Ghrelin and a Novel Preproghrelin Isoform Are Highly Expressed
in Prostate Cancer and Ghrelin Activates Mitogen-Activated
Protein Kinasein Prostate Cancer
Anthony H. Yeh, Penelope L. Jeffery, Russell P. Duncan, Adrian C. Herington, and Lisa K. Chopin
Purpose:There is evidence that the hormone ghrelin stimulates proliferationin the PC3 prostate
cancercelllinealthoughtheunderlyingmechanism(s) remaintobe determined. Anovel, exon 3^
deleted preproghrelin isoform has previously been detected in breast and prostate cancer cells;
however, its characterization, expression, and potential function in prostate cancer tissues are
Experimental Design: Expression of ghrelin and exon 3^ deleted preproghrelin was investi-
gated in prostate cancer cell lines and tissues by reverse transcription-PCR and immunohisto-
chemistry. Proliferation and apoptosis assays were done in the LNCaP prostate cancer cell line
to determine if ghrelin stimulates proliferation and/or cell survival. Stimulation of mitogen-
activated protein kinase (MAPK) pathway activation by ghrelin was determined in PC3 and
LNCaP cells by immunoblotting with antibodies specific for phosphorylated MAPKs.
preproghrelin than normal prostate tissues, and prostate cancer cell lines secrete mature ghrelin
into conditioned medium.Treatment with ghrelin (10 nmol/L), but not theunique COOH-terminal
peptide derived from exon 3^ deleted preproghrelin, stimulates proliferation in the LNCaP cells
(45.0 F 1.7% above control, P < 0.01) and rapidly activates the extracellular signal-regulated
kinase-1/2 MAPK pathway inboth PC3 and LNCaP celllines. Ghrelin, however, does not protect
prostate cancer cells from apoptosis induced by actinomycin D (1 Ag/mL).The MAPK inhibitors
PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as proliferation, in both
Conclusions:These data suggest that these components of the ghrelin axis may have potential
asnovelbiomarkers and/oradjunctivetherapeutic targetsfor prostate cancer.
Ghrelin, a 28-amino-acid n-octanoylated peptide, acts via the
growth hormone secretagogue receptor (GHS-R) to stimulate
growth hormone release (1, 2) and has a range of other
biological actions including stimulation of food intake, control
of energy expenditure, modulation of insulin signaling and
cardiovascular effects (3–7). The finding that ghrelin has a
proliferative effect was first described in the HepG2 hepatoma
cell line (6) and prostate cancer cell lines (8). Subsequently, it
has been shown that growth of other cell types is enhanced by
ghrelin (9–18). We have recently shown that ghrelin also
stimulates proliferation in several breast cancer cell lines (19) in
contrast to earlier studies (20).
Both ghrelin and the GHS-R (a G protein–coupled receptor)
are widely expressed in normal tissues (1, 21–23) as well as in
various tumors, including human pituitary adenomas and
various endocrine neoplasms of the lung, stomach, and
pancreas (24–28). We have previously shown that components
of the ghrelin/GHS-R axis, including an apparent human exon
3–deleted preproghrelin mRNA variant, are expressed in
prostate cancer cell lines (8). The exclusion of the third exon
from the preproghrelin transcript causes a translational
frameshift which results in expression of a unique truncated
proghrelin C-peptide, as well as mature ghrelin, in human and
mouse tissues (29, 30).
Few studies have investigated the signaling mechanism of the
endogenous ligand ghrelin through the GHS-R. In the pituitary,
ghrelin activates the GHS-R, triggering activation of protein
kinase C and a transient increase in intracellular calcium levels,
which in turn induces growth hormone secretion (1). Recent
reports have indicated that ghrelin can also activate mitogen-
activated protein kinase (MAPK) cascades in some tissues
(11, 12, 14, 16, 17, 31, 32). The MAPK signaling pathways play
Human Cancer Biology
Authors’ Affiliation: School of Life Sciences, Queensland University of
Technology, Brisbane, Australia
Received 2/28/05; revised 8/2/05; accepted 8/25/05.
Grant support: National Healthand Medical Research Councilgrant, Queensland
Universityof Technology, and Sir Edward Dunlop Medical Research Foundation.
The costs of publicationof this articlewere defrayedinpart by the paymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicatethis fact.
Note: A.H.Yehand P.L. Jeffery contributedequally to this work.
