INTERNATIONAL JOURNAL OF ONCOLOGY 41: 1587-1592, 2012
Abstract. Androgen receptor (AR) signals have been suggested
to contribute to bladder tumorigenesis and cancer progres-
sion. Activation of epidermal growth factor receptor (EGFR)
also leads to stimulation of bladder tumor growth. However,
crosstalk between AR and EGFR pathways in bladder cancer
remains uncharacterized. We have recently shown that andro-
gens activate the EGFR pathway in bladder cancer cells. The
purpose of this study was to investigate the effects of EGF on
AR activity in bladder cancer. EGF increased AR transcrip-
tional activity by 1.2-, 1.9- and 2.0-fold in UMUC3, 5637-AR
and J82-AR cell lines, respectively, over mock treatment and a
specific EGFR inhibitor, PD168393, antagonized the EGF effect.
Combined treatment of EGF and dihydrotestosterone (DHT)
further induced AR transactivation while an AR antagonist,
hydroxyflutamide (HF), abolished the effect of not only DHT but
also EGF. In growth assays, EGF alone/DHT alone/EGF+DHT
increased cell numbers by 16/12/19%, 6/14/18% and 30/12/38%
in UMUC3-control-shRNA, 5637-AR and J82-AR, respectively,
whereas the effects of EGF were marginal or less significant in
UMUC3-AR-shRNA (8%) or AR-negative 5637-V (<1%) and
J82-V (17%) cells. HF treatment at least partially counteracted
the EGF effect on the growth of AR-positive cells. Western
blotting demonstrated that EGF, especially in the presence of
DHT, upregulated the expression of the p160 coactivator TIF2
and HF again blocked this stimulation. Co-immunoprecipitation
revealed the association between AR and estrogen receptor
(ER)-β or Src in UMUC3 cells and stronger associations with
EGF treatment, implying the involvement of the AR/ER/Src
complex in EGF-increased AR transactivation and cell growth.
Current results, thus, suggest that EGF promotes bladder cancer
cell proliferation via modulation of AR signals. Taken together
with our previous findings, crosstalk between EGFR and AR
pathways can play an important role in the progression of bladder
Epidemiological and clinical evidence has indicated a substan-
tially higher risk of urinary bladder cancer in males yet there is
a tendency showing more aggressive behavior in tumors from
female patients (1,2). Recent experimental data suggest that
urothelial carcinoma, like prostate and breast cancers, is an
endocrine-related neoplasm (reviewed in ref. 3). In particular,
the androgen receptor (AR) and estrogen receptor (ER) signaling
pathways have been shown to contribute to bladder tumorigen-
esis and cancer progression (3-13), which may explain some of
the differences in male versus female bladder cancer.
Activation of the epidermal growth factor (EGF) receptor
(EGFR) family is known to involve the growth and progression
of a variety of malignancies. In bladder cancer, EGFR/ERBB2
is frequently overexpressed, which correlates with higher tumor
grade/stage and poorer prognosis (14-16). Experimental evidence
in bladder cancer has also suggested that the EGFR pathway
plays a critical role in cell proliferation, apoptosis, differen-
tiation, migration and angiogenesis (17-19). Consequently, the
efficacy of targeted therapy directed at EGFR signals has been
assessed in bladder cancer.
The crosstalk between nuclear hormone receptors and
growth factors leads to activation of nuclear receptor-mediated
transcription. Specifically, in prostate cancer cells, AR signals
upregulate EGFR and ERBB2 gene expression, whereas activa-
tion of EGFR and ERBB2 modulates AR functions (20-24). It
has also been shown that the assembly of the EGFR/AR/ER/
Src signaling complex is crucial for proliferation of prostate
and breast cancer cells triggered by androgens, estrogens and/
or EGF (25). In contrast, the relationship between the AR and
EGFR pathways in bladder cancer remains poorly understood.
