Author Manuscript Published OnlineFirst on June 12, 2015; DOI: 10.1158/1078-0432.CCR-14-3163 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
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Autocrine activation of CHRM3 promotes prostate cancer growth and
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castration resistance via CaM/CaMKK-mediated phosphorylation of Akt
3
Naitao Wang, Ming Yao, Jin Xu, Yizhou Quan, Kaiqing Zhang, Ru Yang* and
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Wei-Qiang Gao*
5 6
Authors’ Affiliations:
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State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem
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Cell Research Center, Ren Ji Hospital, School of Biomedical Engineering, Shanghai
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Jiao Tong University，Shanghai, China.
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Running Title: Autocrine cholinergic signaling in prostate cancer
12 13
Financial support: The study is supported by funds to Wei-Qiang Gao from the
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Chinese Ministry of Science and Technology (2012CB966800, 2013CB945600 and
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2012CB967903), the National Natural Science Foundation of China (81130038 and
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81372189), the Science and Technology Commission of Shanghai Municipality
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(Pujiang program), the Shanghai Education Committee Key Discipline and Specialty
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Foundation (J50208), the Shanghai Health Bureau Key Discipline and Specialty
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Foundation and the KC Wong foundation.
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*Corresponding Authors: Wei-Qiang Gao or Ru Yang, Ren Ji Hospital, School
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of Medicine, Shanghai Jiao Tong University, Shanghai, China.
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E-mail: [email protected] or [email protected]
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The authors declare no potential conflicts of interest
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Statement of translational relevance
43
This study aimed to elucidate the comprehensive roles of autocrine cholinergic
44
signaling in prostate cancer growth and castration resistance. Although androgen
45
deprivation is initially effective for the treatment of prostate cancer, patients
46
inevitably develop resistance to androgen deprivation therapy, called castration
47
resistance, which is the major cause of morbidity and mortality. To date, there is
48
no effective therapy available for the treatment of castration resistant prostate
49
cancer (CRPC). The present work demonstrates an autocrine activation of the
50
cholinergic system in prostate cancer. Importantly, a selective cholinergic
51
muscarinic receptor 3 (CHRM3) antagonist, darifenacin, effectively inhibits
52
prostate cancer growth and castration resistance both in vitro and in vivo,
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suggesting a potential therapeutic application of selective CHRM3 antagonists in
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the treatment of prostate cancer, including CRPC.
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Abstract
68
Purpose: Although a previous study reported nerve ending-derived acetylcholine
69
promoted
70
microenvironment of cancer cells, the present study aims to determine whether
71
there is autocrine cholinergic signaling in prostate epithelial cells that promotes
72
prostate cancer growth and castration resistance.
73
Experimental design: In this study, immunohistochemistry (IHC) was performed
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to detect protein expression in mouse prostate tissue sections and human
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prostate cancer tissue sections. Subcutaneously and orthotopically xenografted
76
tumor models were established to evaluate the functions of autocrine cholinergic
77
signaling in regulating prostate cancer growth and castration resistance. Western
78
blotting analysis was performed to assess the autocrine cholinergic signaling-
79
induced signaling pathway.
80
Results: We found the expression of choline acetyltransferase (ChAT), the
81
secretion of acetylcholine and the expression of CHRM3 in prostate epithelial
82
cells, supporting the presence of autocrine cholinergic signaling in the prostate
83
epithelium. In addition, we found that CHRM3 was up-regulated in clinical
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prostate cancer tissues compared to adjacent non-cancer tissues. Over-
85
expression of CHRM3 or activation of CHRM3 by carbachol promoted cell
86
proliferation, migration and castration resistance. On the contrary, blockading
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CHRM3 by shRNA or treatment with darifenacin inhibited prostate cancer growth
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and castration resistance both in vitro and in vivo. Furthermore, we found that
89
autocrine cholinergic signaling caused calmodulin/calmodulin-dependent protein
90
kinase kinase (CaM/CaMKK)-mediated phosphorylation of Akt.
91
Conclusions: These findings suggest that blockade of CHRM3 may represent a
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novel adjuvant therapy for CRPC.
prostate
cancer
invasion
and
metastasis
by
regulating
the
93 94
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Introduction
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Prostate cancer is the most common malignant disease in men in developed
97
countries (1). In 2014, 233,000 new cases of prostate cancer are expected to
98
occur in the United States, which accounts for 27% of incident cases in men (1).
99
Although androgen deprivation is initially effective, the patients inevitably
100
encounter the problem of resistance to androgen deprivation therapy, called
101
castration resistance (2). Discovery of novel strategies to suppress castration
102
resistance is especially helpful for the management of advanced prostate cancer.
103 104
Acetylcholine,
normally
released
from
nerve
endings,
is
a
classical
105
neurotransmitter in the central and peripheral nervous system. A recent study
106
reported the involvement of nerve ending-derived acetylcholine activated
107
cholinergic muscarinic receptor 1 (CHRM1) in mesenchymal cells in the tumor
108
microenvironment to promote prostate cancer invasion and metastasis (3).
109
However, besides nerve ending-derived acetylcholine, there is also wide-spread
110
synthesis of acetylcholine by a variety of non-neuronal cell types, including lung,
111
colon, airway and ovarian epithelial cells (4). Whether there is synthesis of
112
acetylcholine in prostate epithelial cells which might play an autocrine activation
113
role in promoting prostate cancer progression has not yet been studied.
114 115
Muscarinic receptors are G-protein coupled receptors consisting of five members,
116
CHRM1-CHRM5 (5). Activation of muscarinic receptors triggers Ca2+ influx,
117
which causes smooth muscle contraction and glandular secretion. In the 1990s,
118
muscarinic receptor subtypes were firstly defined as conditional oncogenes when
119
they were activated by acetylcholine in NIH-3T3 cells (6). Subsequently,
120
muscarinic receptors have been implicated to be involved in a few types of
121
epithelial cancers including colorectal cancer and small cell lung cancer (7,8). In
122
addition, activation of muscarinic receptors was reported to promote prostate
123
cancer cell proliferation in vitro (9). However, the mechanism and comprehensive
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functions of muscarinic receptors in prostate cancer progression, particularly in
125
CRPC, has not been clearly elucidated.
126 127
In the present study, we found the presence of a functional cholinergic system in
128
prostate epithelial cells. An autocrine activation of CHRM3 that regulated prostate
129
cancer cell growth and castration resistance was observed. Blockade of CHRM3
130
via a specific antagonist or shRNAs could effectively inhibit prostate cancer
131
growth and castration resistance. The cholinergic signaling occurred via the
132
CaM/CaMKK-mediated phosphorylation of Akt. These findings suggest a
133
potential application of muscarinic receptor antagonists in the treatment of
134
prostate cancer.
135 136
Materials and methods
137
Cell culture
138
Human prostate cell lines used in this study: LNCaP, PNT1B (10), BPH1 (a kind
139
gift from Dr. Simon W. Hayward) (11), C4-2B, PC3, PC3-AR+ and PC3-luc (kind
140
gifts from Dr. Jianhua Wang at Shanghai Jiao Tong University School of Medicine)
141
(12,13). Cells were cultured in DMEM or RPMI-1640 supplemented with 10%
142
FBS and P/S (penicillin and streptomycin) at 37°C, 5% CO2. In the cell
143
proliferation assay, cell numbers were determined by hemocytometer.
144 145
Cell transfection
146
Two shRNA sequences for CHRM3 (5’-3’, AGCAGAGACAGTCGGTCATTT and
147
TCGGCAACATCCTGGTAATTG) and scrambled shRNA were cloned into the
148
lentiviral vector pUCTP. The cDNA of CHRM3 was subcloned into the lentiviral
149
expression vector EGH. Transfection efficiencies were determined at both the
150
protein and mRNA levels.
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Cell migration assays
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In the scratch tests, confluent monolayer cells were scraped with a pipette tip.
154
Cells were cultured in standard cell culture medium containing 10% FBS.
155
Photographs were taken immediately and at 24 hours after wounding. To rule out
156
the potential effects of cell proliferation on cell migration, we normalized the cell
157
migration distance with the total number of cells at 24 hours.
