Expression of organic anion-transporting polypeptides 1B3, 1B1, and 1A2 in human pancreatic cancer reveals a new class of potential therapeutic targets.

Abstract

Organic anion-transporting polypeptides (OATPs) are influx transporters that mediate intracellular uptake of selective endogenous and xenobiotic compounds. Identification of new molecular targets and discovery of novel targeted therapies is top priority for pancreatic cancer, which lacks any effective therapy.
We studied expression of OATP 1A2, 1B1, and 1B3 in pancreatic cancer tissue and in cell lines. Formalin-fixed paraffin-embedded biopsy material of 12 human pancreatic cancers was immunohistochemically assessed for protein expression of the three studied influx transporters. Immunohistochemistry was evaluated by experienced pathologists and quantified by use of an automated image analysis system. BxPC-3 and MIA PaCa-2 pancreatic cancer cell lines were used to quantify transcripts of OATP 1B1 and 1B3.
OATP 1A2, 1B1, and 1B3 proteins were found ubiquitously expressed in all studied cases. Quantification performed by HistoQuest system revealed that mean intensity was 53 for 1A2, 45 for 1B1, and 167 for OATP 1B1/1B3 on a range scale 0-250 units. At mRNA level, 1B1 and 1B3 were overexpressed in both studied cancer cell lines but not in normal pancreatic tissue.
OATPs 1A2, 1B1, and 1B3 are highly expressed in pancreatic adenocarcinoma. We suggest that expression of these transporters in pancreatic cancer justify research efforts towards discovery of novel therapeutics targeting OATPs.

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expression of organic anion-transporting
polypeptides 1B3, 1B1, and 1A2 in human
pancreatic cancer reveals a new class of potential
therapeutic targets
DOI: 10.2147/OTT.S16706
Valentinos Kounnis1
elli ioachim2
Martin svoboda3
Andreas Tzakos4
ioannis sainis1
Theresia Thalhammer3
georg steiner5
evangelos Briasoulis1
1cancer Biobank center of the
University of ioannina, greece;
2Pathology Department of hatzikosta
general hospital, ioannina greece;
3Department of Pathophysiology and
Allergy research, Medical University
of Vienna, Austria; 4Department of
chemistry, University of ioannina,
greece; 5Tissuegnostics gmbh,
Vienna, Austria
correspondence: evangelos Briasoulis
interscience Molecular Oncology Lab,
cancer Biobank center, University of
ioannina, greece
Tel +30 265 100 7713
Fax +30 265 100 8087
email ebriasou@me.com
Background: Organic anion-transporting polypeptides (OATPs) are influx transporters that
mediate intracellular uptake of selective endogenous and xenobiotic compounds. Identification
of new molecular targets and discovery of novel targeted therapies is top priority for pancreatic
cancer, which lacks any effective therapy.
Materials and methods: We studied expression of OATP 1A2, 1B1, and 1B3 in pancreatic
cancer tissue and in cell lines. Formalin-fixed paraffin-embedded biopsy material of 12 human
pancreatic cancers was immunohistochemically assessed for protein expression of the three
studied influx transporters. Immunohistochemistry was evaluated by experienced pathologists
and quantified by use of an automated image analysis system. BxPC-3 and MIA PaCa-2
pancreatic cancer cell lines were used to quantify transcripts of OATP 1B1 and 1B3.
Results: OATP 1A2, 1B1, and 1B3 proteins were found ubiquitously expressed in all studied
cases. Quantification performed by HistoQuest system revealed that mean intensity was 53 for
1A2, 45 for 1B1, and 167 for OATP 1B1/1B3 on a range scale 0–250 units. At mRNA level,
1B1 and 1B3 were overexpressed in both studied cancer cell lines but not in normal pancreatic
tissue.
Conclusion: OATPs 1A2, 1B1, and 1B3 are highly expressed in pancreatic adenocarcinoma.
We suggest that expression of these transporters in pancreatic cancer justify research efforts
towards discovery of novel therapeutics targeting OATPs.