We have recently shown that AR activation results in upregula-
Epidermal growth factor induces bladder cancer cell
proliferation through activation of the androgen receptor
KOJI IZUMI1*, YICHUN ZHENG1,2*, YI LI1,2, JACQUELINE ZAENGLE1 and HIROSHI MIYAMOTO1
1Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA;
2Department of Urology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, P.R. China
Received May 3, 2012; Accepted June 25, 2012
Correspondence to: Dr Hiroshi Miyamoto, Department of Pathology
and Laboratory Medicine, University of Rochester Medical Center,
601 Elmwood Avenue, Box 626, Rochester, NY 14642, USA
Abbreviations: AR androgen receptor; ER, estrogen receptor; EGF,
epidermal growth factor; EGFR, epidermal growth factor receptor; FBS,
fetal bovine serum; DHT, dihydrotestosterone; HF, hydroxyflutamide;
shRNA, short hairpin RNA; SDS, sodium dodecylsulfate; PAGE,
polyacrylamide gel electrophoresis; TIF2, transcriptional intermediary
factor 2; ARE androgen response element
Key words: androgen receptor, bladder cancer, epidermal growth
factor, hydroxyflutamide, Src, transcriptional intermediary factor 2
IZUMI et al: EPIDERMAL GROWTH FACTOR AND ANDROGEN RECEPTOR IN BLADDER CANCER
tion of EGFR and ERBB2 expression in bladder cancer cells,
which may play an important role in androgen-mediated tumor
progression (26). In the present study, we investigated whether
EGF could alter AR activity in bladder cancer cells.
Materials and methods
Cell culture and chemicals. Human bladder cancer cell lines,
UMUC3, 5637 and J82, obtained from the American Type
Culture Collection (Manassas, VA, USA) were maintained in
Dulbecco's modified Eagle's medium (Mediatech, Manassas,
VA, USA) supplemented with 10% fetal bovine serum (FBS) at
37˚C in a humidified atmosphere of 5% CO2. Cells were cultured
in phenol-red free medium supplemented with 5% charcoal-
stripped FBS at least 18 h before experimental treatment. We
obtained dihydrotestosterone (DHT) and EGF from Sigma
(St. Louis, MO, USA); hydroxyflutamide (HF) from Schering
(Kenilworth, NJ, USA); and PD168393 from Calbiochem (San
Diego, CA, USA).
Stable cell lines with AR and AR-short hairpin RNA (shRNA).
Cell lines stably expressing a full-length wild-type human AR
(5637-AR and J82-AR) or vector only (5637-V and J82-V) were
established, using a lentivirus vector (pWPI-AR or pWPI-control)
with psPAX2 envelope and pMD2.G packaging plasmids, as we
described previously (11,26). Similarly, stable AR knockdown/
control cell lines (UMUC3-AR-shRNA/UMUC3-control-
shRNA) were established with a retrovirus vector pMSCV/
U6-AR-shRNA or pMSCV/U6-control-shRNA (5,26).
Reporter gene assay. Bladder cancer cells at a density of 50-60%
confluence in 24-well plates were co-transfected with 250 ng of
MMTV-luc reporter plasmid DNA and 2.5 ng of pRL-TK-luc
plasmid DNA, using GeneJuice transfection reagent (Novagen,
Gibbstown, NJ, USA). Six hours after transfection, the medium
was replaced with one supplemented with 5% charcoal-stripped
FBS containing ethanol or ligands (DHT, HF, EGF and/or
PD168393) for 24 h. Cells were harvested, lysed and assayed for
luciferase activity determined using a dual-luciferase reporter
assay kit (Promega, Madison, WI, USA) and luminometer
(TD-20/20; Turner BioSystems, Sunnyvale, CA, USA).