158
In the transwell assays, cells were first starved in serum-free medium for 24
159
hours. Ten thousand-50,000 cells in 100 μL serum free medium were seeded on
160
the top of a transwell chamber (8 μm pore size, 3422, Corning), 500 μL medium
161
containing 10% FBS was added in the bottom of 24-well plates to induce cell
162
migration. Twelve hours later, the culture inserts were fixed with 4%
163
paraformaldehyde and stained in 0.1% crystal violet for 10 minutes. Cells that
164
stayed on the top of the membrane were gently scraped by a cotton swab. To
165
eliminate the effects of cell proliferation on cell migration, we starved PNT1B cells
166
(Lenti-vector and Lenti-CHRM3 transfected PNT1B cells) and PC3 cells
167
(scramble, shRNA1 and shRNA2 transfected PC3 cells) in serum free medium
168
for 24 hours and replaced the medium with standard cell culture medium for 12
169
hours. We measured the cell numbers before and at 12 hours after the
170
replacement of the standard cell culture medium and calculated the cell number
171
changes in both PNT1B cells and PC3 cells (data not shown). Then, we
172
normalized the results in transwell assays with the total number of cells at 12
173
hours.
174 175
In vivo experiments
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For the subcutaneously xenografted tumor models, 1,000,000 cells (50 μL cell
177
suspension + 50 μL matrigel) were injected subcutaneously into the flank regions
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of 6-week-old male BALB/c nude mice. Tumor volume was measured once a
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week (V=0.5ab2; a, the longest side; b, the shortest side). For orthotopical
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implantation assays, 8-week-old male BALB/c nude mice were first castrated.
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Two weeks later, 1,000,000 cells (25 μL cell suspension + 25 μL matrigel) were
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injected into the anterior lobes of the prostate with a 31G insulin syringe.
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Bioluminescent signal was induced by intraperitoneal injection of D-luciferin
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(GoldBio, 1.5 mg D-luciferin per 10 g body weight in saline) and analyzed 10
185
minutes later using a Berthold imaging system. In some assays, darifenacin was
186
injected intraperitoneally at doses of 1 mg/kg/day and 5 mg/kg/day. Vehicle (PBS
187
mixed with DMSO at 1:1) was injected in the control group. Mice were fed in the
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SPF grade animal facility of Ren Ji hospital with controlled temperature and
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humidity. All animal studies were carried out following the guidelines of the Ren Ji
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hospital institutional animal care and ethics committee.
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Clinical prostate cancer samples
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Paraffin embedded human prostate tissue array slides containing fifty-eight spots
194
(29 paired prostate cancer and adjacent non-cancerous tissues) (OD-CT-
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UrPrt03-001) were purchased from Shanghai Outdo Biotech Ltd.. A primary
196
antibody against CHRM3 (Abbiotec) and HRP conjugated secondary antibodies
197
(Jackson ImmunoResearch) were applied. The immunostaining was visualized
198
with DAB (3, 3’-diaminobenzidine). Images were captured using a Leica DM2500
199
microscope under the same exposure conditions and analyzed with the Image-
200
pro Plus 6.0 software (Media Cybernetics).
201 202
Calcium influx detection
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Cells were incubated with Fluo-4 AM (Invitrogen, 2 μM in Hank’s Balanced Salt
204
Solution, HBSS) for 60 minutes at 37°C and incubated for another 15 minutes in
205
fresh HBSS to allow de-esterification of intracellular AM esters. Acetylcholine and
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carbachol were added to the culture medium to trigger a Ca2+ influx. For
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antagonist studies, darifenacin was added 5 minutes before the stimulation of 10
208
μM carbachol. Images were taken with a Leica inverted microscope for 300
209
seconds at intervals of 6 seconds. Data were analyzed with the Image-pro Plus
210
6.0 software.
211 212
Western blot and immunoprecipitation
213
In western blotting assays, cells were lysed in a RIPA buffer (Thermo) with
214
proteinase inhibitors and phosphatase inhibitors (Roche). Total proteins were
215
measured by a BCA method (Thermo). Primary antibodies against GAPDH, E-
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cadherin, N-cadherin, Vimentin (Epitomic), CaMKKα (Santa Cruz), CHRM3
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(Abbiotec), Flag (Sigma), Slug, Akt, pAkt-Ser473, pAkt-Thr308 (CST) and HRP
218
conjugated secondary antibodies were applied. Immunoblots were visualized
219
with an ECL blotting detection kit (Thermo). In the immunoprecipitation assay,
220
cell lysates were incubated with the rabbit Akt antibody or control mouse IgG
221
antibody at 4°C overnight. Protein A-agarose beads (Roche) were added, and
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lysates were further incubated for 1 h at room temperature. Beads were
223
precipitated by centrifugation at 5,000 rpm for 3 minutes and boiled in SDS-page
224
loading buffer for 5 minutes. The samples were then detected according to
225
standard western blotting procedures.
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Immunofluorescent staining
228
Immunofluorescent staining was performed as previously described (14).
229
Antibodies used in this study included Tuj-1 (Sigma), ChAT (Millipore), CHRM3
230
(Abbiotec), Ki67, E-cadherin, N-cadherin, Vimentin (Epitomic) and Cleaved-
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caspase3 (CST). The TUNEL apoptosis detection system (Promega) was used to
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detect apoptotic cells in mouse xenografted tumor sections. Images were taken
233
using a Leica DM2500 microscope.
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Real-time PCR
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Total RNA was obtained and reverse-transcripted to cDNA by the RNeasy plus
237
Mini Kit and the QuantiTect RT Kit (Qiagen). SYBR (Roche) real-time PCR was
238
performed on the 7900HT machine (ABI). The sequences of the primers used in
239
this study are listed in Supplementary Table S1.
240 241
Statistical analysis
242
Data in this study are expressed as the means ± SEM. Immunostaining densities
243
of CHRM3 in matched human prostate cancerous and adjacent non-cancerous
244
tissues were compared by paired Student’s t-test. Cell proliferation, real-time
245
PCR and in vivo tumor xenograft growths were analyzed using a non-parametric
246
Student’s t-test. Ki67, TUNEL and cleaved-caspase3 positive cells were counted
247
in at least three randomly-selected visual fields and analyzed using a
248
nonparametric Student’s t-test. The Kaplan–Meier log-rank test was used for
249
analysis of mouse survival data. Data were analyzed with GraphPad Prism 5
250
software (GraphPad Software). Statistical significance was defined as * P < 0.05,
251
** P < 0.01, *** P < 0.001.
252 253 254 255 256 257 258 259
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Results
261
Presence of functional autocrine cholinergic signaling in the prostate
262
epithelium
263
Autonomic nerves were reported to regulate prostate cancer metastasis by
264
regulating the microenvironment of epithelial cancer cells (3). Immunostaining of
265
mouse prostate sections confirmed dense innervation of Tuj-1 immunoreactive
266
nerve fibers; however, few, if any, fibers could be observed inside the epithelium
267
(Fig.
268
immunoreactivity of ChAT, which is the enzyme necessary for acetylcholine
269
synthesis (Fig. 1B). Importantly, ChAT immunoreactivity in human prostate
270
sections also showed the expression of ChAT in the epithelium (Fig. 1C). In
271
addition, we detected the secretion of acetylcholine from both cancerous and
272
non-cancerous human prostate epithelial cells, which are free of any neuronal
273
innervations (Fig. 1D). These data demonstrate clearly that there is endogenous
274
production and secretion of acetylcholine from prostate cancerous and non-
275
cancerous epithelial cells.
1A).
Instead,
mouse
prostate
epithelial
cells
showed
strong
276 277
A complete, functional cholinergic loop requires the presence of not only
278
acetylcholine but also muscarinic receptors. To determine the expression
279
patterns of muscarinic receptors in the prostate, we profiled gene expression in
280
the Oncomine database and GEO Profiles database. Of five muscarinic
281
cholinergic receptors, only CHRM3 was significantly elevated in prostate cancer
282
samples compared to non-cancer samples (Supplementary Fig. S1). To confirm
283
the Oncomine and GEO data analysis, we performed immunohistochemistry of
284
CHRM3 in the human prostate cancer tissue array and found that CHRM3 was
285
mainly expressed in the prostate epithelium rather than in the mesenchyme (Fig.
286
1E). Notably, CHRM3 was significantly up-regulated in cancer tissues compared
287
to their matched, adjacent non-cancer tissues (Fig. 1F).