Keywords: organic anion-transporting polypeptides, targeted therapy, transporter
Introduction
Pancreatic carcinoma is the most deadly among cancers, ranked fourth as a cause of
cancer-related deaths in the economically developed world.1,2 Characteristically, in
Europe, new diagnoses of pancreatic cancer almost equaled the deaths caused by this
disease in 2008.3
Surgery remains the gold standard for the treatment of pancreatic cancer, if
diagnosed early. However, even in cases of surgically resected tumors, the outcome
remains poor, and adjuvant therapy can offer marginal benefits.2,4 In advanced pancreatic
cancer, the outlook is even worse. Extensive research has failed to produce any therapy
efficient enough to substantially extend the median survival of treated patients beyond
6 months. Currently available therapies remain palliative on their intent.5–7 Therefore,
identification of new molecular targets and discovery of novel targeted therapies is
top priority for pancreatic cancer research.
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Kounnis et al
The organic anion transporting polypeptides (OATPs)
superfamily comprises 11 polypeptide molecules that share
a largely common structure with 12 putative transmembrane
regions and a large extracellular loop between the 9th and
10th transmembrane domains8 (Figure 1). They operate as
influx transporters that mediate the transmembrane uptake
of various endogenous and xenobiotic anion compounds.
Besides their characteristic expression in normal tissues,
OATPs have been found overexpressed in several cancers, and
it was such data that prompted us to undertake investigation of
these transporters as potential therapeutic targets.9 Regarding
pancreatic cancer, Abe et al, showed expression of OATP
1B3 in human pancreatic cancer on mRNA and protein level
in a single case.10 To our knowledge, this is the first study that
systemically assessed the expression profile of three OATPs
(1A2, 1B1, and 1B3) in pancreatic cancer.
Materials and methods
Tissue samples and anti-OATP antibodies
Formalin-fixed paraffin-embedded tissue samples of human
pancreatic cancer were retrieved from the archives of the
Department of Pathology, Chatzikosta General Hospital,
Ioannina, Greece. The patients were diagnosed in the
period 2000–2008. Their median age was 69 years; six were
female and six male. Histologically, eight cases were diag-
nosed as poorly differentiated pancreas adenocarcinomas,
and four cases had intermediate differentiation.
The samples were assessed for expression of OATP 1B1
and 1B1/1B3 by using the mESL and mMDQ antibodies
respectively (PROGEN Biotechnik, Heidelberg, Germany).
Expression of OATP 1A2 was evaluated in 11 samples by
polyclonal anti-OATP 1A2 antibody (Atlas Antibodies AB,
Stockholm, Sweden). A polyclonal anti-OATP 1B3 antibody
(Atlas Antibodies AB, Stockholm, Sweden) was also used
that recognizes C-terminal region of OATP 1B3, on the aim
to monitor the expression of the 1B3 transporter as a single
entity. All antibodies were diluted with Dako REAL™
Antibody Diluent (DAKO, Code S2022) to the final working
concentration (Table 1). The DAKO Autostainer/PT link
system was used for the immunostaining process.
cell lines
Two pancreatic cancer cell lines, BxPC-3 (CRL-1687TM)
and MIA PaCa-2 (CRL-1420TM), were obtained from the
American Tissue Culture Collection (ATCC, Manassas,
VA) to be used in this study. MIA PaCa-2 originated from
a male14 and BxPC-3 from a female donor.15 Cells were
routinely cultured in RPMI-1640 medium (PAN Biotech,
Aidenbach, Germany) supplemented with 10% fetal calf
serum (Invitrogen Life Technologies, Paisley, Scotland)
and 1% penicillin/streptomycin (Invitrogen) under standard
culture conditions.
immunohistochemistry
Tissue sections of 3–4 µm width were cut using a microtome
and applied on microscope slides. Slides were incubated
overnight at 65°C to enable optimal tissue–glass adhesion.
Next, slides were immersed in DAKO’s PT-Link containing
preheated (65°C) target retrieval solution, at pH 9 (DAKO
Code S2375), treated at 93°C for 20 minutes in order to
achieve deparaffinization, rehydration, and heat-induced
epitope-retrieval (HIER). After cooling back to 65°C, slides
were inserted in DAKO Autostainer system for the rest
of the immunohistochemistry procedure. The mESL and
mMDQ antibodies were applied on slides for 70 minutes,
while for the polyclonal anti-OATP1A2 and anti-OATP1B3
antibodies incubation time was set to 60 minutes. The
endogenous peroxidase was blocked using Daco REAL
peroxidase blocking solution (Code S2023) for 10 minutes.
