The significance of Her2 on AR protein stability in the transition of androgen 1
requirement in prostate cancer cells 2
3 4
Fu-Ning Hsu1, Min-Shiou Yang1, Eugene Lin1, 3, Chun-Fu Tseng1, and Ho Lin1, 2* 5
6 7
1Department of Life Sciences, National Chung Hsing University, Taichung 40227;
8
2Graduate Institute of Rehabilitation Science, China Medical University, Taichung 9
40402; 3Department of Urology, Chang Bing Show Chwan Memorial Hospital, 10
Changhua 505, Taiwan 11
12 13
Running head: androgen receptor stability & Her2 activity 14
15 16
Keyword: Her2, androgen receptor, androgen-independent, prostate cancer cells 17
18 19 20 21 22 23 24 25 26 27 28 29 30
Correspondence: *Ho Lin, Ph.D.
31
Department of Life Sciences 32
National Chung Hsing University 33
250 Kuo Kuang Road, 34
Taichung 40227, Taiwan 35
Tel No.: 886-4-2284-0416-617 36
Fax No.: 886-4-2287-4740 37
E-mail: [email protected] 38
Abstract 40
Androgen ablation therapy is the most common strategy to suppress prostate 41
cancer progression; however, tumor cells eventually escape androgen requirement and 42
progress into androgen-independent phase. Androgen receptor (AR) plays a pivotal 43
role in this transition. In order to answer this transition mystery in prostate cancer, we 44
established an androgen-independent prostate cancer cell line (LNCaPdcc) by 45
long-term screening LNCaP cells in androgen-deprived condition to investigate 46
changes of molecular mechanisms before and after androgen withdrawal. We found 47
that LNCaPdcc cells displayed the morphology of neuroendocrine differentiation, less 48
aggressive growth, weaker androgen sensitivity, and lower expression levels of cell 49
cycle-related factors, although the cell cycle distribution was similar to parental 50
LNCaP cells. Interestingly, higher protein expressions of AR, phospho-Ser81-AR, and 51
PSA in LNCaPdcc cells were observed. Moreover, nuclear distribution and protein 52
stability of AR increased in LNCaPdcc cells. On the other hand, LNCaPdcc cells 53
expressed higher levels of Her2, phospho-Her2, and ErbB3 proteins than parental 54
LNCaP cells. Notably, these two cell lines exhibited distinct responses toward Her2 55
activation (by heregulin treatment) and Her2 inhibition (by AG825 or Herceptin 56
treatments) on proliferation. In addition, Her2 inhibitor more effectively caused AR 57
degradation in LNCaPdcc cells. Taken together, our data demonstrate that Her2 plays 58
an important role to support AR protein stability in the transition of androgen 59
requirement in prostate cancer cells. We hope these findings would provide new 60
suggestion on the treatment of hormone-refractory prostate cancer.
61
62
Introduction 63
Prostate cancer is an age-related carcinoma and the most commonly diagnosed 64
malignancy among men (25). Although the prostate specific antigen (PSA), a 65
biomarker of hypertrophy in the prostate gland, helps to identify prostate cancer in the 66
early stages, the disease still causes high mortality. Traditionally, gonadectomy is the 67
main therapeutic procedure for androgen-dependent prostate cancer. Once the cancer 68
escapes from androgen dependence and becomes androgen-independent, radio- or 69
chemo-therapies are subsequently applied. Unfortunately, the treatment of 70
hormone-refractory prostate cancer in this stage is often ineffective and the 71
mechanisms of prostate cancer progression in this stage remain to be elucidated.
72
Therefore, it is imperative to understand the transition of androgen requirement and to 73
develop strategies for prolonging the survival of patients with recurrent and 74
hormone-refractory prostate cancer.
75
The androgen receptor (AR), a member of the steroid receptor family, plays a 76
decisive role in the development of the prostate gland and in the pathogenesis and 77
progression of prostate cancer. AR binds to androgen response elements (AREs) and 78
thereby mediates androgen-regulated gene expression (12). A growing number of 79
clinical investigations show amplifications of AR and AR-regulated genes in 80
hormone-refractory prostate cancer, which suggests that the AR signaling pathway is 81
still activated and important at limiting concentrations of androgen (14). Previous 82
research indicates that the elevated AR expression levels were correlated to resistance 83
to anti-androgen therapy (3). The cross-talk between receptor tyrosine kinases with 84
their cognate ligands and AR signaling in hormone-refractory transition of prostate 85
cancer has also been addressed (6, 11, 26). On the other hand, Her2/ErbB3 signals 86
have been suggested to stabilize AR proteins and to increase the interaction of AR to 87
promote/enhancer regions of AR-regulated gene in androgen-dependent prostate 88
cancer cells (23).