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To determine whether the autocrine acetylcholine could activate muscarinic
290
receptors in epithelial cells, we performed experiments to measure Ca2+ influx,
291
which is a direct indicator of muscarinic receptor activation. We measured Ca2+
292
influx with Fluo-4 AM, a commonly used fluorescent Ca2+ influx indicator, and
293
found that both acetylcholine and its stable analogue carbachol could induce
294
Ca2+ influx. CHRM3-specific antagonist darifenacin could largely reduce Ca2+
295
influx induced by carbachol (Fig. 1G). In addition, conditioned medium from PC3
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cells also triggered Ca2+ influx, suggesting the occurrence of secretion of
297
endogenous non-nerve ending-derived acetylcholine (Fig. 1H). Importantly, the
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Ca2+ influx induced by endogenous acetylcholine could be effectively blocked by
299
the CHRM3 specific inhibitor (Fig. 1H). Taken together, these findings show the
300
presence of functional autocrine cholinergic signaling in prostate epithelium.
301 302
Activation of CHRM3 by endogenous acetylcholine promotes prostate
303
cancer growth
304
To evaluate the role of autocrine cholinergic signaling in prostate cancer growth,
305
we over-expressed CHRM3 in non-tumorigenic PNT1B cells. Over-expression of
306
CHRM3 in PNT1B cells promoted cell growth as time proceeded (Fig. 2A),
307
implicating that over-expressed CHRM3 could be activated by endogenous
308
acetylcholine. In contrast, the knock-down of CHRM3 by shRNA in PC3 cells
309
inhibited cell growth (Fig. 2C). The lentiviral transfection efficiencies were
310
analyzed by western blot (Fig. 2B and 2D). In addition, we treated PC3 cells with
311
carbachol (a stable agonist of muscarinic receptors) and darifenacin (a selective
312
antagonist of CHRM3). While carbachol promoted the proliferation of PC3 and
313
22Rv1 cells, blockade of CHRM3 by darifenacin could effectively reduce cell
314
proliferation (Fig. 2E, F).
315 316
To extend our in vitro studies to an in vivo setting, we implanted CHRM3 knock-
317
down PC3 cells subcutaneously in BALB/c nude mice. We found that knock-
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down of CHRM3 reduced tumor growth in vivo (Fig. 2G). Consistently, CHRM3-
319
specific antagonist darifenacin also inhibited the growth of xenografted PC3 and
320
22Rv1 cells (Fig. 2H, I). Histological examination of PC3 xenografted tumors
321
treated with darifenacin showed a reduced percentage of Ki67 positive,
322
proliferating cells and increased percentage of apoptotic cells (Fig. 2J-L). These
323
findings indicate that autocrine activation of CHRM3 promotes prostate cancer
324
growth.
325 326
Autocrine activation of CHRM3 promotes cell migration through regulating
327
epithelial-mesenchymal transition
328
To evaluate the role of CHRM3 in regulating cell migration, we performed
329
transwell assays and scratch tests. In transwell assays, over-expression of
330
CHRM3 in PNT1B cells increased the number of cells that migrated through the
331
membrane (Fig. 3A, B). In scratch tests, over-expression of CHRM3 promoted
332
the confluence of scratched cells (Supplementary Fig. S2A, B). To rule out the
333
potential effects of cell proliferation on cell migration, we normalized the results in
334
transwell assay and scratch test with the total number of cells at the same
335
detection time point as described in the materials and methods section. On the
336
contrary, knock-down of CHRM3 in PC3 cells inhibited their migration capability
337
both in the transwell assay and in the scratch test (Fig. 3C, D and Supplementary
338
Fig. S2C, D). Similarly, the cell migration results were also normalized to the total
339
number of cells at the same detection time point.
340 341
Epithelial-mesenchymal transition (EMT) is an important process in epithelial
342
cancer progression, which facilitates cell migration and invasion (15). To verify
343
whether the activation of CHRM3 could induce EMT, we over-expressed CHRM3
344
in PNT1B cells. We observed that CHRM3 up-regulation caused the PNT1B cells
345
to become less attached to each other and exhibit a mesenchymal phenotype
346
(Fig. 3E). Immunostaining of these cells with E-cadherin, N-cadherin and
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Vimentin antibodies confirmed that E-cadherin expression was decreased in
348
CHRM3 over-expressing PNT1B cells; N-cadherin and Vimentin expression was
349
sharply increased (Fig. 3E). Western blotting analysis confirmed the decreased
350
E-cadherin protein level and increased Vimentin protein level in CHRM3 over-
351
expressing PNT1B cells (Fig. 3F). Consistently, real-time PCR analysis also
352
showed an increase in mesenchyme-related gene expression, while E-cadherin
353
was down-regulated in these CHRM3 over-expressing PNT1B cells (Fig. 3G). On
354
the contrary, CHRM3 knock-down PC3 cells reversed its mesenchymal status
355
and expressed more E-cadherin and less N-cadherin (Fig. 3H). The
356
immunoblotting analysis also confirmed that CHRM3 knock-down reversed EMT
357
in PC3 cells (Fig. 3I). In addition, real-time PCR analysis of several EMT-related
358
genes also showed that CHRM3-silenced PC3 cells underwent an opposite
359
process of EMT, that is, mesenchymal-epithelial transition (Fig. 3J). These
360
findings together indicate that autocrine activation of CHRM3 promotes prostate
361
cell migration by regulating EMT.
362 363
CHRM3 is up-regulated in castration-resistant prostate cancer cells
364
To evaluate the role of CHRM3 in castration resistance, a very important feature
365
of prostate cancer, we first measured CHRM3 expression in paired CRPC and
366
androgen-dependent prostate cancer cells: C4-2B and LNCaP, PC3 and PC3-
367
AR+. C4-2B was a bone metastatic and castration-resistant subline of androgen-
368
dependent LNCaP cells (16). PC3-AR+ cells were generated through stable
369
expression of full-length human androgen receptor (AR) in PC3 cells (17). Re-
370
expression of AR restored the response to androgens in PC3-AR+ cells (17,18).
371
Real-time PCR analysis revealed higher CHRM3 mRNA levels in castration-
372
resistant C4-2B and PC3 cells than their paired androgen-dependent LNCaP and
373
PC3-AR+ cells, respectively (Fig. 4A). Secondly, we treated LNCaP cells with
374
bicalutamide, a clinically-used androgen deprivation agent, to imitate the
375
androgen deprivation condition in vitro. After several generations, the expression
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(Supplementary Fig. S3A). Thirdly, we subcutaneously implanted PC3-AR+ cells
378
in nude mice. When tumors grew for approximately 1 month, we castrated the
379
recipient mice. We analyzed CHRM3 mRNA levels before and post castration.
380
We found that the expression of CHRM3 was significantly increased after
381
castration (Fig. 4B). These findings are in agreement with the GEO database
382
indicating that androgen deprivation resulted in enhanced CHRM3 expression in
383
LuCaP35 cells (Supplementary Fig. S3B) (19). All of these data together suggest
384
a positive correlation of CHRM3 with castration resistance.
385 386
Over-expression of CHRM3 promotes castration resistant growth
387
To evaluate whether activation of CHRM3 could cause castration resistance in
388
vivo, we established tumor xenografts in castrated nude mice. At first, we wanted
389
to establish in vivo tumor models with LNCaP cells. However, LNCaP cells failed
390
to efficiently and consistently form tumors in normal, un-castrated nude mice
391
even with ten million cells. Then, we engrafted PC3-AR+ and PC3 cells
392
subcutaneously in castrated nude mice. We observed that PC3 cells transfected
393
with control lentivirus indeed formed tumors in androgen deprivation conditions
394
(Fig. 4C). On the contrary, PC3-AR+ cells transfected with control lentivirus failed
395
to do so under the same conditions (Fig. 4C). More importantly, over-expression
396
of CHRM3 in these PC3-AR+ cells caused them to re-gain the castration
397
resistant capability and form tumors under conditions of hormone deprivation
398
condition (Fig. 4C). Consistently, immunofluorescent staining of Ki67 and cleaved
399
caspase-3 in tumor sections showed increased cell proliferation and decreased
400
cell apoptosis, respectively, in CHRM3 over-expressed xenografts (Fig. 4D-G).
401
These results suggest that activation of CHRM3 can enhance castration-resistant
402
growth capability of the androgen-dependent prostate cancer cells.
403 404
Blockade of CHRM3 inhibits the castration resistant growth of PC3-luc cells
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405
To study whether the blockade of CHRM3 could influence castration-resistant
406
growth or the sensitivity of prostate cancer cells under androgen deprivation
407
conditions, we established an orthotopic prostate cancer model with luciferase
408
stably expressing PC3 (PC3-luc) cells under an androgen deprivation paradigm,
409
which is more similar to the in situ castration resistant prostate cancer. Seven
410
weeks after implantation, we examined tumor growth by bioluminescence (Fig.