DAKO’s special engineered Dextran backbone enriched with
peroxidase molecules and goat secondary antibody molecules
Figure 1 ribbon representation of the three-dimensional model of organic
anion-transporting polypeptide 1B3. TM domains, a probable substrate binding
site and the conserved amino acid side chains. The model was built by Modeller
program (san Francisco, cA, UsA) by using the structure template of the Escherichia
coli glycerol-3-phosphate transporter (PDB 1pw4).24,25 in blue color the potential
location of the substrate binding site (the putative translocation pathway) according
to former mutagenesis studies is indicated.26 conserved amino acid side-chains can
also be seen on this figure.

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expression of OATPs and potential therapeutic targets
against rabbit and mouse immunoglobulins (Dako REAL™
EnVision™/HRP, Rabbit/Mouse ENV, Code Code K5007)
was applied on slides for 20 minutes followed by a 5 minute
Dako REAL™ DAB+ Chromogen (DAKO, Code K5007)
detection system and a 2 minute hematoxylene (QS H-3404
Vector Laboratories) treatment. Finally, sections were
dehydrated with alcohol/xylene baths and stabilized with
mounting medium.
immunohistochemistry evaluation
Immunostaining intensity was assessed by two experienced
pathologists, who graded it, using endothelial cells and tissue
macrophages as internal controls (Table 2). Quantitation was
done by HistoQUEST (TissueGnostics, Vienna, Austria)
system in values of gray from 0 to 25011,12 (Figures 2 and 3).
sLcO1A2, sLcO1B1, and sLcO1B3
mRNA expression quantification
Total RNA was isolated from both cell lines grown to
subconfluency using the Trizol reagent (Invitrogen). We also
used human liver RNA (purchased from Stratagene La Jolla,
USA) and human pancreas RNA (purchased from Clontech,
Saint-Germain-en-Laye, France) for control purposes.
The concentration, purity, and integrity of RNA samples
were determined on a Nanodrop ND-1000 (Kisker-Biotech,
Steinfurt, Germany) and by agarose gel electrophoresis.
Reverse transcription of total RNA to cDNA (2 µg) was
done by using the high capacity cDNA reverse transcription
kit (Applied Biosystems, Foster City, USA) as recommended
by the manufacturer. The quantification of SLCO/OATP
mRNA transcripts was done by using TaqMan® real-time
Table 1 Antibodies and technical data used for immunohistochemistry
Ab symbol
OATP
target
Clonality
Host
species
Immunoglobulin
subclass
Working
dilution
Incubation
time (minutes)
Epitope target
Company
Cat #
mMDQ
1B1/1B3
Monoclonal
Mouse
igg1
1:10
70
MDQhQhLnKTAesAsseKKKTrrc for 1B3 (n-terminus) MDQnQhLnKTAeAQPsenKKTrYc for 1B1 (n-terminus)
Progen Biotechnik
651140
mesL
1B1
Monoclonal
Mouse
igM
1:5
70
esLnKnKhFVPsAgADseThc (c-terminus)
Progen Biotechnik
651139
p1A2
1A2
Polyclonal
rabbit
igg
1:250
60
ssVVginTsYegiPQDLYVenDiFADcnVDcncPsKiWDP
VcgnngLsYLsAcLAgceTsigTginMVFQncs
ciQTsg nssAVLgLcDKgPDc (c-terminus)
Atlas Antibodies
hPA027537
p1B3
1B3
Polyclonal
rabbit
igg
1:250
60
QgKDTKAsDnerKVMDeAnLeFLnngehFV
PsAgTDsKTcnLD MQDnAAA (c-terminus)
Atlas Antibodies
hPA004943
Abbreviation: OATP, organic anion-transporting polypeptide.