89
Here, we established an androgen-independent prostate cancer cell line named 90
LNCaPdcc by incubating LNCaP cells in androgen-deprived condition for a long 91
period (eight months). We try to take advantage of this popular strategy of cell model 92
to answer how prostate cancer cells maintain AR protein levels and activation in 93
androgen free environment. Indeed, we observed several characteristics obviously 94
changed after androgen deprivation. Importantly, our data showed that LNCaPdcc 95
cells were more sensitive to Her2 inhibition with increase of AR degradation than 96
parental LNCaP cells. These findings suggest that Her2 activation might be an 97
important support of AR protein stability in prostate cancer cells under adaptation of 98
androgen deprivation.
99
100
Materials and Methods 101
Materials 102
R1881 (Methyltrienolone; NLP-005) was purchased from PerkinElmer (Boston, 103
MA, USA); Cycloheximide (CHX; C1988) from Sigma (Missouri, USA); MG-132 104
(474791) from Calbiochem (San Diego, CA, USA); Recombinant human heregulin β1 105
(396-HB) from R&D Systems, Inc. (Minneapolis, MN, USA ); AG825 (121765) 106
from Calbiochem and Herceptin from Roche Applie Science (Mannheim, Germany).
107
Antibodies used for immunoblotting were indicated: Cdk1 (sc-54, Santa Cruz 108
Biotechnology, Santa Cruz, CA, USA), Cyclin A (sc-751, Santa Cruz), Cyclin B1 109
(sc-752, Santa Cruz), Cyclin D1 (sc-20044, Santa Cruz), β-actin (MAB1501, 110
Millipore, Temecula, CA, USA), phospho-Ser81-AR (07-541, Upstate, Lake Placid, 111
NY, USA), AR (sc-13062 and sc-7305, Santa Cruz), PSA (sc-7316, Santa Cruz), 112
α-tubulin (05-829, Upstate), PARP (06-557, Upstate), phospho-Tyr1221/1222-Her2 113
(2249, Cell Signaling, Danvers, MA, USA), Her2 (C-18, Santa Cruz; OP-15, 114
Calbiochem) and ErbB3 (sc-285 and 7309, Santa Cruz). Secondary antibodies were 115
peroxidase-conjugated anti-mouse or anti-rabbit (Jackson ImmunoResearch 116
Laboratory, West Grove, PA, USA).
117
118
Cell Culture 119
Human prostate carcinoma cell lines derived from lymph node carcinoma of the 120
prostate (LNCaP clone FGC (fast growing colony), BCRC 60088) (13) were 121
purchased from Food Industry Research and Development Institute, Taiwan. LNCaP 122
cells were maintained in complete medium: phenol red-positive RPMI-1640 culture 123
medium (Gibco, Carlesbad, CA, USA) supplemented with 1.5 g/L sodium bicarbonate 124
(NaHCO3) (Sigma), 10% fetal bovine serum (FBS) (Gibco), and 125
penicillin/streptomycin (P/S) (100 IU/mL and 100 μg/mL, respectively) (Gibco). Cells 126
were cultured at 37 oC in a humidified atmosphere with 5% CO2 (18). Cells were 127
routinely passaged by trypsin/EDTA (0.05% and 0.02%, respectively) (Gibco) twice a 128
week in the ratio 1:3. LNCaPdcc cells, a subline from LNCaP cells, was designed to 129
be an in vitro model for investigating the progression of androgen-independent 130
prostate cancer (7). LNCaPdcc cells were established by domesticating LNCaP cells 131
in a long-term androgen-ablated condition over 14 passages. To deprive cells of 132
steroid hormones, FBS was incubated with dextran-coated charcoal (dcc) (Sigma) by 133
rotating at a low speed at 4 °C for 12-16 h. The charcoal-FBS mixture was then 134
centrifuged twice at 500 g for 10 min. Then the supernatant was stored at –20 °C until 135
use. LNCaPdcc cells were grown in phenol red-free RPMI-1640 medium (Sigma) 136
plus 10% dcc-stripped FBS, 1.5 g/L NaHCO3, and P/S (100 IU/mL, 100 μg/mL) at 37 137
oC in a humidified atmosphere at 5% CO2. Cells were split once a week in the ratio 138
1:2. All experiments on LNCaPdcc were performed between passage 25 and 45.
139
140
Cell Viability Assay 141
The modified colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 142
bromide (MTT) assay was manipulated to quantify the viability of LNCaP and 143
LNCaPdcc cancer cells. Yellow MTT compound (Sigma) is converted by living cells 144
into blue formazen, which is soluble in isopropanol. The intensity of blue staining in 145
culture medium is proportional to the number of living cells and measured by using an 146
optical density reader (Athos-2001, Australia) at 570 nm (background, 620 nm) (1, 18, 147
19).
