constitutively
activated AKT1. In keeping with this, a recent study
demonstrated that AKT1 sensitized cells to reactive
oxygen species-mediated apoptosi s by increasing
intracellular levels of reactive oxygen species
through an increase in oxygen consumption and
the inhibition of FoxO transcription factors.
31
In this
study, rapamycin, a well-known MTOR inhibitor,
rather sensitized cells to reactive oxygen species-
mediated cell death, because it paradoxically acti-
vated AKT1 via the inhibition of a negative feedback
loop. In addition, Lu et al
32
observed that stimula-
tion of the Fas receptor induced rapid phosphoryla-
tion of AKT1, and a parallel increase in cell
apoptosis in epidermal C141 cells.
It is questionable how AKT1 is assigned a dual
role in both cell survival and cell death. Ther e are
some molecules that can trigger such opposing
pathways. For example, well-described oncogenes
such as Myc, Ras, and E2F1, which deliver
strong mitogenic signals, have also been reported
to cause cell death.
33,34
In addition, insulin-like
growth factor, which is a well-known mitogenic and
antiapoptotic factor for many cells, was proven to
significantly inhibit DNA synthesis and cell prolif-
eration in a human lung adenocarcinoma cell line,
possibly via the sustained activation of AKT1 and
the induction of p21.
35
Therefore, it is possible that
the outcome of AKT 1 activation may vary depend-
ing on the type of stimulus and the microenviron-
ment, or activa ted AKT1 may direct signals in an
organ (or tumor)-specific manner. For instance,
BCL2, which is known to block apoptosis, promotes
cell death when BCL2 interacts with nuclear orphan
receptor Nur77.
36
Nur77 binding induces a BCL2
conformational change, resulting in conversion of
BCL2 as an opposing phenotype. In another study,
insulin-like growth factor-1 and the adenovirus-
mediated overexpression of wild-type class I PIK3CA
accelerates the accumulation of autophagic vacuoles
and subsequent autophagic cell death during glucose
deprivation.
37
From this point of view, abnormally
activated AKT signaling pathway by other than loss of
PTEN in the current study could possibly change its
downstream targets and result in the opposite out-
come, or AKT1 might show a different action
specifically in intrahepatic cholangiocarcinomas.
We observed that p-MTOR expression was
associated with well-to-moderately differentiated
tumors, and that p-MTOR was inversely correlated
with metastatic disease. A positive correlation
between p-MTOR and better survival was recently
described in early-stage lung adenocarcinoma
38
and
gastric adenocarcinoma,
39
and the association of
MTOR and the intestinal type histology in gastric
adenocarcinoma was also observed. Similar findings
for p-AKT1 have also been reported. Shah et al
40
reported that p-AKT1 was strongly correlated
with well-differentiated tumors, and that p-AKT1
expression in non-small cell lung cancer was a
favorable independent prognostic factor. In a study
by Cappuzzo et al,
41
p-AKT1 was associated
with bronchioloalceolar carcinoma, whic h is a
well-differentiated form of adenocarcinoma, and
p-AKT1 positivity was associated with a reduced
risk of disease progression. Taken together, there are
tumors with high p-AKT1 or p-MTO R expression
that remain well differentiated, seldom metastasize,
and thus are associated with a better survival.
This suggests the possibility that the AKT1–MTOR
pathway may have a pivotal role in the develop-
ment of certain tumors rather than in disease
progression. Lee et al
42
supported this hypothesis
by demonstrating that there was no difference in
the p-AKT1 levels between primary non-small cell
lung cancer and the corresponding metastatic node.
AKT1’s role in intrahepatic cholangiocarcinoma
DLeeet al 137
Modern Pathology
(2012) 25, 131139
Another notable finding in the present study is
that the overexpression of PTEN was not associated
with the overexpression of p-AKT1 and other
downstream proteins, excep t for p-EIF4EBP1, in
intrahepatic cholangiocarcinoma, whereas all the
other proteins except for PTEN showed correlated
expression levels with one another. This indicates
that PTEN mutation or loss of PTEN expression does
not necessarily lead to activation of the AKT1
pathway in intrahepatic cholangiocarcinoma. As
there are numerous signaling molecules involved
in the PTEN–AKT1 pathway with frequent cross-
talk, it is not surprising that the expression of these
signaling proteins could be closely correlated
in some conditions, but not in others. A previous
report described that a high level of p-AKT1
expression occurred independent of the presence
of PTEN mutations in endometrial cancers.
43
In a
report regarding non-small cell lung cancer by
Yoshizawa et al,
23
there was a lack of association
between p-AKT1 and p-MTO R expression. It is
striking that Panigrahi et al
44
found that the level
of PTEN expression was even pos itively correlated
with the level of the p-AKT1 expression in breast
cancer. Moreover, RPS6KB2 and eIF4E can be
activated by pathways that are independent of
MTOR, including the Ras-Raf-MEK-ERK pathway.
45
Taken together, our results indicate that the activa-
tion of AKT1 signaling pathway in intrahepatic
cholangiocarcinoma may occur via alternative
pathways that are independent of PTEN, yet the
conventional model of the AKT1–MTOR–RPS6KB2/
EIF4EBP1 pathway, except for the upstream effect
of PTEN, appears to be valid in this type of tumor.
The treatment of intrahepatic cholangiocarcino-
mas is challenging because of the aggressive nature
of the disease, critical location of the tumor, and lack
of effective adjuvant treatment modalities. Complete
surgical resection is currently the mainstay of
treatment, and it is the only therapeutic option with
a chance of cure for patients with intrahepatic
cholangiocarcinoma. However, only a limited num-
ber of patients with early-stage disease are eligible for
curative surgery. The palliative treatments that
mainly consist of systemic chemotherapy are of
limited benefit as cholangiocarcinomas respond
poorly to the existing therapies.
46
Therefore, further
clinical and preclinical trials are necessary to develop
novel therapeutic options based on novel targets and
tumor markers. In addition, it is becoming increas-
ingly clear that different therapeutic approaches for
intrahepatic and extrahepatic cholangiocarcinoma
may be necessary, because the intra- and extrahepatic
bile ducts have different embryological origins.
In summary, the aberrant expressions of p-AKT1
and p-MTOR in intrahepatic cholangiocarcinoma
were associated with a favorable prognosis, possibly
through its downstream signaling proteins
(p-RPS6KB2 or p-EIF4EBP1), in a PTEN-indepen-
dent manner. We also demonstrated that PTEN
overexpression was an independent favorable prog-
nostic factor for patients with intrahepatic cholan-
giocarcinoma. In addition, the overexpression of
p-MTOR was more frequently observed in well-to-
moderately differentiated tumors, and in tumors
without metastasis. These data may indicate that
dysregulation of the AKT1 pathway may have an
important role in the development of intrahepatic
cholangiocarcinoma, but not necessarily during the
progression of the disease, and that the AKT1
pathway may be regulated by an alternative path-
way, independent of PTEN in intrahepatic cholan-
giocarcinoma. Therapeutic strategies targeting
AKT1 signaling pathway in intrahepatic cholangio-
carcinoma warrants further investigation in light
of these new findings.
Acknowledgement
We thank Mr Hyunsub Lee for his technical assistance.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/
modpathol)
AKT1’s role in intrahepatic cholangiocarcinoma
DLeeet al 139
Modern Pathology
(2012) 25, 131139
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