Table 2 immunohistochemical expression of OATPs. score scale
(0, 1+, 2+, 3+) reflecting antibody product expression intensity
Patient
number
Sex AgeDifferentiationp1A2 mESLmMDQ
1
2
3
4
5
6
7
8
9
10
11
12
F
F
F
M
M
F
F
F
M
M
M
M
71
67
74
56
80
55
78
70
72
67
65
70
intermediate
Poor
Poor
Poor
intermediate
Poor
Poor
Poor
intermediate
Poor
Poor
intermediate
+
++
+++
+++
++
++
+++
+++
+++
++
++
nA
+
+
+
+
+
+
+
+
+
+
+
+
+++
+++
++
++
++
++
++
+++
++
++
++
++
Abbreviations: F, female; M, male; nA, not applicable; OATP, organic anion-
transporting polypeptide.

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Kounnis et al
PCR method (Applied Biosystems). The following TaqMan
assays were used: hs00272374_m1 for OATP 1A2 and
hs00251986_m1 for OATP 1B3. For accurate normalization
of real time RT-PCR data we included four reference genes
in the analysis, namely 18S rRNA (PN 4310893E; Applied
Biosystems), ACTB (PN 4326315E; Applied Biosystems),
CYC1 (PrimerDesign Ltd, Southampton, UK), and UBC
(PrimerDesign Ltd). The target gene amplification mixture
contained 5 µL 2× TaqMan® Gene Expression PCR Master
Mix (Applied Biosystems), 0.5 µL of the appropriate Gene
Expression Assay, 10 ng template cDNA diluted in 2.5 µL
nuclease free water, and 2 µL nuclease free water. 5´–3'
exonuclease activity of the DNA polymerase was measured
with the ABI 7900HT Fast real time PCR system was
equipped with SDS 2.3 software (Applied Biosystems).
All samples were amplified in duplicates. Results were
imported into Prsim GraphPad for further analysis. Specimens
exhibiting cycle threshold (Ct) values higher than 38 were
considered to be negative, and comparable cDNA amounts
in the experimental samples were calculated according to
Hellemans et al.13 Values are expressed as n-fold difference
according to a calibrator set to value 1. As a calibrator, RNA
pooled from a panel of human tissue samples and cell lines
analyzed was taken, and expression values in the calibrator
were set to 1.
Results
immunohistochemistry
The three studied polypeptides were found ubiquitously
expressed in all studied biopsy cases. Both methods used
confirmed extensive immunostaining of the entire cancer cell
tissue with the antibodies used (Table 2; Figures 2 and 4).
Specifically, OATP 1A2 expression signal was weak
in one sample and moderate to strong in all others. The
HistoQUEST quantification analysis returned a mean intensity
of 53.88 units with a standard error (SE) of 7.19. OATP 1B1
A
B
C
D
Figure 4 immunohistochemical staining of pancreatic adenocarcinoma tissue
sample from a 77-year-old female patient diagnosed with poorly differentiated
adenocarcinoma of the head of the pancreas. A) p1A2 antibody (×40), B) mesL
antibody (×100), C) mMDQ antibody (×200), D) p1B3 antibody (×40).
HistoQUEST (TissueGnostics)
OATP expression evaluation
OATP1A2 OATP1B1
OATP1B1/1B3
0
50
100
150
200
250
Mean intensity
(values of grey)
Figure 2 OATP immunohistochemical expression intensity as was assessed by
histoQUesT (Tissuegnostics) automated immunohistochemistry analysis system.
The Y-axis represents mean intensity measured in values of grey scale from 0 to 250.
Abbreviation: OATP, organic anion-transporting polypeptide.
A
C
EF
D
B
Figure 3 histoQUesT (Tissuegnostics) expression analysis. Pictures A, C, and E
represent the identification and segmentation of cells and nuclei. Pictures B, D, and F
demonstrate the diaminobenzidine signal intensity for each cell in the studied area.