411
5A). Stable silencing of CHRM3 reduced tumor growth when compared with the
412
scrambled shRNA transfected group (Fig. 5B, C). When these primary
413
xenografted tumor sections were processed for immunofluorescent staining with
414
anti-Ki67 antibody to detect the proliferating cells, we observed decreased Ki67
415
positive nuclei in CHRM3 knock-down tumors (Fig. 5D, E). Similarly, when the
416
tumor recipient mice were treated with vehicle or darifenacin at a dosage of 1
417
mg/kg/day, darifenacin significantly inhibited castration resistant growth of PC3-
418
luc cells when compared to the vehicle group (Fig. 5F-H). In addition, darifenacin
419
improved the survival status in tumor-bearing recipient mice (Fig. 5I).
420
Immunostaining of Ki67 also showed decreased cell proliferation in darifenacin
421
treated tumors (Fig. 5J). These findings together confirm that blockade of
422
CHRM3 enhances the sensitivity of androgen-independent PC3-luc cells to
423
androgen deprivation.
424 425
Activation of CHRM3 promotes CaM/CaMKK-dependent phosphorylation of
426
Akt.
427
Next, we wanted to explore the mechanism of autocrine cholinergic signaling in
428
regulating prostate cancer growth and castration resistance. Previous studies
429
reported that Ca2+ influx could promote the phosphorylation of Akt (20-22).
430
Because our data had shown that activation of muscarinic receptors stimulated
431
Ca2+ influx in prostate cancer cells, we next wanted to determine whether the
432
autocrine activation of cholinergic signaling could enhance calcium signaling-
433
mediated phosphorylation of Akt. As shown in Fig. 6A, over-expression of
434
CHRM3 in LNCaP cells increased the phosphorylation of Akt due to the 16 Downloaded from clincancerres.aacrjournals.org on July 23, 2015. © 2015 American Association for Cancer Research.
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production of endogenous acetylcholine from the LNCaP cells (Fig. 6A). On the
436
contrary, silencing of CHRM3 in PC3 cells decreased the phosphorylation of Akt
437
(Fig. 6B). These findings indicate that the autocrine cholinergic signaling could
438
promote Akt phosphorylation.
439
To confirm that Akt phosphorylation induced by the autocrine cholinergic
440
signaling is calcium-signaling dependent, we first treated PC3 cells with the
441
CHRM3-specific antagonist darifenacin and the calmodulin-selective antagonist
442
W-7. Western blotting analysis revealed that both darifenacin and W-7 could
443
effectively inhibit Akt phosphorylation stimulated by carbachol (Fig. 6C). Next, we
444
treated PC3 cells with CaMKK antagonist STO-609 to determine whether the
445
downstream signaling of CaM was involved in Akt phosphorylation. STO-609
446
could also effectively block Akt phosphorylation induced by carbachol (Fig. 6D).
447
Furthermore, co-immunoprecipitation showed a direct binding of Akt to CaMKKα
448
(Fig. 6E). These data suggest that autocrine cholinergic signaling promotes
449
prostate cancer growth and castration resistance through CaM/CaMKK-mediated
450
activation of Akt (Fig. 6F).
451 452
Discussion
453
Although a previous study reported a role for neuronal cholinergic signaling in
454
prostate cancer metastasis (3), the present study demonstrates several different
455
and novel findings. First, we found the autonomous expression of ChAT and
456
synthesis of acetylcholine in prostate epithelial cells, suggesting the presence of
457
autocrine cholinergic signaling in the prostate epithelium. Second, we detected
458
an up-regulation of CHRM3 in human prostate cancer tissues compared to their
459
adjacent non-cancer tissues, implicating that CHRM3 might be an additional
460
diagnostic marker of prostate cancer. Third, different from the parasympathetic
461
cholinergic signaling that regulated the microenvironment to promote prostate
462
cancer metastasis (3), we found that direct over-expression or knock-down of
463
CHRM3 in the prostate cells significantly promoted or inhibited cell migration
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464
through the regulation of EMT. Finally, we found that activation or inhibition of
465
CHRM3 promoted or inhibited prostate cancer growth and castration resistance
466
both in vitro and in vivo. Thus, our data strongly indicate that there is an
467
autocrine activation of CHRM3 in prostate cancer epithelial cells.
468 469
Our study shows, for the first time, the secretion of autocrine acetylcholine from
470
prostate epithelial cells and cancer cells. Immunostaining indicated the
471
expression of ChAT in both mouse and human prostate epithelia. In addition, we
472
detected the production of acetylcholine by human prostate epithelial cells,
473
including both cancer cells and non-cancer cells. The concentration of autocrine
474
acetylcholine in the cell culture medium is approximately 2~4 μM, which is
475
sufficient to stimulate Ca2+ influx that can be induced by 0.1 μM of acetylcholine
476
or carbachol (Supplementary Fig. S4). Considering that in the microenvironment
477
of prostate cancer, the concentration of acetylcholine in prostate cancer tissues
478
may be even higher than the acetylcholine secreted into the medium due to
479
higher cell density, such autocrine acetylcholine signaling likely functions in the
480
prostate in vivo. Further support for our autocrine cholinergic signaling model
481
also comes from the results reported previously in other tissue adenocarcinoma
482
cells, such as colon cancer cells and small cell lung cancer cells (7,8).
483 484
The present study provides important insights into the mechanism for the
485
biological effects of activation of muscarinic receptors. First, either the CHRM3-
486
specific inhibitor darifenacin or the calmodulin-selective antagonist W-7 can
487
effectively inhibit Akt phosphorylation stimulated by carbachol. Second, CaMKK
488
antagonist STO-609, which is a downstream signaling component of CaM, also
489
effectively blocks Akt phosphorylation induced by activation of CHRM3. Third,
490
there is direct binding between Akt and CaMKK based on co-immunoprecipitation
491
assays. Fourth, calcium signaling has been shown to mediate Ca2+ influx-
492
induced Akt phosphorylation (22-24). Finally, a blockade of Akt activity has been
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493
shown to suppress castration-resistant growth in both mouse models and clinical
494
settings (25-28). These findings together suggest that autocrine cholinergic
495
signaling promotes prostate cancer growth and castration resistance through the
496
CaM/CaMKK-mediated activation of Akt (Fig. 6F).
497 498
Castration resistance is a major challenge in prostate cancer treatment. However,
499
an effective approach for targeting castration resistant prostate cancer is
500
currently unavailable. In this study, we found that CHRM3 was upregulated in
501
CRPC cells compared to matched androgen-dependent cells. While over-
502
expression of CHRM3 was sufficient to cause androgen-dependent PC3-AR+
503
cells to form tumors in castrated mice, stable silencing of CHRM3 inhibited the
504
castration resistant growth of PC3 cells in orthotopic xenografts. Notably, our
505
study showed that the CHRM3-specific antagonist darifenacin was effective to
506
inhibit PC3 cell growth in castrated nude mice. Given that specific antagonists of
507
CHRM3 have been widely used in clinical conditions such as OAB (overactive
508
bladder) and COPD (chronic obstructive pulmonary diseases), clinical trials with
509
such CHRM3 antagonists are warranted and may hold promise for the treatment
510
of primary prostate cancer as well as castration-resistant prostate cancer.
511 512 513 514 515 516 517 518
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519
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604
Figure legends
605
Figure 1. Presence of a functional autocrine cholinergic system in the prostate
606
epithelium. A, Immunofluorescent staining of Tuj-1 in 2-week-old mouse prostate
607
sections. B, Immunofluorescent images showing a selective epithelial expression
608
pattern of ChAT in the mouse prostate. C, Immunohistochemistry of ChAT in
609
human prostate sections. D, Detection of the release of acetylcholine from
610
various human prostate cell lines. E, Immunohistochemistry of CHRM3 in human
611
prostate cancer tissue arrays. F, Staining intensities of CHRM3 quantified by IOD
612
(Integral optical density); data are analyzed by paired t-test. G, Ca2+ influx
613
detected by Fluo-4 AM in PC3 cells. Acetylcholine (10 μM) and carbachol (10 μM)
614
are used to trigger Ca2+ influx. To counteract Ca2+ influx induced by carbachol,
615
darifenacin (10 μM) is added 5 minutes before the addition of carbachol. H,
616
Detection of Ca2+ influx induced by endogenous acetylcholine. Conditioned
617
medium is collected from PC3 cells cultured in a serum free medium, in which
618
neostigmine (10 μM) was added to inhibit the degradation of acetylcholine. Data
619
are analyzed with Student’s t-test, * P
1
Autocrine activation of CHRM3 promotes prostate cancer growth and
2
castration resistance via CaM/CaMKK-mediated phosphorylation of Akt
3
Naitao Wang, Ming Yao, Jin Xu, Yizhou Quan, Kaiqing Zhang, Ru Yang* and
4
Wei-Qiang Gao*
5 6
Authors’ Affiliations:
7
State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem
8
Cell Research Center, Ren Ji Hospital, School of Biomedical Engineering, Shanghai
9
Jiao Tong University，Shanghai, China.