Cell proliferation assay. We used the MTT (methyl thiazolyl
diphenyl tetrazolium bromide) assay to assess cell viability, as
described previously (26,27). Briefly, cells (3x103) seeded in
96-well tissue culture plates were incubated with medium supple-
mented with charcoal-stripped FBS in the presence or absence of
ligands (DHT, HF and EGF). The media were refreshed every
24 h. After 96 h of treatment, 10 µl MTT (Sigma) stock solution
(5 mg/ml) was added to each well with 0.1 ml of medium for 4 h
at 37˚C. The medium was replaced with 100 µl DMSO followed
by incubation for 5 min at room temperature. The absorbance
was then measured at a wavelength of 570 nm with background
subtraction at 655 nm.
Western blotting. Protein extraction and western blotting were
performed, as described previously (27) with minor modifica-
tions. Briefly, equal amounts of protein (20 µg) obtained from cell
extracts were separated in a 10% sodium dodecylsulfate (SDS)-
polyacrylamide gel electrophoresis (PAGE) and transferred to
polyvinylidene difluoride membrane (Millipore, Billerica, MA,
USA) by electroblotting using a standard protocol. Specific
antibody binding was detected, using an anti-AR antibody (clone
N20; diluted 1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA,
USA), an anti-transcriptional intermediary factor 2 (TIF2) anti-
body (clone 29/TIF2; diluted 1:1,000; BD Bioscience, Franklin
Lakes, NJ, USA), or an anti-GAPDH antibody (clone 6C5; diluted
1:1,000; Santa Cruz Biotechnology), with horseradish peroxidase
detection system (SuperSignal West Pico Chemiluminescent
Substrate; Thermo Scientific, Rockford, IL, USA).
Co-immunoprecipitation. UMUC3 cells were treated with
mock (ethanol) or EGF for 24 h and protein (500 µg) from the
cell lysates was incubated with 2 µg of anti-AR antibody (N20)
or normal rabbit IgG (Santa Cruz Biotechnology) for 16 h at
4˚C with agitation. To each sample we added 20 µl of protein
A/G-agarose beads (Santa Cruz Biotechnology), incubated for
1 h and washed thrice with radio-immunoprecipitation assay
buffer. Then, the complex was resolved on a 10% SDS-PAGE,
transferred to the membrane and blotted with an anti-ERβ
antibody (clone 14C8; diluted 1:500; Abcam, Cambridge, MA,
USA) or an anti-v-Src antibody (clone 327; diluted 1:1,000;
Statistical analysis. Student's t-test was used to analyze differ-
ences in relative luciferase activity and relative cell number
between the two groups. P<0.05 was considered statistically
EGF mediates AR transactivation via EGFR. Because previous
studies showed ligand-independent activation of AR transcrip-
tion by EGF in prostate cancer cells (20-22), we first assessed
the effects of EGF and a specific EGFR inhibitor PD168393
on AR transactivation in bladder cancer lines. In AR-positive
UMUC3 and AR-negative 5637 and J82 with a full-length
AR stably expressed by lentivirus, luciferase activity was
determined in the cell extracts with transfection of a plasmid
(MMTV-luc) containing an androgen response element (ARE)
as a reporter of AR-mediated transcriptional activity. As shown
in Fig. 1, EGF treatment increased luciferase activity by 1.2-,
1.9- and 2.0-fold in UMUC3 (p=0.013), 5637-AR (p=0.036) and
J82-AR (p=0.050), respectively, over mock treatment. PD168393
showing only marginal activity (in UMUC3 and 5637-AR) or
some agonist effect (1.5-fold in J82-AR) could antagonize the