148
149
Immunoblotting and Fractionation Analyses 150
Cell lysates were obtained in lysis buffer (50 mM Tris-HCl [pH 8.0], 0.5%
151
Nonidet P-40 [NP-40], 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl 152
fluoride [PMSF], 2 mM sodium orthovanadate [Na3VO4], and protease inhibitor 153
cocktail [Roche Applied Science]). Lysates were then analyzed for immunoblotting 154
using methods modified from those previously described (1, 18, 19). To isolate 155
subcellular proteins, cells were collected and washed in PBS/Na3VO4. Pelleted cells 156
were resuspended in hypotonic buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM 157
EDTA, 0.1 mM EGTA, 0.5% NP-40, 1 mM PMSF, 2 mM Na3VO4, and protease 158
inhibitor cocktail). Nuclei were pelleted and the supernatant was harvested as the 159
cytosolic fraction. The nuclear pellet was washed three times with hypotonic buffer 160
before lysing in nuclear extraction buffer (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 161
mM EDTA, 1 M EGTA, 20% glycerol, 1 mM PMSF, 2 mM Na3VO4, and protease 162
inhibitor cocktail) in a procedure modified from those described previously (1, 18, 19).
163
Protein samples were analyzed by direct immunoblotting (25-35 μg/lane) or blotting 164
after immunoprecipitation (0.5-1 mg /immunoprecipitation). ECL detection reagent 165
(PerkinElmer Life Science) was used to visualize the immunoreactive proteins on 166
membranes (polyvinylidene difluoride, PVDF; Perkin Elmer Life Science) after 167
transfer using a Trans-Blot SD (Bio-Rad, Berkeley, CA, USA).
168
169
Trypan Blue Assay 170
LNCaP and LNCaPdcc cells were seeded in a 24-well plate in the complete 171
culture medium. Cells were trypsinized, stained with 0.2% trypan blue (Sigma), and 172
counted by hemocytometer to distinguish the live and dead cells.
173
174
Analysis of Cell Cycle Distribution 175
Propidium iodide staining was used for DNA content measurement. Cancer cells, 176
trypsinized and fixed in 70% ethanol, were washed once with PBS and treated with 177
RNase A for 30 minutes, followed by staining with propidium iodide (0.1% sodium 178
citrate, 0.1% Triton X-100, and 20 μl/mL propidium iodide). DNA content was 179
measured by using flow cytometry (FACS Calibur, Germany). Percentage of cells in 180
each phase of the cell cycle was analyzed by the software, Cell Quest.
181
182
Statistics 183
All values are given as the mean ± standard error of the mean (SEM). Student’s 184
t-test was used in the cell proliferation. A difference between two means was 185
considered statistically significant when p < 0.05.
186
Results 187
Comparisons of characteristics between LNCaPdcc and parental LNCaP cells 188
The LNCaPdcc cells displayed the dendritic-like morphology in neuroendocrine 189
differentiation as compared with parental LNCaP cells (Fig. 1A). In addition, growth 190
curves of two cell lines were determined by cell counting (Fig. 1B). The comparison 191
of doubling time (inset table of fig. 1B) showed that LNCaPdcc cells grew much 192
slower than parental LNCaP cells. By using the flow cytometry, the differences of cell 193
cycle distribution between parental LNCaP and LNCaPdcc cells were identified. The 194
data showed that S phase distribution of LNCaPdcc cells was obviously higher than 195
parental LNCaP cells although G1 and G2/M phase distributions of two cell lines 196
were similar (Fig. 2A). Therefore, it is interesting to understand the levels of cell 197
cycle-related proteins expressed in two cell lines. The results revealed that the protein 198
levels of Cdk1, cyclin A, cyclin B1, and cyclin D1 were all lower in LNCaPdcc cells 199
(Fig. 2B), which might explain why LNCaPdcc cells grew slowly and stuck in S 200
phase.
201
202
AR-related features in two cell lines 203
Compared to parental LNCaP cells, LNCaPdcc cells expressed higher protein 204
levels of phospho-Ser81-AR, AR, and PSA (AR-regulated gene) (Fig. 3A). In addition, 205
the protein fractionation was utilized to investigate the subcellular distribution of AR 206
proteins. Interestingly, compared to parental LNCaP cells, LNCaPdcc cells contained 207
higher levels of nuclear AR protein (Fig. 3B), indicating that AR in LNCaPdcc cells 208
are still activated even in the absence of androgen. Then, cycloheximide (CHX) was 209
used to block protein synthesis and the degradation of existing protein was then 210
monitored. The result exhibited that AR protein in LNCaPdcc cells was more stable 211
than that in parental LNCaP cells (Fig. 3C). Subsequently, the cell proliferation in 212
response to androgen treatment was investigated by using MTT assay. Parental 213
LNCaP cell proliferation was sensitive to synthetic androgen R1881 under 214
steroid-deprived condition, especially at the limiting concentrations (0.1 and 1 nM) of 215
androgen. However, the proliferation of LNCaPdcc cells was inhibited by these 216
concentrations (Fig. 4). The result illustrates that the proliferation of LNCaPdcc cells 217
is androgen-independent.