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expression of OATPs and potential therapeutic targets
was found to be weakly expressed in all 12 cases with mean
intensity 45.10 units (SE 3.15). Immunostaining for mMDQ
(OATP 1B1/1B3) was proved the most intense. Nine cases
demonstrated moderate expression, three cases stained strong,
and HistoQUEST assessment showed a mean intensity of
167.90 units (SE 8.27). Interestingly, the anti-OATP 1B3 p1B3
antibody failed to detect expression of its nominal target in
the same cancer tissue material (Figure 4), although it stained
normal human liver tissue, which was used in this case as a
positive control for p1B3 antibody (Figure 5).
cell lines
OATP 1B1 and 1B3 mRNA expression in the two cell lines
was comparable to that in normal liver, which was taken as
a control, because both these transporters are considered
“liver-specific”. Their mRNA expression, however, in normal
pancreas was either undetectable (OATP 1B1) or 30–60 times
lower than in normal liver (OATP 1B3) (Figure 6).
Discussion
The need for discovery of new molecular targets and
the development of innovative targeted therapeutics in
pancreatic cancer is indisputably illustrated by the fact that
of the 36 orphan-designated products for pancreatic cancer
by the United States Food and Drug Administration, none
received marketing approval on this indication during the
last 25 years.16 We considered that expression of organic
anion-transporting polypeptides in a wide range of cancers
along with their unique capacity for energy-independent
intracellular transport of xenobiotics makes them reasonable
targets for potential development of novel cancer therapeutics
against hard-to-treat cancers.17–20
We opted to investigate the expression of OATP 1A2,
1B1, and 1B3 in pancreatic cancer, because of their
common and distinctive ability to facilitate intracellular
uptake of microcystin cyclopeptides, which are natural
toxins produced from cyanobacteria.21 Microcystins exert
cytotoxic effects through depletion of glutathione, generation
of reactive oxygen species, and strong inhibition of protein
phosphatases 1 and 2A.22 We surmise that these natural
toxins are of potential pharmacological interest because they
offer opportunities to engineer analogs optimized to induce
selective cancer toxicity in OATP expressing tumors.9
The OATPs investigated in this study were all found
to be ubiquitously expressed in pancreatic cancer: OATP
1A2, 1B1, and 1B3 protein expression was documented in
biopsy samples and OATP 1B1 and 1B3 mRNA in cell lines.
Quantification analysis showed an increased expression of
1B1/1B3 compared with 1A2 and 1B1, which indicates
enhanced expression potential for OATP 1B3 in these tumors.
It should be noted that the inability to detect OATP 1B3
C-terminal region with p1B3 antibody opposes the strong
positive mMDQ signal in the same samples, which confirmed
the presence of the 24 amino acid N-terminal epitope of OATP
1B3. Interestingly, we obtained similar results in a colon
cancer study in which we identified an abundant presence of
1B3 protein when studied with mMDQ monoclonal antibody
but not with the p1B3 antibody.23 We speculate that these
findings might possibly be related to unknown mutation near
the C-terminal end of 1B3 polypeptide in cancers. This is
currently under investigation in our laboratory.
Finally, we consider that demonstration of OATP 1B3 and
1B1 mRNA expression in BxPC-3 and MIA PaCa-2 human
pancreatic cell lines flags them as appropriate candidates for
in-vitro studying of OATP targeted anticancer compounds.
Acknowledgments
This research was funded by the “Heraklitos II” program of
the Operational Program for Education and Initial Vocational
A
B
Figure 5 immunostaining with p1B3 of normal liver tissue with moderate steatosis
of a 75-year-old individual who underwent subtotal hepatectomy because of a car
accident trauma, shows cell membrane signal with increasing intensity towards
central vein regions. A) ×40, B) ×200.
0
2
Relative expression
4
BxPC-3
MIA PaCa-2
Liver
6
8
10
OATP 1B3 mRNA expression
Figure 6 OATP 1B3 mrnA expression in BxPc-3, MiA Paca-2 cancer cell lines and
in human liver. Values are expressed as n-fold difference to the calibrator (pooled
from the human tissue samples and cell lines) taken as value 1.
Abbreviation: OATP, organic anion-transporting polypeptide.

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Kounnis et al
Training of the Hellenic Ministry of Education under the
3rd Community Support Framework and the European
Social Fund.
Special thanks are given to University of Ioannina Cancer
Biobank Center and the Pathology Department of Hatzikosta
General Hospital in Ioannina for supporting this research,
and Ms Sofia Kokouva for most valuable help.
Disclosure
The authors report no conflicts of interest in this work.
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