10 11
Running Title: Autocrine cholinergic signaling in prostate cancer
12 13
Financial support: The study is supported by funds to Wei-Qiang Gao from the
14
Chinese Ministry of Science and Technology (2012CB966800, 2013CB945600 and
15
2012CB967903), the National Natural Science Foundation of China (81130038 and
16
81372189), the Science and Technology Commission of Shanghai Municipality
17
(Pujiang program), the Shanghai Education Committee Key Discipline and Specialty
18
Foundation (J50208), the Shanghai Health Bureau Key Discipline and Specialty
19
Foundation and the KC Wong foundation.
20
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21
*Corresponding Authors: Wei-Qiang Gao or Ru Yang, Ren Ji Hospital, School
22
of Medicine, Shanghai Jiao Tong University, Shanghai, China.
23
E-mail: [email protected] or [email protected]
24 25
The authors declare no potential conflicts of interest
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
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42
Statement of translational relevance
43
This study aimed to elucidate the comprehensive roles of autocrine cholinergic
44
signaling in prostate cancer growth and castration resistance. Although androgen
45
deprivation is initially effective for the treatment of prostate cancer, patients
46
inevitably develop resistance to androgen deprivation therapy, called castration
47
resistance, which is the major cause of morbidity and mortality. To date, there is
48
no effective therapy available for the treatment of castration resistant prostate
49
cancer (CRPC). The present work demonstrates an autocrine activation of the
50
cholinergic system in prostate cancer. Importantly, a selective cholinergic
51
muscarinic receptor 3 (CHRM3) antagonist, darifenacin, effectively inhibits
52
prostate cancer growth and castration resistance both in vitro and in vivo,
53
suggesting a potential therapeutic application of selective CHRM3 antagonists in
54
the treatment of prostate cancer, including CRPC.
55 56 57 58 59 60 61 62 63 64 65 66
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67
Abstract
68
Purpose: Although a previous study reported nerve ending-derived acetylcholine
69
promoted
70
microenvironment of cancer cells, the present study aims to determine whether
71
there is autocrine cholinergic signaling in prostate epithelial cells that promotes
72
prostate cancer growth and castration resistance.
73
Experimental design: In this study, immunohistochemistry (IHC) was performed
74
to detect protein expression in mouse prostate tissue sections and human
75
prostate cancer tissue sections. Subcutaneously and orthotopically xenografted
76
tumor models were established to evaluate the functions of autocrine cholinergic
77
signaling in regulating prostate cancer growth and castration resistance. Western
78
blotting analysis was performed to assess the autocrine cholinergic signaling-
79
induced signaling pathway.
80
Results: We found the expression of choline acetyltransferase (ChAT), the
81
secretion of acetylcholine and the expression of CHRM3 in prostate epithelial
82
cells, supporting the presence of autocrine cholinergic signaling in the prostate
83
epithelium. In addition, we found that CHRM3 was up-regulated in clinical
84
prostate cancer tissues compared to adjacent non-cancer tissues. Over-
85
expression of CHRM3 or activation of CHRM3 by carbachol promoted cell
86
proliferation, migration and castration resistance. On the contrary, blockading
87
CHRM3 by shRNA or treatment with darifenacin inhibited prostate cancer growth
88
and castration resistance both in vitro and in vivo. Furthermore, we found that
89
autocrine cholinergic signaling caused calmodulin/calmodulin-dependent protein
90
kinase kinase (CaM/CaMKK)-mediated phosphorylation of Akt.
91
Conclusions: These findings suggest that blockade of CHRM3 may represent a
92
novel adjuvant therapy for CRPC.
prostate
cancer
invasion
and
metastasis
by
regulating
the
93 94
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95
Introduction
96
Prostate cancer is the most common malignant disease in men in developed
97
countries (1). In 2014, 233,000 new cases of prostate cancer are expected to
98
occur in the United States, which accounts for 27% of incident cases in men (1).
99
Although androgen deprivation is initially effective, the patients inevitably
100
encounter the problem of resistance to androgen deprivation therapy, called
101
castration resistance (2). Discovery of novel strategies to suppress castration
102
resistance is especially helpful for the management of advanced prostate cancer.
103 104
Acetylcholine,
normally
released
from
nerve
endings,
is
a
classical
105
neurotransmitter in the central and peripheral nervous system. A recent study
106
reported the involvement of nerve ending-derived acetylcholine activated
107
cholinergic muscarinic receptor 1 (CHRM1) in mesenchymal cells in the tumor
108
microenvironment to promote prostate cancer invasion and metastasis (3).
109
However, besides nerve ending-derived acetylcholine, there is also wide-spread
110
synthesis of acetylcholine by a variety of non-neuronal cell types, including lung,
111
colon, airway and ovarian epithelial cells (4). Whether there is synthesis of
112
acetylcholine in prostate epithelial cells which might play an autocrine activation
113
role in promoting prostate cancer progression has not yet been studied.
114 115
Muscarinic receptors are G-protein coupled receptors consisting of five members,
116
CHRM1-CHRM5 (5). Activation of muscarinic receptors triggers Ca2+ influx,
117
which causes smooth muscle contraction and glandular secretion. In the 1990s,
118
muscarinic receptor subtypes were firstly defined as conditional oncogenes when
119
they were activated by acetylcholine in NIH-3T3 cells (6). Subsequently,
120
muscarinic receptors have been implicated to be involved in a few types of
121
epithelial cancers including colorectal cancer and small cell lung cancer (7,8). In
122
addition, activation of muscarinic receptors was reported to promote prostate
123
cancer cell proliferation in vitro (9). However, the mechanism and comprehensive
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124
functions of muscarinic receptors in prostate cancer progression, particularly in
125
CRPC, has not been clearly elucidated.
126 127
In the present study, we found the presence of a functional cholinergic system in
128
prostate epithelial cells. An autocrine activation of CHRM3 that regulated prostate
129
cancer cell growth and castration resistance was observed. Blockade of CHRM3
130
via a specific antagonist or shRNAs could effectively inhibit prostate cancer
131
growth and castration resistance. The cholinergic signaling occurred via the
132
CaM/CaMKK-mediated phosphorylation of Akt. These findings suggest a
133
potential application of muscarinic receptor antagonists in the treatment of
134
prostate cancer.
135 136
Materials and methods
137
Cell culture
138
Human prostate cell lines used in this study: LNCaP, PNT1B (10), BPH1 (a kind
139
gift from Dr. Simon W. Hayward) (11), C4-2B, PC3, PC3-AR+ and PC3-luc (kind
140
gifts from Dr. Jianhua Wang at Shanghai Jiao Tong University School of Medicine)
141
(12,13). Cells were cultured in DMEM or RPMI-1640 supplemented with 10%
142
FBS and P/S (penicillin and streptomycin) at 37°C, 5% CO2. In the cell
143
proliferation assay, cell numbers were determined by hemocytometer.
144 145
Cell transfection
146
Two shRNA sequences for CHRM3 (5’-3’, AGCAGAGACAGTCGGTCATTT and
147
TCGGCAACATCCTGGTAATTG) and scrambled shRNA were cloned into the
148
lentiviral vector pUCTP. The cDNA of CHRM3 was subcloned into the lentiviral
149
expression vector EGH. Transfection efficiencies were determined at both the
150
protein and mRNA levels.
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151 152
Cell migration assays
153
In the scratch tests, confluent monolayer cells were scraped with a pipette tip.
154
Cells were cultured in standard cell culture medium containing 10% FBS.
155
Photographs were taken immediately and at 24 hours after wounding. To rule out
156
the potential effects of cell proliferation on cell migration, we normalized the cell
157
migration distance with the total number of cells at 24 hours.
