ONCOLOGY LETTERS 14: 999-1004, 2017

Overexpression of the transmembrane protein BST-2
induces Akt and Erk phosphorylation in bladder cancer
YOSHINORI SHIGEMATSU1,2, NAOHIDE OUE1, YURI NISHIOKA1, NAOYA SAKAMOTO1,
KAZUHIRO SENTANI1, YOHEI SEKINO1,2, SHOICHIRO MUKAI1,
JUN TEISHIMA2, AKIO MATSUBARA2 and WATARU YASUI1
Departments of 1Molecular Pathology and 2Urology, Hiroshima University Instituteof Biomedical and Health Sciences, Hiroshima 734‑8551, Japan
Received May 31, 2016; Accepted March 7, 2017
DOI: 10.3892/ol.2017.6230
Abstract. Bladder cancer, the majority of which is urothelial carcinoma (UC), is a common malignancy worldwide.
Genes encoding transmembrane/secretory proteins expressed
specifically in certain cancers may be ideal biomarkers forcancer diagnosis and may represent therapeutic targets. In
the present study, the expression and function of the bonemarrow stromal cell antigen 2 (BST2) gene was analyzed in
UC. Reverse transcription‑quantitative polymerase chain reaction demonstrated that expression of BST2 in normal tissue
samples was the highest in liver tissue. However, expressionof BST2 in UC tissue samples was higher than in normal liver.
Immunohistochemical analysis revealed weak or no stainingof BST‑2 in non‑neoplastic mucosa, whereas UC tissue
exhibited stronger and more extensive staining compared with
non‑neoplastic mucosa. BST‑2 staining was observed mainly
on UC cell membranes. In total, 28 (41%) of 69 UC cases werepositive for BST‑2. UC cases positive for BST‑2 were more
frequently T2/3/4 cases [so‑called muscle‑invasive bladdercancer (MIBC)] than Ta/is/1 cases (P=0.0001). However,Kaplan‑Meier analysis demonstrated no association between
BST‑2 expression and survival. BST2 small interfering RNA(siRNA)‑transfected T24 cells exhibited significantly reduced
cell growth relative to negative control siRNA‑transfected
T24 cells. The levels of phosphorylated Akt and extracellularsignal‑regulated kinase were lower in BST2 siRNA‑transfected T24 cells than in control cells. These results suggest
the involvement of BST‑2 in the pathogenesis of UC. Since
BST‑2 is expressed on the cell membrane, BST‑2 may be agood therapeutic target for MIBC.

Correspondence to: Dr Naohide Oue, Department of MolecularPathology, Hiroshima University Institute of Biomedical and Health
Sciences, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima 734‑8551, JapanE‑mail: naoue@hiroshima‑u.ac.jp

Key words: bone marrow stromal cell antigen 2, urothelial
carcinoma, bladder cancer, Akt, Erk

Introduction
Bladder cancer, the majority of which is urothelial carcinoma(UC), is a common malignancy worldwide. The prognosis ofpatients with UC is poor when the disease includes muscle
invasion. Metastatic UC is almost uniformly fatal (1). However,progress in systemic therapies for muscle‑invasive bladdercarcinoma (MIBC) has been stagnant for decades, with few
new systemic therapies being evaluated, until recently (2).
Therefore, there is an urgent requirement for new potential
therapeutic targets in UC.
Better knowledge of the changes in gene expression that
occur during carcinogenesis may lead to improvements indiagnosis, treatment and prevention of cancer. Genes encoding
transmembrane/secretory proteins expressed specifically incertain cancers may be ideal biomarkers for cancer diagnosis,
and may represent therapeutic targets. Escherichia coli ampicillin secretion trap (CAST), a signal sequence trap method
developed by Ferguson et al (3), is a unique large‑scale
analysis method that is useful to identify genes encoding
transmembrane/secretory proteins. Using the CAST method,
overexpression of the transmembrane protein bone marrowstromal cell antigen‑2 (BST‑2) was detected in gastrointestinal
cancer (4). Furthermore, knockdown of the BST2 gene inhibits
gastric cancer cell growth, suggesting that BST‑2 could bea useful therapeutic target for gastric cancer (4). BST‑2 is alipid raft‑associated type II transmembrane glycoprotein thatis overexpressed on multiple myeloma cells (5,6). Immunotherapy with a monoclonal antibody against BST‑2 reduces
tumor size and improves survival in a multiple myeloma
mouse model (7). Such monoclonal antibody against BST‑2
induces antibody‑dependent cellular cytotoxicity, suggestingthat it may be effective for a wide range of human malignancies (7). In addition to gastrointestinal cancer, high levels of
BST‑2 have been reported in neoplastic B cells (6), and in
ovarian (8), breast (9), endometrial (10) and lung cancer (11).
However, the expression of BST‑2 has not been investigatedin UC to date.
In the present study, the expression and distribution of
BST‑2 was examined in UC by immunohistochemistry, andpotential correlations with clinicopathological factors wereanalyzed. In addition, the effects of BST2 knockdown on