EGF effect on AR transcription. In AR-knockdown UMUC3-
AR-shRNA and AR-negative lines (5637, 5637-V, J82 and
J82-V), EGF and/or PD168393 showed marginal effects on AR
transcriptional activity (data not shown). These results suggest
that EGF induces AR transactivation via EGFR in an androgen-
Antiandrogen blocks EGF-induced AR transactivation. We
next assessed the effect of EGF, in conjunction with androgen
and/or antiandrogen, on AR transcriptional activity in bladder
cancer cells. As shown in Fig. 2A, DHT treatment increased
AR transcription by 25% (lanes 1 vs. 3, p=0.032) and addi-
tion of EGF further induced it by 35% (lanes 1 vs. 4, p=0.001;
lanes 3 vs. 4, p=0.103) in UMUC3. Interestingly, HF showing
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 1587-1592, 2012
only marginal activity (lanes 1 vs. 5) abolished the effects of not
only DHT (lanes 3 vs. 7, p=0.077) but also EGF (lanes 2 vs. 6,
p=0.061) and EGF+DHT (lanes 4 vs. 8, p=0.082). Similarly, in
5637-AR (Fig. 2B) and J82-AR (Fig. 2C), DHT (lane 3) induced
AR transcription to 52- and 7.4-fold, respectively and EGF in
the presence of DHT (lanes 4 vs. 3) enhanced it to 78- (p=0.035)
and 30-fold (p=0.054), respectively. HF showing some agonist
activities (lanes 1 vs. 5) in 5637-AR (15-fold)/J82-AR (1.8-fold),
which were much higher (vs. 1.7-fold)/similar (vs. 2.1-fold)
compared to EGF stimulations (lane 2), could block the effects
of DHT (lanes 3 vs. 7, p=0.005/p=0.164) and EGF+DHT (lanes
4 vs. 8, p=0.009/p=0.013). Again, in UMUC3-AR-shRNA,
5637(-V) and J82(-V) cells, EGF, DHT and/or HF showed
marginal effects on AR transcription (data not shown). These
findings suggest that EGF and androgen cooperatively induce
AR transactivation that is sufficiently inhibited by an anti-
EGF stimulates cell growth via AR signaling. We then
performed the MTT assay to investigate the effects of EGF
androgen and antiandrogen on cell proliferation of bladder
cancer lines with vs. without AR (i.e., UMUC3-control-shRNA
vs. UMUC3-AR-shRNA, 5637-AR vs. 5637-V and J82-AR vs.
J82-V). As shown in Fig. 3A, in UMUC3-control-shRNA, treat-
ment of EGF, DHT and EGF+DHT increased cell growth by
16% (p=0.020), 12% (p=0.195) and 19% (p=0.009), respectively,
over mock treatment and HF treatment appeared to restore the
growth to the basal levels. In UMUC3-AR-shRNA, DHT effect
was marginal (2%) and the effects of EGF (8%, p=0.039) and
EGF+DHT (11%, p=0.040) were less significant compared to
those in UMUC3-control-shRNA. In 5637-AR, treatment of
EGF, DHT and EGF+DHT induced cell growth by 6% (p=0.558),
14% (p=0.016) and 19% (p=0.050), respectively and HF almost
completely abolished the stimulation (Fig. 3B). In 5637-V, only
marginal effects of EGF, DHT and/or HF on cell numbers
were seen. In J82-AR, treatment of EGF, DHT and EGF+DHT
induced cell growth by 30% (p=0.001), 12% (p=0.179) and
38% (p<0.001), respectively (Fig. 3C). Interestingly, HF was
able to antagonize the DHT effect but only partially blocked the
EGF effect. As expected, in J82-V, DHT did not increase cell
growth, while EGF and EGF+DHT, although less significant,
induced it by 17% (p=0.010) and 20% (p=0.043), respectively.
Additionally, HF failed to block the EGF effect in J82-V cells.
These results suggest that EGF promotes bladder cancer cell
proliferation at least partially through the AR pathway.