158
In the transwell assays, cells were first starved in serum-free medium for 24
159
hours. Ten thousand-50,000 cells in 100 μL serum free medium were seeded on
160
the top of a transwell chamber (8 μm pore size, 3422, Corning), 500 μL medium
161
containing 10% FBS was added in the bottom of 24-well plates to induce cell
162
migration. Twelve hours later, the culture inserts were fixed with 4%
163
paraformaldehyde and stained in 0.1% crystal violet for 10 minutes. Cells that
164
stayed on the top of the membrane were gently scraped by a cotton swab. To
165
eliminate the effects of cell proliferation on cell migration, we starved PNT1B cells
166
(Lenti-vector and Lenti-CHRM3 transfected PNT1B cells) and PC3 cells
167
(scramble, shRNA1 and shRNA2 transfected PC3 cells) in serum free medium
168
for 24 hours and replaced the medium with standard cell culture medium for 12
169
hours. We measured the cell numbers before and at 12 hours after the
170
replacement of the standard cell culture medium and calculated the cell number
171
changes in both PNT1B cells and PC3 cells (data not shown). Then, we
172
normalized the results in transwell assays with the total number of cells at 12
173
hours.
174 175
In vivo experiments
176
For the subcutaneously xenografted tumor models, 1,000,000 cells (50 μL cell
177
suspension + 50 μL matrigel) were injected subcutaneously into the flank regions
178
of 6-week-old male BALB/c nude mice. Tumor volume was measured once a
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179
week (V=0.5ab2; a, the longest side; b, the shortest side). For orthotopical
180
implantation assays, 8-week-old male BALB/c nude mice were first castrated.
181
Two weeks later, 1,000,000 cells (25 μL cell suspension + 25 μL matrigel) were
182
injected into the anterior lobes of the prostate with a 31G insulin syringe.
183
Bioluminescent signal was induced by intraperitoneal injection of D-luciferin
184
(GoldBio, 1.5 mg D-luciferin per 10 g body weight in saline) and analyzed 10
185
minutes later using a Berthold imaging system. In some assays, darifenacin was
186
injected intraperitoneally at doses of 1 mg/kg/day and 5 mg/kg/day. Vehicle (PBS
187
mixed with DMSO at 1:1) was injected in the control group. Mice were fed in the
188
SPF grade animal facility of Ren Ji hospital with controlled temperature and
189
humidity. All animal studies were carried out following the guidelines of the Ren Ji
190
hospital institutional animal care and ethics committee.
191 192
Clinical prostate cancer samples
193
Paraffin embedded human prostate tissue array slides containing fifty-eight spots
194
(29 paired prostate cancer and adjacent non-cancerous tissues) (OD-CT-
195
UrPrt03-001) were purchased from Shanghai Outdo Biotech Ltd.. A primary
196
antibody against CHRM3 (Abbiotec) and HRP conjugated secondary antibodies
197
(Jackson ImmunoResearch) were applied. The immunostaining was visualized
198
with DAB (3, 3’-diaminobenzidine). Images were captured using a Leica DM2500
199
microscope under the same exposure conditions and analyzed with the Image-
200
pro Plus 6.0 software (Media Cybernetics).
201 202
Calcium influx detection
203
Cells were incubated with Fluo-4 AM (Invitrogen, 2 μM in Hank’s Balanced Salt
204
Solution, HBSS) for 60 minutes at 37°C and incubated for another 15 minutes in
205
fresh HBSS to allow de-esterification of intracellular AM esters. Acetylcholine and
206
carbachol were added to the culture medium to trigger a Ca2+ influx. For
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207
antagonist studies, darifenacin was added 5 minutes before the stimulation of 10
208
μM carbachol. Images were taken with a Leica inverted microscope for 300
209
seconds at intervals of 6 seconds. Data were analyzed with the Image-pro Plus
210
6.0 software.
211 212
Western blot and immunoprecipitation
213
In western blotting assays, cells were lysed in a RIPA buffer (Thermo) with
214
proteinase inhibitors and phosphatase inhibitors (Roche). Total proteins were
215
measured by a BCA method (Thermo). Primary antibodies against GAPDH, E-
216
cadherin, N-cadherin, Vimentin (Epitomic), CaMKKα (Santa Cruz), CHRM3
217
(Abbiotec), Flag (Sigma), Slug, Akt, pAkt-Ser473, pAkt-Thr308 (CST) and HRP
218
conjugated secondary antibodies were applied. Immunoblots were visualized
219
with an ECL blotting detection kit (Thermo). In the immunoprecipitation assay,
220
cell lysates were incubated with the rabbit Akt antibody or control mouse IgG
221
antibody at 4°C overnight. Protein A-agarose beads (Roche) were added, and
222
lysates were further incubated for 1 h at room temperature. Beads were
223
precipitated by centrifugation at 5,000 rpm for 3 minutes and boiled in SDS-page
224
loading buffer for 5 minutes. The samples were then detected according to
225
standard western blotting procedures.
226 227
Immunofluorescent staining
228
Immunofluorescent staining was performed as previously described (14).
229
Antibodies used in this study included Tuj-1 (Sigma), ChAT (Millipore), CHRM3
230
(Abbiotec), Ki67, E-cadherin, N-cadherin, Vimentin (Epitomic) and Cleaved-
231
caspase3 (CST). The TUNEL apoptosis detection system (Promega) was used to
232
detect apoptotic cells in mouse xenografted tumor sections. Images were taken
233
using a Leica DM2500 microscope.
234
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235
Real-time PCR
236
Total RNA was obtained and reverse-transcripted to cDNA by the RNeasy plus
237
Mini Kit and the QuantiTect RT Kit (Qiagen). SYBR (Roche) real-time PCR was
238
performed on the 7900HT machine (ABI). The sequences of the primers used in
239
this study are listed in Supplementary Table S1.
240 241
Statistical analysis
242
Data in this study are expressed as the means ± SEM. Immunostaining densities
243
of CHRM3 in matched human prostate cancerous and adjacent non-cancerous
244
tissues were compared by paired Student’s t-test. Cell proliferation, real-time
245
PCR and in vivo tumor xenograft growths were analyzed using a non-parametric
246
Student’s t-test. Ki67, TUNEL and cleaved-caspase3 positive cells were counted
247
in at least three randomly-selected visual fields and analyzed using a
248
nonparametric Student’s t-test. The Kaplan–Meier log-rank test was used for
249
analysis of mouse survival data. Data were analyzed with GraphPad Prism 5
250
software (GraphPad Software). Statistical significance was defined as * P < 0.05,
251
** P < 0.01, *** P < 0.001.
252 253 254 255 256 257 258 259
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260
Results
261
Presence of functional autocrine cholinergic signaling in the prostate
262
epithelium
263
Autonomic nerves were reported to regulate prostate cancer metastasis by
264
regulating the microenvironment of epithelial cancer cells (3). Immunostaining of
265
mouse prostate sections confirmed dense innervation of Tuj-1 immunoreactive
266
nerve fibers; however, few, if any, fibers could be observed inside the epithelium
267
(Fig.
268
immunoreactivity of ChAT, which is the enzyme necessary for acetylcholine
269
synthesis (Fig. 1B). Importantly, ChAT immunoreactivity in human prostate
270
sections also showed the expression of ChAT in the epithelium (Fig. 1C). In
271
addition, we detected the secretion of acetylcholine from both cancerous and
272
non-cancerous human prostate epithelial cells, which are free of any neuronal
273
innervations (Fig. 1D). These data demonstrate clearly that there is endogenous
274
production and secretion of acetylcholine from prostate cancerous and non-
275
cancerous epithelial cells.
1A).
Instead,
mouse
prostate
epithelial
cells
showed
strong
276 277
A complete, functional cholinergic loop requires the presence of not only
278
acetylcholine but also muscarinic receptors. To determine the expression
279
patterns of muscarinic receptors in the prostate, we profiled gene expression in
280
the Oncomine database and GEO Profiles database. Of five muscarinic
281
cholinergic receptors, only CHRM3 was significantly elevated in prostate cancer
282
samples compared to non-cancer samples (Supplementary Fig. S1). To confirm
283
the Oncomine and GEO data analysis, we performed immunohistochemistry of
284
CHRM3 in the human prostate cancer tissue array and found that CHRM3 was
285
mainly expressed in the prostate epithelium rather than in the mesenchyme (Fig.
286
1E). Notably, CHRM3 was significantly up-regulated in cancer tissues compared
287
to their matched, adjacent non-cancer tissues (Fig. 1F).