218
219
Her2-related features in two cell lines 220
According to previous research, there is a correlation between AR and Her2 221
signals in androgen-dependent prostate cancer cells (23). Therefore, the protein 222
expressions of Her2 and its activation partner, ErbB3, in two cell lines were 223
investigated. The data showed that LNCaPdcc cells expressed higher levels of 224
phospho-Y1221/1222-Her2, Her2, and ErbB3 (Fig. 5A). Since Her2 activation was 225
correlated to its phosphorylation status, these data imply that Her2 is more active in 226
LNCaPdcc cells than parental cells. In addition, parental LNCaP and LNCaPdcc cells 227
were both treated with 10 ng/mL of heregulin (HRG, ligand of Her2/ErbB3) in a 228
time-course manner. HRG-induced Her2 activation in LNCaPdcc cells could sustain 229
for 24 hours after treatment; however, that activation in parental cells simply dropped 230
since 1 hour after treatment (Fig. 5B). In order to understand the physiological 231
functions of Her2 in different cell lines, the effects of Her2 inhibitors on proliferation 232
of two cell lines were evaluated by MTT assay. AG825 and Herceptin (monoclonal 233
antibody of Her2 for clinical use) were treated to both cell lines. Parental LNCaP cell 234
proliferation displayed weak response to both Her2 inhibitors whereas LNCaPdcc cell 235
proliferation was significantly declined by Her2 inhibition (Fig. 5C). It might be due 236
to the high levels of Her2 in LNCaPdcc cells. Accordingly, Her2 in LNCaPdcc cells 237
might take more charge on LNCaPdcc proliferation comparing to parental cells.
238
239
AR stability in LNCaPdcc cells depends on high Her2 activation 240
AR is a short half-life protein and tends to be degraded through the 241
ubiquitin-proteasome pathway (27). It has been reported that AR proteins can be 242
stabilized under Her2/ErbB3 activation (23). In addition, our data indicated that AR 243
protein levels were correlated to Her2 activation in both cell lines (data not shown). In 244
order to determine whether Her2 is involved in the increase of AR stability in 245
LNCaPdcc cells (Fig. 3C), Her2 inhibition (by AG825) was performed and AR 246
stability in two cell lines was monitored. The results showed that Her2 inhibition 247
accelerated AR degradation in LNCaPdcc cells (Fig. 6A), although the initial level of 248
AR protein in LNCaPdcc cells was still higher than that in parental cells (time=0, Fig.
249
6A). After 9-hour treatment of CHX, the AR degradation percentage of LNCaPdcc 250
(quantitative ratio) was 79% which is much higher than 32% of parental cells.
251
Furthermore, Ser81 phosphorylation of AR has been reported to be responsible for 252
itself stability (21). Corresponding to previous research (23), Her2 inhibitor 253
effectively reduced AR Ser81 phosphorylation in both cell lines (Fig. 6B).
254
Interestingly, LNCaPdcc cells were more sensitive to AG825 treatment on the 255
inhibition of AR Ser81 phosphorylation (40% inhibition in LNCaPdcc cells versus 256
20% inhibition in parental cells). Taken together, higher Her2 activation might make 257
more contribution to AR protein stability through Ser81 site phosphorylation in 258
LNCaPdcc cells.
259
260
Discussion 261
Prostate carcinoma is a leading cause of death in male malignancy. Since the 262
prostate is an androgen-dependent gland, androgen ablation therapy is the most 263
frequent strategy used to suppress prostate tumor pathogenesis. Nevertheless, cancer 264
cells eventually escape the androgen requirement and progress to an 265
androgen-independent phenotype. The cure for the hormone-refractory prostate cancer 266
remains a main clinical challenge. In the progression of prostate cancer, AR emerges 267
as an important determinant. AR protein controls cell cycle, cell proliferation, 268
inhibition of apoptosis, regulation of angiogenic growth factors, and stimulation of 269
cellular migration among other functions (5). In order to investigate the roles of AR 270
activity in prostate cancer progression following androgen withdrawal, the authors 271
established LNCaPdcc subline by long-term screening LNCaP cells in an 272
androgen-stripped condition. The LNCaPdcc cells revealed a dendritic-like 273
morphology (Fig. 1A) and a lower growth rate (Fig. 1B) indicating the adaptation of 274
LNCaPdcc cells to androgen-free condition.
275
276
Interestingly, AR proteins of LNCaPdcc cells were even more active in the 277
absence of androgen because higher levels of AR Ser81 phosphorylation, PSA 278
proteins (Fig. 3A) and nuclear AR proteins (Fig. 3B) in LNCaPdcc cells were 279
observed. It might be due to the excessive recruitment of coactivators (10) or crosstalk 280
with several polypeptide growth factors as well as cognate receptors (22, 28) in the 281
transition of prostate cancer. On the other hand, cyclin D1 was reported to interact 282
predominantly with the N-terminal domain of AR and this interaction depends on the 283
presence of the AR 23FxxLF27 motif, which is also important for interaction between 284
the N- and C-termini of AR. Through this motif, cyclin D1 protein prevents the 285
interaction between the two termini of AR, consequently inhibiting AR activity (2).