EGF increases AR and TIF2 expression. To further investigate
how EGF influences AR signals, we examined AR expression
by western blotting. In UMUC3, AR expression was increased
by DHT (4.4-fold) and further enhanced by addition of EGF
(6.4-fold), whereas no significant effect of EGF or HF was seen
in the absence of DHT (Fig. 4A). HF clearly antagonized the
effects of DHT with or without EGF. In J82-AR, EGF appeared
to increase AR expression both in the presence (2.8-fold) and
Figure 1. Effects of EGF on AR transactivation. Bladder cancer cells (A, UMUC3; B, 5637-AR; C, J82-AR) were transfected with MMTV-Luc and were then
cultured for 24 h in the presence of ethanol (mock), 100 ng/ml EGF and/or 1 µM PD168393, as indicated. Luciferase activity analyzed in a luminometer is presented
relative to that of mock treatment in each cell line (first lanes; set as 1-fold). Each value represents the mean + SD from at least three independent experiments.
Figure 2. Effects of androgen and antiandrogen on EGF-mediated AR transactivation. Bladder cancer cells (A, UMUC3; B, 5637-AR; C, J82-AR) were transfected
with MMTV-Luc and were then cultured for 24 h in the presence of ethanol (mock), 100 ng/ml EGF, 10 nM DHT and/or 10 µM HF, as indicated. Luciferase activity
analyzed in a luminometer is presented relative to that of mock treatment in each cell line (first lanes; set as 1-fold). Each value represents the mean + SD from at
least three independent experiments. *p<0.05; **p<0.01.
IZUMI et al: EPIDERMAL GROWTH FACTOR AND ANDROGEN RECEPTOR IN BLADDER CANCER
absence (1.5-fold) of DHT and HF abolished these effects
(Fig. 4C). In contrast, only marginal effects of EGF and/or DHT
on AR expression were observed in 5637-AR (Fig. 4B).
Because EGF was shown to induce AR transcription by
upregulating TIF2 expression in prostate cancer cells (21), we
then determined the levels of TIF2 expression in bladder cancer
cell lines upon treatment with EGF, androgen and/or antian-
drogen. As shown in middle panels of Fig. 4, EGF increased TIF2
expression in the presence (1.5- to 1.8-fold) and absence (1.2- to
1.3-fold) of DHT. DHT alone increased TIF2 expression only in
5637-AR (1.4-fold) and showed marginal effects in UMUC3 and
J82-AR. In addition, HF abrogated EGF- and/or DHT-enhanced
TIF2 expression in all these three lines.
EGF induces AR association with ER and Src. Previous studies
in prostate and breast cancers demonstrated that EGF induced
AR/ER/Src association, resulting in activation of Src signaling
(25,28) and that Src signals phosphorylated tyrosine residue
Figure 3. Effects of EGF on cell viability. Bladder cancer cells (A, UMUC3-
control-shRNA/AR-shRNA; B, 5637-AR/vector; C, J82-AR/vector) were
cultured for 4 days in the presence of ethanol (mock), 100 ng/ml EGF, 10 nM
DHT and/or 10 µM HF, as indicated. Cell viability was assayed with MTT
and growth induction is presented relative to cell number with mock treatment
estimated by measuring the absorbance at a wavelength of 570 nm with a back-
ground subtraction at 655 nm (first lanes; set as 1-fold). Each value represents
the mean + SD from at least three independent experiments. *p<0.05; **p<0.01.
Figure 4. Effects of EGF on AR and TIF2 protein expression. Bladder cancer
cells (A, UMUC3; B, 5637-AR; C, J82-AR) were cultured for 24 h in the
presence of ethanol (mock), 100 ng/ml EGF, 10 nM DHT and/or 10 µM HF,
as indicated. Equal amounts of protein extracted from each cell line were
immunoblotted for AR (110 kDa, upper), TIF2 (160 kDa, middle), or GAPDH
(37 kDa, lower) as indicated. Densitometry values for specific bands stan-
dardized by GAPDH that are relative to those of mock treatment (first lanes;
set as 1-fold) are included below the lanes.