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SHIGEMATSU et al: BST-2 EXPRESSION IN BLADDER CANCER

cell growth activity were evaluated using RNA interference(RNAi) or forced expression of BST2 in UC cell lines.

Table I. Association between BST‑2 expression and clinicopathological characteristics in bladder cancer.

Materials and methods

BST‑2 expression,n (%)
	‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
Characteristic
Positive Negative P‑value

Tissue samples. Using a retrospective study design, 77 primarytumors were collected from patients diagnosed with UC, who
underwent surgery between April 2003 and March 2007 at
Hiroshima University Hospital (Hiroshima, Japan). All patient
samples were obtained with consent, and the present study
was approved by the Ethical Committee for Human Genome
Research of Hiroshima University (Hiroshima, Japan). All
patients underwent curative resection. Only patients without
preoperative radiotherapy or chemotherapy and withoutclinical evidence of distant metastasis were enrolled in the
study. Operative mortality was defined as mortality within
30 days of patients leaving the hospital, and these patientswere removed from the analysis. Postoperative follow‑up
was scheduled every 1, 2 or 3 months during the first 2 years
after surgery, and every 6 months thereafter, unless more
frequent follow‑ups were deemed necessary. Chest X‑ray,chest computed tomography scan and serum chemistries
were performed at every follow‑up visit. Follow‑ups of thepatients were conducted by the physician until mortality or
until the date of the last documented contact. Tumor staging
was performed according to the tumor‑node‑metastasis classification system (12).
For reverse transcription‑quantitative polymerase chain
reaction (RT‑qPCR), 8 UC samples were used. The samples
were frozen immediately in liquid nitrogen and stored at ‑80˚Cuntil use. A total of 14 types of normal tissue samples [namely
heart (catalog no. 636532), lung (catalog no. 636524), stomach(catalog no. 636578), small intestine (catalog no. 636539),
colon (catalog no. 636553), liver (catalog no. 636531), pancreas(catalog no. 636577), kidney (catalog no. 636529), bone marrow(catalog no. 636591), leukocytes (catalog no. 636592), spleen(catalog no. 636525), skeletal muscle (catalog no. 636547),
brain (catalog no. 636530) and spinal cord (catalog no. 636554)]
were purchased from Clontech Laboratories, Inc. (Mountainview, CA, USA).
Fo r i m mu n o h i st o ch e m ic a l a n a lysi s, a r ch iva lformalin‑fixed, paraffin‑embedded tissues from 69 patients
who had undergone surgical excision for UC were used. All
69 patients with UC were treated by cystectomy between April
2003 and March 2007 at the Hiroshima University Hospital(Hiroshima, Japan).
RT‑qPCR analysis. Total RNA was extracted with an
RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), and 1 µg
total RNA was converted to complementary DNA (cDNA)
using a First Strand cDNA Synthesis kit (GE Healthcare Life
Sciences, Chalfont, UK). Quantitation of BST2 messenger
RNA (mRNA) levels was performed by quantitative
fluorescence detection as described previously (13). PCR
was conducted using a SYBR-Green PCR Core Reagentskit (Applied Biosystems; Thermo Fisher Scientific, Inc.,
Waltham, MA, USA). Real‑time detection of the emission
intensity of SYBR Green bound to double‑stranded DNA
was performed with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific,