288
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289
To determine whether the autocrine acetylcholine could activate muscarinic
290
receptors in epithelial cells, we performed experiments to measure Ca2+ influx,
291
which is a direct indicator of muscarinic receptor activation. We measured Ca2+
292
influx with Fluo-4 AM, a commonly used fluorescent Ca2+ influx indicator, and
293
found that both acetylcholine and its stable analogue carbachol could induce
294
Ca2+ influx. CHRM3-specific antagonist darifenacin could largely reduce Ca2+
295
influx induced by carbachol (Fig. 1G). In addition, conditioned medium from PC3
296
cells also triggered Ca2+ influx, suggesting the occurrence of secretion of
297
endogenous non-nerve ending-derived acetylcholine (Fig. 1H). Importantly, the
298
Ca2+ influx induced by endogenous acetylcholine could be effectively blocked by
299
the CHRM3 specific inhibitor (Fig. 1H). Taken together, these findings show the
300
presence of functional autocrine cholinergic signaling in prostate epithelium.
301 302
Activation of CHRM3 by endogenous acetylcholine promotes prostate
303
cancer growth
304
To evaluate the role of autocrine cholinergic signaling in prostate cancer growth,
305
we over-expressed CHRM3 in non-tumorigenic PNT1B cells. Over-expression of
306
CHRM3 in PNT1B cells promoted cell growth as time proceeded (Fig. 2A),
307
implicating that over-expressed CHRM3 could be activated by endogenous
308
acetylcholine. In contrast, the knock-down of CHRM3 by shRNA in PC3 cells
309
inhibited cell growth (Fig. 2C). The lentiviral transfection efficiencies were
310
analyzed by western blot (Fig. 2B and 2D). In addition, we treated PC3 cells with
311
carbachol (a stable agonist of muscarinic receptors) and darifenacin (a selective
312
antagonist of CHRM3). While carbachol promoted the proliferation of PC3 and
313
22Rv1 cells, blockade of CHRM3 by darifenacin could effectively reduce cell
314
proliferation (Fig. 2E, F).
315 316
To extend our in vitro studies to an in vivo setting, we implanted CHRM3 knock-
317
down PC3 cells subcutaneously in BALB/c nude mice. We found that knock-
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318
down of CHRM3 reduced tumor growth in vivo (Fig. 2G). Consistently, CHRM3-
319
specific antagonist darifenacin also inhibited the growth of xenografted PC3 and
320
22Rv1 cells (Fig. 2H, I). Histological examination of PC3 xenografted tumors
321
treated with darifenacin showed a reduced percentage of Ki67 positive,
322
proliferating cells and increased percentage of apoptotic cells (Fig. 2J-L). These
323
findings indicate that autocrine activation of CHRM3 promotes prostate cancer
324
growth.
325 326
Autocrine activation of CHRM3 promotes cell migration through regulating
327
epithelial-mesenchymal transition
328
To evaluate the role of CHRM3 in regulating cell migration, we performed
329
transwell assays and scratch tests. In transwell assays, over-expression of
330
CHRM3 in PNT1B cells increased the number of cells that migrated through the
331
membrane (Fig. 3A, B). In scratch tests, over-expression of CHRM3 promoted
332
the confluence of scratched cells (Supplementary Fig. S2A, B). To rule out the
333
potential effects of cell proliferation on cell migration, we normalized the results in
334
transwell assay and scratch test with the total number of cells at the same
335
detection time point as described in the materials and methods section. On the
336
contrary, knock-down of CHRM3 in PC3 cells inhibited their migration capability
337
both in the transwell assay and in the scratch test (Fig. 3C, D and Supplementary
338
Fig. S2C, D). Similarly, the cell migration results were also normalized to the total
339
number of cells at the same detection time point.
340 341
Epithelial-mesenchymal transition (EMT) is an important process in epithelial
342
cancer progression, which facilitates cell migration and invasion (15). To verify
343
whether the activation of CHRM3 could induce EMT, we over-expressed CHRM3
344
in PNT1B cells. We observed that CHRM3 up-regulation caused the PNT1B cells
345
to become less attached to each other and exhibit a mesenchymal phenotype
346
(Fig. 3E). Immunostaining of these cells with E-cadherin, N-cadherin and
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347
Vimentin antibodies confirmed that E-cadherin expression was decreased in
348
CHRM3 over-expressing PNT1B cells; N-cadherin and Vimentin expression was
349
sharply increased (Fig. 3E). Western blotting analysis confirmed the decreased
350
E-cadherin protein level and increased Vimentin protein level in CHRM3 over-
351
expressing PNT1B cells (Fig. 3F). Consistently, real-time PCR analysis also
352
showed an increase in mesenchyme-related gene expression, while E-cadherin
353
was down-regulated in these CHRM3 over-expressing PNT1B cells (Fig. 3G). On
354
the contrary, CHRM3 knock-down PC3 cells reversed its mesenchymal status
355
and expressed more E-cadherin and less N-cadherin (Fig. 3H). The
356
immunoblotting analysis also confirmed that CHRM3 knock-down reversed EMT
357
in PC3 cells (Fig. 3I). In addition, real-time PCR analysis of several EMT-related
358
genes also showed that CHRM3-silenced PC3 cells underwent an opposite
359
process of EMT, that is, mesenchymal-epithelial transition (Fig. 3J). These
360
findings together indicate that autocrine activation of CHRM3 promotes prostate
361
cell migration by regulating EMT.
362 363
CHRM3 is up-regulated in castration-resistant prostate cancer cells
364
To evaluate the role of CHRM3 in castration resistance, a very important feature
365
of prostate cancer, we first measured CHRM3 expression in paired CRPC and
366
androgen-dependent prostate cancer cells: C4-2B and LNCaP, PC3 and PC3-
367
AR+. C4-2B was a bone metastatic and castration-resistant subline of androgen-
368
dependent LNCaP cells (16). PC3-AR+ cells were generated through stable
369
expression of full-length human androgen receptor (AR) in PC3 cells (17). Re-
370
expression of AR restored the response to androgens in PC3-AR+ cells (17,18).
371
Real-time PCR analysis revealed higher CHRM3 mRNA levels in castration-
372
resistant C4-2B and PC3 cells than their paired androgen-dependent LNCaP and
373
PC3-AR+ cells, respectively (Fig. 4A). Secondly, we treated LNCaP cells with
374
bicalutamide, a clinically-used androgen deprivation agent, to imitate the
375
androgen deprivation condition in vitro. After several generations, the expression
376
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377
(Supplementary Fig. S3A). Thirdly, we subcutaneously implanted PC3-AR+ cells
378
in nude mice. When tumors grew for approximately 1 month, we castrated the
379
recipient mice. We analyzed CHRM3 mRNA levels before and post castration.
380
We found that the expression of CHRM3 was significantly increased after
381
castration (Fig. 4B). These findings are in agreement with the GEO database
382
indicating that androgen deprivation resulted in enhanced CHRM3 expression in
383
LuCaP35 cells (Supplementary Fig. S3B) (19). All of these data together suggest
384
a positive correlation of CHRM3 with castration resistance.
385 386
Over-expression of CHRM3 promotes castration resistant growth
387
To evaluate whether activation of CHRM3 could cause castration resistance in
388
vivo, we established tumor xenografts in castrated nude mice. At first, we wanted
389
to establish in vivo tumor models with LNCaP cells. However, LNCaP cells failed
390
to efficiently and consistently form tumors in normal, un-castrated nude mice
391
even with ten million cells. Then, we engrafted PC3-AR+ and PC3 cells
392
subcutaneously in castrated nude mice. We observed that PC3 cells transfected
393
with control lentivirus indeed formed tumors in androgen deprivation conditions
394
(Fig. 4C). On the contrary, PC3-AR+ cells transfected with control lentivirus failed
395
to do so under the same conditions (Fig. 4C). More importantly, over-expression
396
of CHRM3 in these PC3-AR+ cells caused them to re-gain the castration
397
resistant capability and form tumors under conditions of hormone deprivation
398
condition (Fig. 4C). Consistently, immunofluorescent staining of Ki67 and cleaved
399
caspase-3 in tumor sections showed increased cell proliferation and decreased
400
cell apoptosis, respectively, in CHRM3 over-expressed xenografts (Fig. 4D-G).
401
These results suggest that activation of CHRM3 can enhance castration-resistant
402
growth capability of the androgen-dependent prostate cancer cells.
403 404
Blockade of CHRM3 inhibits the castration resistant growth of PC3-luc cells
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405
To study whether the blockade of CHRM3 could influence castration-resistant
406
growth or the sensitivity of prostate cancer cells under androgen deprivation
407
conditions, we established an orthotopic prostate cancer model with luciferase
408
stably expressing PC3 (PC3-luc) cells under an androgen deprivation paradigm,
409
which is more similar to the in situ castration resistant prostate cancer. Seven
410
weeks after implantation, we examined tumor growth by bioluminescence (Fig.