286
Our data revealed that cyclin D1 proteins dramatically declined in LNCaPdcc cells 287
(Fig. 2B), illustrating that the decrease of cyclin D1 levels might help to increase AR 288
activation. In addition, we found that the proliferation of LNCaPdcc cells was not 289
dependent on androgen (Fig. 4). It has been reported that AR in LNCaP cell line is a 290
T877A mutant that can be activated not only by androgens but also by non-androgenic 291
steroid hormones and anti-androgens (31). Our unpublished data showed that parental 292
LNCaP cell proliferation was significantly stimulated by estradiol bezoate (EB, 293
synthetic estrogen) in dose-dependent manner while LNCaPdccdisplayed insensitive 294
to EB.
295
296
According to previous study, AR is a short half-life protein in the absence of 297
androgen (10) and tends to be degraded through the ubiquitin-proteasome pathway 298
(27). Ubiquitin-proteasome degradation is important to transcriptional regulation (20) 299
and ubiquitin-ligase E6-associated protein may be a cofactor of steroid receptors (24).
300
Therefore, it is of interest to investigate what delays AR degradation in LNCaPdcc 301
cells (Fig. 3C). In addition to ligand-dependent regulation, post-translational 302
modification of AR has also been extensively discussed (8). The existence of AR 303
Ser81 phosphorylation is correlated to protein stability (21). On the other hand, the 304
Her2/ErbB3 axis has been reported to provide signals to AR which protects AR 305
protein stability (23). It also demonstrates that the androgen-induced Ser81 306
phosphorylation of AR is declined by a small molecule Her2 inhibitor PKI-166 (23).
307
Additionally, our findings indicated that AR protein levels seem to be positively 308
regulated by Her2 activity but not by epidermal growth factor receptor (EGFR) 309
activation (data not shown). These results suggest the existence of a specific and 310
enhanced regulation between Her2 activation and AR stability in LNCaPdcc cells. In 311
addition, our findings also indicated that AR Ser81 phosphorylation was inhibited by 312
Her2 inhibitors (Fig. 6B), which suggests that AR Ser81 site is a downstream 313
substrate of Her2 pathway. As regards to Her2-downstream serine-threonine kinases, 314
Akt/protein kinase B (PKB) has been reported not to be the kinase that responds to 315
AR Ser81 phosphorylation due to the analysis of phosphorylation consensus sequence 316
sites (23). Although the Ser81 site occurs in the consensus sequence of protein kinase 317
C (PKC), PKC inhibitors fail to reduce AR Ser81 phosphorylation (9). Several 318
kinases are implied or predicted to be the candidates responding to AR Ser81 319
phosphorylation such as Cdk1, Cdk5 (4), and Erk (29). However, Cdk1 activation is 320
inhibited by Her2 via phosphorylation on tyrosine 15 site (30). Moreover, Cdk1 321
proteins diminished in our LNCaPdcccells (Fig. 2B), illustrating that the increasing 322
levels of Her2-dependent AR Ser81 phosphorylation might be irrelevant to Cdk1 323
activity. On the contrary, we have reported that Cdk5 activity is elevated by Her2 324
activation through Tyr15 phosphorylation in thyroid cancer cells (16). In addition, 325
Cdk5 is also reported to modulate androgen production (17) and cell fate of prostate 326
cancer (15, 18) by us. With regards to Erk, we found that both phospho-Erk and Erk 327
levels increased in LNCaPdcccells as compared to those in parental LNCaP cells 328
(data not shown). The specific kinases regulated by Her2 and responsible for Ser81 329
phosphorylation of AR need to be further investigated.
330
331
According to the results in Fig. 3, LNCaPdcccells displayed higher level of AR 332
Ser81 phosphorylation and longer half-time of AR proteins in androgen-stripped 333
environment. Coincidentally, LNCaPdcccells expressed higher levels of 334
phospho-Her2 and Her2 proteins (Fig. 5A). By using Her2 inhibitor, Her2 in 335
LNCaPdcc cells was more sensitive to its inhibitor and resulted in the drops of either 336
AR Ser81 phosphorylation or AR protein stability (Fig. 6). These results suggest that 337
Her2 not only plays a role of growth factor receptor, but also protects AR protein 338
stability through Ser81 phosphorylation in LNCaPdcc cells after cells escape the 339
androgen requirement.