Age, years
≤70
>70
Sex
Male
FemaleT classification
Ta/is/1
T2/3/4
Cellular atypism classification
Low gradeHigh grade
Lymphatic invasion
Positive
Negative
Vascular invasion
Positive
Negative

16 (40)12 (41)24 (60)17 (59)0.908323 (40)5 (42)

34 (60)7 (58)

0.93276 (18)
22 (63)28 (82)13 (37)0.00019 (30)
19 (49)21 (70)20 (51)0.116413 (46)
13 (35)15 (54)24 (65)0.84709 (60)
17 (34)6 (40)
33 (66)

0.0714

BST‑2, bone marrow stromal cell antigen‑2.

Inc.). β ‑Actin‑specific PCR products were amplified fromthe same RNA samples and served as an internal control.
Quantitation of ACTB mRNA levels was performed by quantitative fluorescence detection as described previously (13).
RT‑qPCR reactions were performed in triplicate for eachsample primer set, and the mean of the three experiments
was used as the relative quantification value (14). Primer
sequences for BST2 were forward, 5'‑CAG AAG GGC
TTT CAG GAT GT‑3' and reverse, 5'‑TTC TCA GTC GCTCCA CCT CT‑3'. Primer sequences for ACTB were forward,
5'‑TCA CCG AGC GCG GCT‑3' and reverse, 5'‑TAA TGTCAC GCA CGA TTT CCC‑3'. The thermocycling conditions
were used as described previously (13).
Immunohistochemistry. Immunohistochemical analysis was
performed with the EnVison+ Rabbit Peroxidase Detection
System (Dako; Agilent Technologies GmbH, Waldbronn,
Germany) as described previously (15). As the primary
antibody, a rabbit polyclonal anti‑BST‑2 antibody was used(dilution, 1:50; catalog no. HPA017060, Sigma‑Aldrich; Merck
KGaA, Darmstadt, Germany) (4). A result was considered
positive if ≥10% of cancer cells were stained. When <10% ofcancer cells were stained, the immunostaining was considerednegative.
Cell lines. Two cell lines derived from human UC (T24 andKMBC2) were used. Both cell lines were purchased from

ONCOLOGY LETTERS 14: 999-1004, 20171001

according to the manufacturer's protocol. KMBC2 cells wereselected as the cell line express low levels of BST‑2.
To examine cell growth, MTT assays were performed. The
cells were seeded at a density of 2,000 cells per well in 96‑wellplates. Cell growth was examined after 1, 2 and 4 days. Three
independent experiments were performed. The mean and standard error were calculated for each experiment.
Western blot analysis. Cells were lysed as described previously (17). The lysates (40 µg protein) were solubilized in
Laemmli sample buffer (catalog no. S3401, Sigma‑Aldrich;
Merck KGaA) by boiling and then subjected to 10%
SDS‑PAGE, followed by electrotransfer onto a nitrocellulose membrane. An anti‑BST‑2 monoclonal antibody(catalog no. H00000684‑B02P) was purchased from Abnova(Taipei, Taiwan) (4). Peroxidase‑conjugated anti‑mouse
immunoglobulin G was used as the secondary antibody (4).
Immunocomplexes were visualized with the Amersham ECL
Western Blotting Detection kit (GE Healthcare Life Sciences).
β ‑Actin (catalog no. A5441; Sigma‑Aldrich; Merck KGaA)
was also stained as a loading control (4).

Figure 1. Expression of BST2 in UC. (A) RT‑qPCR analysis of BST2 expression in 14 types of normal tissue and 8 UC samples. RT‑qPCR reactions were
performed in triplicate for each sample, and the mean of three experiments
was used as the relative quantification value. (B) Immunohistochemical
analysis of BST‑2 in non‑neoplastic mucosa and UC. Original magnification,x100. (C) Immunostaining of BST‑2 in UC. BST‑2 staining was observed on
the cell membranes of UC cells. Original magnification, x400. UC, urothelial
carcinoma; BST2, bone marrow stromal cell antigen 2; mRNA, messenger
RNA; RT‑qPCR, reverse transcription quantitative polymerase chain reaction.