Figure 5. Effects of EGF on AR/ER/Src association. UMUC3 cells were cul-
tured for 24 h in the presence of ethanol (mock) or 100 ng/ml EGF. Cell lysates
were immunoprecipitated with anti-AR antibody or normal rabbit IgG and
were then immunoblotted for AR (110 kDa), ERβ (56 kDa), or Src (60 kDa),
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 1587-1592, 2012
of AR, provoking its transactivation and cell proliferation
(29). We therefore investigated whether EGF induced AR/ER/
Src complex formation in UMUC3 which is ERα-negative/
ERβ-positive (figure not shown). As shown in Fig. 5, both Src and
ERβ were co-immunoprecipitated with AR in bladder cancer
cells. Furthermore, EGF treatment facilitated the association of
AR with ERβ or Src.
Dysregulation of the EGFR family is well known to associate
with bladder cancer (14-16). AR signals have also been implicated
in bladder carcinogenesis and tumor progression (3,5,7,9-13).
Nonetheless, crosstalk between the AR and EGFR pathways
remains unclear in bladder cancer, although it has been widely
studied in prostate cancer (20-24). We have recently shown that
AR signals increase EGFR and ERBB2 expression and activity,
suggesting androgen-mediated bladder cancer progression via
the regulation of the EGFR/ERBB2 pathways (26). In the present
study, we provided evidence suggesting that EGF could regulate
cell proliferation by activating AR signals in bladder cancer.
In prostate cancer, accumulating evidence has indicated
that EGFR/ERBB2 signals induce AR transactivation in an
androgen-dependent and -independent manner (20-22). In
bladder cancer cells, we here showed that EGF could activate
AR transcription and PD168393, a specific inhibitor of EGFR,
restored this EGF effect. These data suggest that EGF androgen-
independently induces EGFR-mediated ARE reporter activity
in bladder cancer. However, it was shown that the effect of EGF
on AR transcription might be almost negligible compared to the
induction by androgens in prostate cancer (20,21). Similarly, in
bladder cancer lines 5637-AR and J82-AR where a wild-type
AR was stably overexpressed, the effect of EGF was less signifi-
cant than that of DHT. On the other hand, in UMUC3 cells that
possess endogenous AR, EGF effect (20% increase) is similar to
the relatively insignificant effect of DHT (25% increase). In addi-
tion, PD168393 displayed agonist effects [1.5-fold (vs. 2.0-fold
by EGF or 7.4-fold by DHT)] on AR transcription in J82-AR
via unknown mechanisms. It was described in prostate cancer
cells that PD168393 upregulated AR target gene expression in
the presence of androgen, possibly via blocking basal activity of
EGFR or ERBB2 (30). Importantly, as shown in prostate cancer
(21), a combination of EGF and androgen further induced AR
transcriptional activity in all the three bladder cancer lines tested
and the AR antagonist HF completely abolished AR transactiva-
tion induced by EGF, androgen, or both at least in UMUC3. We
could not evaluate antagonistic effects of HF on EGF-induced
AR transcription due to the considerable agonist activity of HF
which was even higher than that of EGF in 5637-AR. Thus, our
results support the possibility that EGF mediates AR transcrip-
tional activity through the EGFR and AR pathways in bladder
Consistent with previous findings shown by others and us
(5,7,9,26) androgens promoted AR-positive bladder cancer cell
proliferation that was blocked by antiandrogens. These effects
of androgens were suggested to be at least partially medi-
ated through the EGFR pathway (26). In the present study, as
expected, EGF increased the growth of AR-positive cells and,
less significantly, that of AR-knockdown/negative cells. In
AR-positive lines, combined treatment with EGF and androgen
further induced cell proliferation. Of note were inhibitory effects
of the AR antagonist on EGF- and EGF+androgen-increased cell
growth. Specifically, on the growth of 5637-derived lines, EGF
and/or DHT showed only marginal effects (5637-V) and HF
almost completely abolished EGF-mediated effects (5637-AR).