Requests for reprints: Lisa K. Chopin, School of Life Sciences, Queensland
University ofTechnology, G.P.O. Box 2434, Brisbane 4001, Australia. Phone: 61-7-
3864-2667; Fax: 61-7-3864-1534; E-mail: email@example.com.
F2005 American Association for Cancer Research.
www.aacrjournals.orgClin Cancer Res 2005;11(23) December1, 20058295
an important role in mammalian cells where they modulate
many cellular events including cell proliferation, differentia-
tion, and development (33). The GHS-R can cross-talk with
the MAPK pathway to promote cell proliferation in several
cancer cell lines, including HepG2 hepatoma cells, pancreatic
adenocarcinoma cells, and GH3 rat pituitary somatotroph cells
(6, 10, 12). Similarly, activation of MAPK pathways could be
involved in the increased proliferation observed in prostate
cancer cells after ghrelin treatment (8).
The data presented here provide the first evidence that
ghrelin and exon 3–deleted preproghrelin are highly expressed
in prostate carcinoma specimens compared with normal
prostate tissues and that the unique COOH-terminal peptide
derived from exon 3–deleted preproghrelin is cleaved in the
LNCaP prostate cancer cell line. We also show that the PC3 and
LNCaP prostate cancer cell lines secrete mature ghrelin and that
ghrelin initiates cross-talk with the MAPK signaling cascade to
promote cell proliferation in prostate cancer cell lines. These
findings suggest that the ghrelin/GHS-R axis could play an
important role in prostate cancer.
Materials and Methods
gen-dependent LNCaP and ALVA-41 human prostate cancer cell lines
were obtained from the American Type Culture Collection (Rockville,
MD). All cells were cultivated in RPMI 1640 (Invitrogen, Carlsbad, CA)
with 10% fetal bovine serum (Invitrogen), penicillin G (50 units/mL),
and streptomycin sulfate (50 AL/mL; Invitrogen) in 80-cm2tissue
culture flasks (Nagle Nunc International, Roskilde, Denmark) at 37jC
in a humidified atmosphere containing 5% CO2and 95% air. Cells
were tested to be free from Mycoplasma contamination (American Type
Culture Collection Mycoplasma detection kit).
Reverse transcriptase-PCR. Reverse transcription of prostate cancer
cell line mRNA and normal prostate mRNA (Clontech, Palo Alto, CA)
using Superscript II reverse transcriptase (Invitrogen) was done as
previously described (8). Reverse transcription-PCR to detect full-length
preproghrelin and exon 3–deleted preproghrelin was done in 50-AL
reactions containing 1 unit of Red Hot Polymerase (Integrated Sciences,
Melbourne, Australia), 10? PCR buffer (Integrated Sciences), 100
Amol/L deoxynucleotide triphosphates (Roche, Basel, Switzerland),
100 pmol/L sense (5V -gcccacctgtctgcaacc-3V ) and antisense primers
(5V -tgaacatttattcgcctcctg-3V ; Proligo, Armidale, Australia) from ghrelin
exons 2 and 5, respectively, and 2 AL of cDNA or sterile distilled water
(no template negative control). Thermal cycling consisted of 5 minutes
of denaturation at 95jC, 40 cycles of 30 seconds at 95jC, 30 seconds at
annealing temperature (50jC), 1-minute extension at 72jC, followed
by a final 10-minute extension at 72jC on a PTC-200 Thermal cycler
(MJ Research, Watertown, MA). DNA sequencing of the purified PCR
product was carried out at the Australia Genome Research Facility
(Brisbane, Australia) using the ABI PRISM BigDye Terminator Cycle
Sequencing Kit protocol (Applied Biosystems, Foster City, CA).
Sequences were identified using BLAST software (Entrez).
Immunohistochemistry for ghrelin and exon 3–deleted preproghrelin
expression in prostate tissue sections.