Statistical methods. Associations between clinicopathologicalparameters and BST‑2 expression were analyzed by the Fisher's exact test. Kaplan‑Meier survival curves were constructed
for BST‑2‑positive and ‑negative patients. Survival rates werecompared between BST‑2‑positive and ‑negative groups.
Differences between survival curves were evaluated for statistical significance by the log‑rank test. P<0.05 was considered
to indicate a statistically significant difference. SPSS version
8.0 software was used for these analyses (SPSS Inc., Chicago,
IL, USA).
Results

the Japanese Collection of Research Bioresources Cell Bank
(Osaka, Japan), and were maintained in RPMI 1640 medium(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing
10% fetal bovine serum (BioWhittaker; Lonza, Basel, Switzerland) at 37˚C in a humidified atmosphere with 5% CO2.
RNAi, expression vector and cell growth assay. Smallinterfering RNA (siRNA) oligonucleotides targeting BST2
and a negative control siRNA were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Two independent
BST2 siRNA oligonucleotide sequences were used (catalog
nos. 251993co3 and 251993co5). Transfection was performed
using Lipofectamine RNAiMAX (Invitrogen; Thermo
Fisher Scientific, Inc.) as described previously (16). Briefly,
60 pmol siRNA and 10 µl Lipofectamine RNAiMAX weremixed in 1 ml RPMI 1640 medium (10 nmol/l final concentration). After 20 min of incubation, the mixture was added
to the cells (100,000 cells/ml), and then the cells were plated
in culture dishes. At 48 h post‑transfection, the cells wereanalyzed.
For constitutive expression of the BST2 gene, cDNA wasamplified by PCR and then subcloned into the pcDNA3.1 vector(Invitrogen; Thermo Fisher Scientific, Inc.). The resultant
pcDNA‑BST2 expression vector was transfected into KMBC2
cells with FuGENE6 (Roche Diagnostics, Basel, Switzerland)

Expression of BST2 in UC. RT‑qPCR analysis of BST2 was
first performed in 14 types of normal tissue samples and 8
UC tissue samples (Fig. 1A). All 8 UC tissue samples wererandomly selected. Among the various normal tissue samples,abundant BST2 mRNA expression was detected in normallung, stomach, liver and bone marrow samples. The expression of BST2 in these normal tissue samples was highest in theliver. However, expression of BST2 in UC tissue samples washigher than in normal liver.
Next, im munohistochem istr y was per for med on
69 UC tissue samples. In non‑neoplastic mucosa, weakor no staining of BST‑2 was observed in urothelial andstromal cells, whereas UC tissue exhibited stronger andmore extensive staining compared with non‑neoplastic
mucosa (Fig. 1B). BST‑2 staining was observed mainly on
UC cell membranes (Fig. 1C). Numerous UC cases displayed
heterogeneity of BST‑2 staining, and the percentage of
BST‑2‑stained UC cells ranged from 0 to 70%. A tendency
for upregulation of BST‑2 was not observed at the invasive
front. In total, 28 (41%) of 69 UC cases were positive for
BST‑2 expression.
Next, the association of BST‑2 staining with clinicopathological characteristics was examined (Table I). UC
cases positive for BST‑2 were more frequently T2/3/4 cases(so‑called MIBC) than Ta/is/1 cases (P=0.0001). However,1002

SHIGEMATSU et al: BST-2 EXPRESSION IN BLADDER CANCER

Figure 2. Effect of BST2 inhibition in T24 urothelial carcinoma cells.
(A) Western blot analysis of BST‑2, Akt, pAkt, Erk1/2 and pErk1/2 in lysatesof T24 cells transfected with BST2 siRNA or negative control siRNA.
β‑Actin was used as a loading control. (B) Effect of BST2 knockdown on thegrowth of T24 cells. Cell viability was assessed by MTT assays at days 1, 2
and 4 after seeding on 96‑well plates. Bars and error bars indicate the meanand standard error, respectively, of three independent experiments. siRNA,
small interfering RNA; p, phosphorylated; Erk, extracellular signal‑regulated
kinase; BST2, bone marrow stromal cell antigen 2; O.D., optical density.
*
P<0.001.