411
5A). Stable silencing of CHRM3 reduced tumor growth when compared with the
412
scrambled shRNA transfected group (Fig. 5B, C). When these primary
413
xenografted tumor sections were processed for immunofluorescent staining with
414
anti-Ki67 antibody to detect the proliferating cells, we observed decreased Ki67
415
positive nuclei in CHRM3 knock-down tumors (Fig. 5D, E). Similarly, when the
416
tumor recipient mice were treated with vehicle or darifenacin at a dosage of 1
417
mg/kg/day, darifenacin significantly inhibited castration resistant growth of PC3-
418
luc cells when compared to the vehicle group (Fig. 5F-H). In addition, darifenacin
419
improved the survival status in tumor-bearing recipient mice (Fig. 5I).
420
Immunostaining of Ki67 also showed decreased cell proliferation in darifenacin
421
treated tumors (Fig. 5J). These findings together confirm that blockade of
422
CHRM3 enhances the sensitivity of androgen-independent PC3-luc cells to
423
androgen deprivation.
424 425
Activation of CHRM3 promotes CaM/CaMKK-dependent phosphorylation of
426
Akt.
427
Next, we wanted to explore the mechanism of autocrine cholinergic signaling in
428
regulating prostate cancer growth and castration resistance. Previous studies
429
reported that Ca2+ influx could promote the phosphorylation of Akt (20-22).
430
Because our data had shown that activation of muscarinic receptors stimulated
431
Ca2+ influx in prostate cancer cells, we next wanted to determine whether the
432
autocrine activation of cholinergic signaling could enhance calcium signaling-
433
mediated phosphorylation of Akt. As shown in Fig. 6A, over-expression of
434
CHRM3 in LNCaP cells increased the phosphorylation of Akt due to the 16 Downloaded from clincancerres.aacrjournals.org on July 23, 2015. © 2015 American Association for Cancer Research.
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435
production of endogenous acetylcholine from the LNCaP cells (Fig. 6A). On the
436
contrary, silencing of CHRM3 in PC3 cells decreased the phosphorylation of Akt
437
(Fig. 6B). These findings indicate that the autocrine cholinergic signaling could
438
promote Akt phosphorylation.
439
To confirm that Akt phosphorylation induced by the autocrine cholinergic
440
signaling is calcium-signaling dependent, we first treated PC3 cells with the
441
CHRM3-specific antagonist darifenacin and the calmodulin-selective antagonist
442
W-7. Western blotting analysis revealed that both darifenacin and W-7 could
443
effectively inhibit Akt phosphorylation stimulated by carbachol (Fig. 6C). Next, we
444
treated PC3 cells with CaMKK antagonist STO-609 to determine whether the
445
downstream signaling of CaM was involved in Akt phosphorylation. STO-609
446
could also effectively block Akt phosphorylation induced by carbachol (Fig. 6D).
447
Furthermore, co-immunoprecipitation showed a direct binding of Akt to CaMKKα
448
(Fig. 6E). These data suggest that autocrine cholinergic signaling promotes
449
prostate cancer growth and castration resistance through CaM/CaMKK-mediated
450
activation of Akt (Fig. 6F).
451 452
Discussion
453
Although a previous study reported a role for neuronal cholinergic signaling in
454
prostate cancer metastasis (3), the present study demonstrates several different
455
and novel findings. First, we found the autonomous expression of ChAT and
456
synthesis of acetylcholine in prostate epithelial cells, suggesting the presence of
457
autocrine cholinergic signaling in the prostate epithelium. Second, we detected
458
an up-regulation of CHRM3 in human prostate cancer tissues compared to their
459
adjacent non-cancer tissues, implicating that CHRM3 might be an additional
460
diagnostic marker of prostate cancer. Third, different from the parasympathetic
461
cholinergic signaling that regulated the microenvironment to promote prostate
462
cancer metastasis (3), we found that direct over-expression or knock-down of
463
CHRM3 in the prostate cells significantly promoted or inhibited cell migration
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464
through the regulation of EMT. Finally, we found that activation or inhibition of
465
CHRM3 promoted or inhibited prostate cancer growth and castration resistance
466
both in vitro and in vivo. Thus, our data strongly indicate that there is an
467
autocrine activation of CHRM3 in prostate cancer epithelial cells.
468 469
Our study shows, for the first time, the secretion of autocrine acetylcholine from
470
prostate epithelial cells and cancer cells. Immunostaining indicated the
471
expression of ChAT in both mouse and human prostate epithelia. In addition, we
472
detected the production of acetylcholine by human prostate epithelial cells,
473
including both cancer cells and non-cancer cells. The concentration of autocrine
474
acetylcholine in the cell culture medium is approximately 2~4 μM, which is
475
sufficient to stimulate Ca2+ influx that can be induced by 0.1 μM of acetylcholine
476
or carbachol (Supplementary Fig. S4). Considering that in the microenvironment
477
of prostate cancer, the concentration of acetylcholine in prostate cancer tissues
478
may be even higher than the acetylcholine secreted into the medium due to
479
higher cell density, such autocrine acetylcholine signaling likely functions in the
480
prostate in vivo. Further support for our autocrine cholinergic signaling model
481
also comes from the results reported previously in other tissue adenocarcinoma
482
cells, such as colon cancer cells and small cell lung cancer cells (7,8).
483 484
The present study provides important insights into the mechanism for the
485
biological effects of activation of muscarinic receptors. First, either the CHRM3-
486
specific inhibitor darifenacin or the calmodulin-selective antagonist W-7 can
487
effectively inhibit Akt phosphorylation stimulated by carbachol. Second, CaMKK
488
antagonist STO-609, which is a downstream signaling component of CaM, also
489
effectively blocks Akt phosphorylation induced by activation of CHRM3. Third,
490
there is direct binding between Akt and CaMKK based on co-immunoprecipitation
491
assays. Fourth, calcium signaling has been shown to mediate Ca2+ influx-
492
induced Akt phosphorylation (22-24). Finally, a blockade of Akt activity has been
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493
shown to suppress castration-resistant growth in both mouse models and clinical
494
settings (25-28). These findings together suggest that autocrine cholinergic
495
signaling promotes prostate cancer growth and castration resistance through the
496
CaM/CaMKK-mediated activation of Akt (Fig. 6F).
497 498
Castration resistance is a major challenge in prostate cancer treatment. However,
499
an effective approach for targeting castration resistant prostate cancer is
500
currently unavailable. In this study, we found that CHRM3 was upregulated in
501
CRPC cells compared to matched androgen-dependent cells. While over-
502
expression of CHRM3 was sufficient to cause androgen-dependent PC3-AR+
503
cells to form tumors in castrated mice, stable silencing of CHRM3 inhibited the
504
castration resistant growth of PC3 cells in orthotopic xenografts. Notably, our
505
study showed that the CHRM3-specific antagonist darifenacin was effective to
506
inhibit PC3 cell growth in castrated nude mice. Given that specific antagonists of
507
CHRM3 have been widely used in clinical conditions such as OAB (overactive
508
bladder) and COPD (chronic obstructive pulmonary diseases), clinical trials with
509
such CHRM3 antagonists are warranted and may hold promise for the treatment
510
of primary prostate cancer as well as castration-resistant prostate cancer.
511 512 513 514 515 516 517 518
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519
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Figure legends
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Figure 1. Presence of a functional autocrine cholinergic system in the prostate
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epithelium. A, Immunofluorescent staining of Tuj-1 in 2-week-old mouse prostate
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sections. B, Immunofluorescent images showing a selective epithelial expression
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pattern of ChAT in the mouse prostate. C, Immunohistochemistry of ChAT in
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human prostate sections. D, Detection of the release of acetylcholine from
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various human prostate cell lines. E, Immunohistochemistry of CHRM3 in human
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prostate cancer tissue arrays. F, Staining intensities of CHRM3 quantified by IOD
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(Integral optical density); data are analyzed by paired t-test. G, Ca2+ influx
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detected by Fluo-4 AM in PC3 cells. Acetylcholine (10 μM) and carbachol (10 μM)
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are used to trigger Ca2+ influx. To counteract Ca2+ influx induced by carbachol,
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darifenacin (10 μM) is added 5 minutes before the addition of carbachol. H,
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Detection of Ca2+ influx induced by endogenous acetylcholine. Conditioned
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medium is collected from PC3 cells cultured in a serum free medium, in which
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neostigmine (10 μM) was added to inhibit the degradation of acetylcholine. Data
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are analyzed with Student’s t-test, * P