These findings indicate that EGF-induced cell proliferation
involves the AR pathway in bladder cancer. Nonetheless, in
J82-derived lines, EGF retained its effect on cell growth without
AR (J82-V) and HF failed to completely inhibit EGF-increased
cell proliferation (J82-AR), suggesting the involvement of those
other than the AR pathway.
It has been reported that EGF is capable of inducing AR
transcription and protein expression in androgen-independent
prostate cancer cells (21). Others also described negative
regulation of AR expression and activity by EGFR signaling
in prostate cancer (30,31). In bladder cancer cells, we previ-
ously showed increases in the expression of endogenous AR by
androgen treatment (26), which was inconsistent with the results
demonstrated by Boorjian et al (9). We also showed no signifi-
cant increases in exogenously overexpressed AR (5637-AR) by
DHT or in endogenous and exogenous ARs by EGF (26). We
confirmed our previous findings in the three lines tested and
further showed EGF-enhanced AR overexpression in the pres-
ence of androgen in UMUC3 and J82-AR, but not in 5637-AR.
The mechanism underlying this discrepancy in the response to
the treatment of EGF+DHT between levels of exogenous AR
expression in 5637 versus J82 remains uncertain. Repeatedly,
the AR expression increased by androgen with or without EGF
in bladder cancer cells was abolished by an AR antagonist.
EGF has been shown to enhance the expression or phosphor-
ylation of TIF2, one of the p160 nuclear receptor coactivators,
leading to an increase in AR transactivation in prostate cancer
cells (21). Indeed, the expression of major AR coactivators,
including TIF2, was detected in bladder cancer cell lines as
well as in AR-positive and even AR-negative bladder tumor
specimens and TIF2 knockdown resulted in a decrease in
androgen-mediated cell proliferation (9). We here found that
TIF2 was considerably (e.g., ≥1.5-fold) augmented in the pres-
ence of EGF and DHT in bladder cancer cells, while EGF or
DHT alone could lead to marginal/only slight increases in TIF2
expression. Interestingly, like our results in AR expression/
activity and cell proliferation, EGF-induced TIF2 upregulation
was abolished by the antiandrogen. Although detailed mecha-
nisms need to be clarified, these results may imply that elevated
levels of TIF2 contribute to EGF/androgen-enhanced AR trans-
activation in bladder cancer cells.
In hormone-responsive cells expressing both AR and ER
(α and/or β), such as prostate and breast cancers, AR/ER/Src
association plays a crucial role in activation of Src signals trig-
gered by EGF and/or sex hormones (25,28). It was noteworthy
that either AR or ER antagonist sufficiently inhibited this
EGF-mediated association and subsequent stimulatory effects
(28). It has also been shown that Src mediates EGF-induced
AR tyrosine phosphorylation in prostate cancer cells, which
leads to an increase in AR transcriptional activity (29). Indeed,
in many bladder cancer tissue specimens, AR and ER(s) were
found to be co-expressed (3,10,12). In this study, we showed
associations of AR with ERβ and Src in UMUC3 which were
enhanced by EGF treatment. These findings suggest that EGF
activates Src via assembling the AR/ER/Src complex, resulting
IZUMI et al: EPIDERMAL GROWTH FACTOR AND ANDROGEN RECEPTOR IN BLADDER CANCER Download full-text
in AR transactivation and cell proliferation in bladder cancer.
This may also justify the drastic inhibition of EGF-induced
effects accomplished by antiandrogen treatment.
In conclusion, EGF could increase AR transcriptional activity
and cell proliferation in bladder cancer. These EGF effects were
likely mediated through the AR pathway involving upregulation
of TIF2 expression as well as activation of Src signals due to
forming an AR/ER/Src complex. These results, together with our
previous findings, not only shed light on crosstalk between the
AR and EGFR pathways in bladder cancer but also enhance the
feasibility of androgen deprivation interfering with this crosstalk
as a potential therapeutic approach.
H.M. was supported by the Department of Defense Prostate
Cancer Research Program (W81XWH-09-1-0305).
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