Figure 3. Effect of BST2 overexpression in KMBC2 urothelial carcinoma
cells. (A) Western blot analysis of BST‑2, Akt, pAkt, Erk1/2 and pErk1/2
in lysates of BST2‑transfected and empty vector‑transfected KMBC2 cells.
β‑Actin was used as a loading control. (B) Effect of BST‑2 on the growth of
KMBC2 cells. Cell viability was assessed by MTT assays at days 1, 2 and 4
after seeding on 96‑well plates. Bars and error bars indicate the mean andstandard error, respectively, of three independent experiments. Erk, extracellular signal‑regulated kinase; p, phosphorylated; BST2, bone marrow stromal
cell antigen 2; O.D., optical density. *P<0.001.

Kaplan‑Meier analysis demonstrated no association between
BST‑2 expression and survival (P=0.4602) (data not shown).
Effect of BST2 inhibition on cell growth. Previously, it wasdemonstrated that inhibition of BST2 by siRNA reducescell growth, and that the levels of phosphorylated epidermal
growth factor receptor (EGFR), Akt and extracellularsignal‑regulated kinase (Erk) are lower in BST2 siRNA‑transfected gastric cancer cells than in control cells (4). However,the biological function of BST‑2 has not been investigatedin UC cells thus far. Therefore, the present study examinedthe effect of BST2 inhibition on cell growth using two
different siRNA sequences (catalog nos. 251993co3 and251993co5; Invitrogen; Thermo Fisher Scientific, Inc.). T24
UC cells were selected, which express detectable levels of
BST‑2 protein (Fig. 2A). BST‑2 expression was substantiallysuppressed by treatment with siRNA1 and siRNA2, as shownby western blotting (Fig. 2B). Next, cell growth was analyzed
by MTT assays, which revealed that BST2 siRNA1‑ andsiRNA2‑transfected T24 cells exhibited significantly reduced
cell growth relative to negative control siRNA‑transfected
T24 cells (Fig. 2B).
Since EGFR activates the Ras‑mitogen‑activatedprotein kinase kinase‑Erk and Akt‑phosphatidylinositol‑
4,5‑bisphosphate 3‑kinase signaling pathways, thus leading tocancer cell proliferation and survival (18), the effect of BST2
inhibition on EGFR signaling was analyzed in the present
study. The results indicated that the levels of phosphorylated
Akt and Erk were lower in BST2 siRNA1‑ and siRNA2‑transfected T24 cells than in control cells (Fig. 2A).
Effect of forced expression of BST2 on cell growth. To further
investigate the biological significance of BST‑2, the KMBC2
UC cell line was stably transfected with a vector expressing
BST2. KMBC2 cells were selected due to their low expression
levels of BST‑2 compared with T24 cells (Fig. 2A). Clones
were selected with G418 and examined for BST‑2 expression by western blotting. Two clones, KMBC2‑BST2‑1 andKMBC2‑BST2‑2, expressed BST‑2 protein at significantly
higher levels than KMBC2 cells transfected with the empty
vector (Fig. 3A). To determine the effect of BST‑2 on cell
growth, MTT assays were performed. BST2‑transfected
KMBC2 cells exhibited significantly increased cell growth
relative to empty vector‑transfected KMBC2 cells (Fig. 3B).
The effect of BST2 overexpression on EGFR signaling was
also analyzed. The levels of phosphorylated Akt and Erk werehigher in BST2‑transfected KMBC2 cells compared withempty vector‑transfected KMBC2 cells (Fig. 3A).
Discussion
Platinum‑based chemotherapy is currently the standard
treatment in previously untreated patients with metastatic
UC and is associated with a median overall survival of
15 months (19). The prognosis for patients who relapse
following platinum‑based chemotherapy is poor, with mediansurvival times ranging from 5 to 7 months, and no known
life‑prolonging treatments are available (20). Therefore, anovel therapeutic target is required for UC. In the present
study, the expression of BST‑2, a lipid raft‑associated type IItransmembrane glycoprotein, was analyzed in UC. Amongthe various normal tissue samples examined, expression of
BST2 was the highest in the liver. However, expression of
BST2 in UC tissue samples was higher than in normal liver,suggesting that BST‑2 is a therapeutic target with fewer
adverse effects compared with other anticancer drugs forvarious cancer types, including UC. Immunohistochemical
analysis revealed BST‑2 expression on the cell membrane,
and 41% of UC cases were positive for BST‑2. UC cases
positive for BST‑2 were more frequently T2/3/4 cases than