340
341
In conclusion, we used a new-established prostate cancer cell subline, LNCaPdcc, 342
to elucidate different characteristics and protein expressions comparing to parental 343
LNCaP cells. LNCaPdcc cells display features of androgen-independent prostate 344
cancer. We found that, in LNCaPdcc cells, Her2 activation becomes more important to 345
protect AR protein from degradation through Ser81 phosphorylation and subsequently 346
modulates cell proliferation. We hope our findings would be helpful in understanding 347
the transition of androgen deprivation. Besides, we also suggest that Her2-AR axis 348
would become a diagnostic and therapeutic target in hormone-refractory prostate 349
cancer in the near future.
350
351
Acknowledgements 352
The authors thank Dr. Shih-Lan Hsu and Ms. Mei-Chun Liu (Department of 353
Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan) for 354
their full support; Dr. Ying-Ming Liou (National Chung Hsing University, Taiwan) for 355
technical support.
356
357
Grants 358
This work was supported by grants NSC97-2320-B-005-002-MY3 and 359
NSC96-2628-B-005-013-MY3 from the National Science Council and in part by the 360
Taiwan Ministry of Education under the ATU plan (to H. Lin, National Chung Hsing 361
University).
362
363
Disclosures 364
The authors have no conflicts of interest to declare.
365
References 366
1. Berger R, Lin DI, Nieto M, Sicinska E, Garraway LA, Adams H, Signoretti 367
S, Hahn WC, and Loda M. Androgen-dependent regulation of Her-2/neu in 368
prostate cancer cells. Cancer Res 66: 5723-5728, 2006.
369
2. Burd CJ, Petre CE, Moghadam H, Wilson EM, and Knudsen KE. Cyclin 370
D1 binding to the androgen receptor (AR) NH2-terminal domain inhibits 371
activation function 2 association and reveals dual roles for AR corepression.
372
Mol Endocrinol 19: 607-620, 2005.
373
3. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, 374
and Sawyers CL. Molecular determinants of resistance to antiandrogen therapy.
375
Nat Med 10: 33-39, 2004.
376
4. Chen S, Xu Y, Yuan X, Bubley GJ, and Balk SP. Androgen receptor 377
phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 378
1. Proc Natl Acad Sci U S A 103: 15969-15974, 2006.
379
5. Culig Z, and Bartsch G. Androgen axis in prostate cancer. J Cell Biochem 99:
380
373-381, 2006.
381
6. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J, Hittmair A, 382
Bartsch G, and Klocker H. Androgen receptor activation in prostatic tumor cell 383
lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal 384
growth factor. Cancer Res 54: 5474-5478, 1994.
385
7. Culig Z, Hoffmann J, Erdel M, Eder IE, Hobisch A, Hittmair A, Bartsch G, 386
Utermann G, Schneider MR, Parczyk K, and Klocker H. Switch from 387
antagonist to agonist of the androgen receptor bicalutamide is associated with 388
prostate tumour progression in a new model system. Br J Cancer 81: 242-251, 389
1999.
390
8. Gelmann EP. Molecular biology of the androgen receptor. J Clin Oncol 20:
391
3001-3015, 2002.
392
9. Gioeli D, Ficarro SB, Kwiek JJ, Aaronson D, Hancock M, Catling AD, 393
White FM, Christian RE, Settlage RE, Shabanowitz J, Hunt DF, and Weber 394
MJ. Androgen receptor phosphorylation. Regulation and identification of the 395
phosphorylation sites. J Biol Chem 277: 29304-29314, 2002.
396
10. Gregory CW, He B, Johnson RT, Ford OH, Mohler JL, French FS, and 397
Wilson EM. A mechanism for androgen receptor-mediated prostate cancer 398
recurrence after androgen deprivation therapy. Cancer Res 61: 4315-4319, 2001.
399
11. Grossmann ME, Huang H, and Tindall DJ. Androgen receptor signaling in 400
androgen-refractory prostate cancer. J Natl Cancer Inst 93: 1687-1697, 2001.
401
12. Heinlein CA, and Chang C. Androgen receptor in prostate cancer. Endocr Rev 402
25: 276-308, 2004.
403
13. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, 404
Mirand EA, and Murphy GP. LNCaP model of human prostatic carcinoma.
405
Cancer Res 43: 1809-1818, 1983.
406
14. Isaacs JT, and Isaacs WB. Androgen receptor outwits prostate cancer drugs.
407
Nat Med 10: 26-27, 2004.
408
15. Lin H. The versatile roles of cyclin-dependent kinase 5 in human diseases.
409
Adaptive Medicine 1 22-25, 2009.
410
16. Lin H, Chen MC, Chiu CY, Song YM, and Lin SY. Cdk5 regulates STAT3 411
activation and cell proliferation in medullary thyroid carcinoma cells. J Biol 412
Chem 282: 2776-2784, 2007.
413
17. Lin H, Chen MC, and Ku CT. Cyclin-dependent kinase 5 regulates 414
steroidogenic acute regulatory protein and androgen production in mouse 415
Leydig cells. Endocrinology 150: 396-403, 2009.
416
18. Lin H, Juang JL, and Wang PS. Involvement of Cdk5/p25 in 417
digoxin-triggered prostate cancer cell apoptosis. J Biol Chem 279: 29302-29307, 418
2004.
419
19. Lin H, Lin TY, and Juang JL. Abl deregulates Cdk5 kinase activity and 420
subcellular localization in Drosophila neurodegeneration. Cell Death Differ 14:
421
607-615, 2007.
422
20. Lipford JR, and Deshaies RJ. Diverse roles for ubiquitin-dependent 423
proteolysis in transcriptional activation. Nat Cell Biol 5: 845-850, 2003.
424
21. Liu S, Yuan Y, Okumura Y, Shinkai N, and Yamauchi H. Camptothecin 425
disrupts androgen receptor signaling and suppresses prostate cancer cell growth.
426
Biochem Biophys Res Commun 394: 297-302, 2010.
427
22. Marcelli M, Ittmann M, Mariani S, Sutherland R, Nigam R, Murthy L, 428
Zhao Y, DiConcini D, Puxeddu E, Esen A, Eastham J, Weigel NL, and 429
Lamb DJ. Androgen receptor mutations in prostate cancer. Cancer Res 60:
430
944-949, 2000.
431
23. Mellinghoff IK, Vivanco I, Kwon A, Tran C, Wongvipat J, and Sawyers CL.
432
HER2/neu kinase-dependent modulation of androgen receptor function through 433
effects on DNA binding and stability. Cancer Cell 6: 517-527, 2004.
434
24. Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, and 435
O'Malley BW. The Angelman syndrome-associated protein, E6-AP, is a 436
coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19:
437
1182-1189, 1999.
438
25. Rhim JS, and Kung HF. Human prostate carcinogenesis. Crit Rev Oncog 8:
439
305-328, 1997.
440
26. Scher HI, Sarkis A, Reuter V, Cohen D, Netto G, Petrylak D, Lianes P, Fuks 441
Z, Mendelsohn J, and Cordon-Cardo C. Changing pattern of expression of 442
the epidermal growth factor receptor and transforming growth factor alpha in 443
the progression of prostatic neoplasms. Clin Cancer Res 1: 545-550, 1995.
444
27. Sheflin L, Keegan B, Zhang W, and Spaulding SW. Inhibiting proteasomes in 445
human HepG2 and LNCaP cells increases endogenous androgen receptor levels.
446
Biochem Biophys Res Commun 276: 144-150, 2000.
447
28. Shi XB, Ma AH, Xia L, Kung HJ, and de Vere White RW. Functional 448
analysis of 44 mutant androgen receptors from human prostate cancer. Cancer 449
Res 62: 1496-1502, 2002.
450
29. Shigemura K, Isotani S, Wang R, Fujisawa M, Gotoh A, Marshall FF, Zhau 451
HE, and Chung LW. Soluble factors derived from stroma activated androgen 452
receptor phosphorylation in human prostate LNCaP cells: Roles of ERK/MAP 453
kinase. Prostate 2009.
454
30. Tan M, Jing T, Lan KH, Neal CL, Li P, Lee S, Fang D, Nagata Y, Liu J, 455
Arlinghaus R, Hung MC, and Yu D. Phosphorylation on tyrosine-15 of 456
p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance 457
to taxol-induced apoptosis. Mol Cell 9: 993-1004, 2002.
458
31. Veldscholte J, Ris-Stalpers C, Kuiper GG, Jenster G, Berrevoets C, 459
Claassen E, van Rooij HC, Trapman J, Brinkmann AO, and Mulder E. A 460
mutation in the ligand binding domain of the androgen receptor of human 461
LNCaP cells affects steroid binding characteristics and response to 462
anti-androgens. Biochem Biophys Res Commun 173: 534-540, 1990.
463 464 465 466
Figure Legends 467
Fig. 1. Comparisons of morphology and cell growth between parental LNCaP 468
and LNCaPdcc cells. A: The morphology of two cell lines was photographed in 16X 469
and 160X magnification. B: LNCaP cells were seeded into 24-well plates at a density 470
of 4 × 104 cells/well in phenol red-positive RPMI-1640 culture medium (10% serum).
471
After 24 hours, the cell counting were carried out every day and lasted for six days by 472
trypan blue staining assay (n = 4). The LNCaPdcc cells were seeded into 24-well 473
plates at a density of 5 × 104 cells/well in phenol red-negative RPMI-1640 culture 474
medium (10% charcoal-stripped serum). After 48 hours, the cell counting were carried 475
out every two days and lasted for 12 days (n = 4). The values of error bars indicated 476
the mean ± standard error of the mean (SEM).