ONCOLOGY LETTERS 14: 999-1004, 2017

Ta/is/1 cases. Taken together, these results suggest that BST‑2
serves an important role in the pathogenesis of UC and is agood therapeutic target for T2/3/4 UC (also known as MIBC)
cases.
Previously, our group reported that 36% of gastriccancer cases, 46% of colorectal cancer cases and 27%
of esophageal cancer cases were positive for BST‑2 (4).
Furthermore, high levels of BST‑2 have been reported in
ovarian cancer (8), neoplastic B cells (6), breast cancer (9),endometrial cancer (10) and lung cancer (11), indicating that
BST‑2 expression is a common event in human malignancies.
Ozaki et al (7) reported that a monoclonal antibody against
BST‑2 induces antibody‑dependent cellular cytotoxicity, andthat immunotherapy using this anti‑BST‑2 antibody reduces
tumor size and improves survival in a multiple myeloma
mouse model. Therefore, immunotherapy using a similaranti‑BST‑2 antibody may also improve the survival of UC
patients.
In the present study, BST2 siRNA‑transfected T24 UC
cells exhibited significantly reduced cell growth relative tonegative control siRNA‑transfected T24 UC cells in MTT
assays. Furthermore, BST2‑transfected KMBC2 UC cells
displayed significantly increased cell growth relative toempty vector‑transfected KMBC2 UC cells in MTT assays.
The levels of phosphorylated Akt and Erk were lower in
BST2 siRNA‑transfected T24 UC cells than in control cells.
It was also demonstrated that the levels of phosphorylated Akt
and Erk were higher in BST2‑transfected KMBC2 UC cells
than in empty vector‑transfected KMBC2 UC cells. Since
the phosphorylation of Akt and Erk inhibits apoptosis (21),these results suggest that apoptosis could be induced in
BST2‑transfected KMBC2 UC cells. EGFR overexpressionin UC is correlated with high tumor grade, muscle invasiveness, tumor recurrence and overall survival (22). Although
the underlying mechanisms remain unclear, it is possiblethat a monoclonal antibody against BST‑2 could inhibit
EGFR signaling and induce apoptosis in UC cells. The safety
and efficacy of a humanized anti‑BST‑2 antibody has been
investigated in a phase I/II clinical study on patients withrelapsed or refractory multiple myeloma and has shown mildand manageable side effects (23). Thus, the efficacy of thishumanized anti‑BST‑2 antibody should be examined in aclinical study on patients with metastatic UC or those who
relapse following platinum‑based chemotherapy.
In summary, the present study demonstrated overexpression of BST‑2 in UC, particularly in T2/3/4 UC (MIBC) cases.
Since a humanized anti‑BST‑2 antibody is currently available,
the efficacy of such antibody should be examined in a clinicalstudy. Although lower levels of phosphorylated Akt and Erk
were detected in BST2 siRNA‑transfected UC cells than incontrol cells, the underlying mechanisms remain unclear.
Identification of BST‑2 signaling will further improve theunderstanding of the basic biology of BST‑2.
Acknowledgements
The authors thank Mr. Shinichi Norimura (Hiroshima University, Hiroshima, Japan) for his excellent technical assistance andadvice. The present study was supported by Grants‑in‑Aid for
Scientific Research (B‑15H04713) and Challenging Exploratory

1003

Research (grant nos. 26670175 and 16K15247) from the Japan
Society for the Promotion of Science. The present study was
also supported by the Takeda Science Foundation.
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