477
478
Fig. 2. Analyses of cell cycle distribution and cell cycle-relate protein expressions 479
in both cell lines. A: Cells were stained by propidium iodide for 30 min and followed 480
by the analysis of flow cytometry as described in “Materials and Methods” (n=3). The 481
figure indicated the average distribution of cell cycle. The values of error bars are 482
given as the mean ± SEM. B: Immunoblotting was performed and specific antibodies 483
were utilized to investigate the expression levels of proteins indicated. β-actin served 484
as an internal control.
485
486
Fig. 3. Comparisons of AR-related proteins, AR subcellular distribution, and AR 487
stability between two cell lines. A: Immunoblotting was performed and specific 488
antibodies were utilized to investigate the levels of protein expression and 489
phosphorylation. B: Protein fractionation was performed on LNCaP and LNCaPdcc 490
cell lysates. AR proteins were immunoblotted in both nuclear (N) and cytosolic (C) 491
fractions. PARP and α-tubulin served as markers for the cytosolic and nuclear 492
fractions, respectively. C: Cycloheximide (CHX) (10 ng/mL) was treated on LNCaP 493
and LNCaPdcc cells for 0, 2, 4, and 8 hours in respective culture conditions. The 494
endogenous AR protein degradation was monitored by immunoblotting.
495
496
Fig. 4. Difference of androgen sensitivity on proliferation of two cell lines. The 497
cells were seeded separately into 96-well plates at densities of 1.5 × 104 cells/well 498
(LNCaP) and 2 × 104 cells/ well (LNCaPdcc) in steroid-deprived medium. After 48 499
hours, the R1881 (synthetic androgen) was added to the medium at the concentration 500
of 0, 0.1, 1, and 10 nM for four days. Cell proliferation was analyzed by using MTT 501
assay (n = 8). Control value of cell proliferation was set at 100%. The values of error 502
bars are given as the mean ± SEM. **, P < 0.01 versus control group of LNCaP cells;
503
##, P < 0.01 and #, P < 0.05 versus control group of LNCaPdcc cells.
504
505
Fig. 5. Comparisons of Her2-related issues between two cell lines. A:
506
Immunoblotting was performed and specific antibodies were utilized to investigate 507
the levels of protein expression and phosphorylation in LNCaP and LNCaPdcc cells.
508
B: HRG was treated on both cell lines at the concentration of 10 ng/mL in a 509
time-course manner (0, 1, 12, and 24 hours) under serum-free condition.
510
Immunoblotting was performed and specific antibodies were utilized to investigate 511
the levels of phosphorylation and protein expression. C: The cells were seeded 512
separately into 96-well plates as described in Fig 4. After cells attached, AG825 (25 513
μM) and Herceptin (20 ng/mL) added in respective complete medium were treated to 514
cells. Cell proliferation was analyzed by using MTT assay (n=8). Control value of cell 515
proliferation was set at 100%. The values of error bars are given as the mean ± SEM.
516
**, P < 0.01 versus control group of LNCaPdcc cells.
517
518
Fig. 6. Comparisons of Her2 activity-dependent AR protein stability between two 519
cell lines. A: AG825 (25 μM) was treated to LNCaP and LNCaPdcc cells for 24 hours.
520
AR protein degradation was monitored by immunoblotting after different time 521
intervals of CHX treatment (10 ng/mL, 0, 3, 6, and 9 hours). B: AG825 (25 μM, 24 522
hours) was treated on both cell lines. The levels of phospho-Ser81 AR and AR protein 523
were detected by immunoblotting while β-actin served as an internal control. The 524
numbers below the gel images represent the relative levels of protein expressions after 525
quantification.
526 527
16X
160X
LNCaP LNCaPdcc
A
Cell Number x 10
4LNCaPdcc LNCaP Doubling Time LNCaP 30.84 hr LNCaPdcc 77.32 hr
B
0 10 20 30 40 50
0 2 4 6 8 10 12
L N C a P
L N C a P d c c
Cyclin D1 Cyclin B1 Cyclin A Cdk1 B
0 20 40 60 80
G1 S G2/M Phase
Distribution of Cell Cycle (%)
LNCaP LNCaPdcc
* *
A
E-actin AR PSA A
L N C a P LN
CaP dcc
L N C a P LN
CaP dcc
D-tubulin AR
PARP B
p-S81A-AR
nuclear cytosolic
AR E-actin
CHX 8 4 2 0 0 2 4 8 (h)
LNCaP LNCaPdcc
L N C a P LN
CaP dcc
C
0 75 100 125
0 0.1 1 10
Cell Proliferation (%)
R1881
Ϯ LNCaP ϭ LNCaPdcc
(nM)
* * *
*
#
# #
A
B
C
AR
E-actin
CHX 9 6 3 0
LNCaP LNCaPdcc
0 3 6 9 (h) + AG825
0.68 0.97 0.91 1.0 1.0 0.48 0.36 0.21
AR E-actin p-S81-AR
AG825 - + - +
LNCaP LNCaPdcc
1.0 0.8 1.0 0.6