tion of ghrelin and the exon 3–deleted preproghrelin variant were
detected using immunohistochemistry on paraffin-embedded prostate
tissue sections. Twenty-six prostate cancer and 11 benign prostatic
hypertrophy tissue specimens were obtained with ethical approval
(Queensland University of Technology ethics approval 1992H) and
normal prostate sections were purchased from Peterborough Hospital
Tissue Bank (Peterborough, United Kingdom). Tissue sections were
microwaved in citric acid buffer for 10 minutes (pH 6) to enhance
antigen retrieval, then incubated overnight with decreasing concen-
Androgen-independent PC3 and DU-145 and andro-
Protein expression and localiza-
trations of primary antibody diluted in 0.01 mol/L PBS containing 1%
bovine serum albumin (BSA; Sigma, St Louis, MO). Immunodetection
was done using the Envision plus diaminobenzamine staining
kit (DAKO, Kyoto, Japan) according to the instructions of the
manufacturer. Polyclonal anti-ghrelin antibodies were raised in rabbits
(Institute for Medical and Veterinary Sciences, Adelaide, Australia)
against the mature Ser-3 n-octanoylated human ghrelin peptide
(GSSFLSPEHQRVQQRKESKKPPAKLQPR) conjugated to diphtheria
toxin (Mimotopes, Melbourne, Australia) and affinity purified as
previously described (8). Antibodies were also raised against the
putative 16-amino-acid COOH-terminal peptide encoded by the exon
3–deleted preproghrelin variant (COOH-terminal D3 peptide;
RPQPTSDRPQALLTSL; Mimotopes) and affinity purified. For both
antibodies, negative controls included omission of primary antibody
and preabsorption of antibody with excess peptide (1 mg/mL) to which
the antibody was raised.
Western blot analysis of prostate cancer cell line lysate and conditioned
media. To establish if PC3 and LNCaP cell lines secrete ghrelin into
conditioned media, Western blot analysis was undertaken. PC3 or
LNCaP cells (3 ? 106seeded into 80-cm2tissue culture flasks) were
allowed to attach overnight at 37jC. They were incubated in serum-
free RPMI 1640, 0.1% BSA, and 20 mmol/L 4-(2-aminoethyl)benzene-
sulfonyl fluoride hydrochloride (Sigma) for 24 hours and conditioned
medium was collected and frozen at ?80jC. Medium was concentrated
using Centriprep Centrifugal filter units with Ultracel-YM membranes
and a 3-kDa nominal molecular weight cutoff (Millipore, Bedford, MA)
was boiled for 5 minutes at 100jC in 2? Tricine sample buffer [200
mmol/L Tris-HCl (pH 6.8), 2% SDS, 40% glycerol, 0.04% Coomassie
blue G-250, and 350 mmol/L DTT] and electrophoresed through a
10% to 20% Tricine gradient gel (Bio-Rad, Hercules, CA). The protein
was electrotransferred to a Protran nitrocellulose membrane (Schleicher
& Schuell, Dassel, Germany) for 1 hour at 4jC at 200 mA in transfer
buffer (10 mmol/L NaHCO3, 3 mmol/L Na2CO3, 20% methanol,
pH 9.9). After the membrane was blocked overnight with 1% BSA in
PBS, it was incubated with the polyclonal anti-ghrelin antibody diluted
in TBS-0.05% Tween 20 containing 1% BSA for 1 hour at room
temperature. After several brief washes in TBS-0.05% Tween 20, the
membrane was incubated with horseradish peroxidase–conjugated
antirabbit secondary antibody (1:100,000 dilution; Jackson Immuno-
Research, West Grove, PA) at room temperature for 1 hour. After several
brief washes, chemiluminescent SuperSignal West Femto Maximum
Sensitivity Substrate (Pierce, Rockford, IL) was applied and the
membrane was exposed to X-ray film and developed (Agfa-Gavaert,
electrophoresed concurrently with the samples as positive and negative
controls. Western blot analysis on LNCaP prostate cancer cell line,
human stomach carcinoma, and normal human stomach lysates
(Pierce) was also done as previously described (8) to determine if
COOH-terminal D3 peptide is cleaved from the rest of the exon 3–
deleted preproghrelin peptide and to ensure the specificity of the anti–
COOH-terminal D3 peptide antibody.
Mitogen-activated protein kinase activation assays.
cells were plated in a six-well plate at a seeding density of 6 ? 105
per well, grown to 70% to 80% confluency, and then serum deprived
overnight at 37jC. Mature n-octanoylated ghrelin peptide (or COOH-
terminal D3 peptide) was dissolved in 1? PBS, 0.05% BSA and added
to RPMI 1640 with antibiotics, 0.1% BSA, and 20 mmol/L 4-(2-
aminoethyl)benzenesulfonyl fluoride hydrochloride. Cells were trea-
ted with various concentrations of ghrelin or the COOH-terminal D3
peptide (0, 1, 10, 100, and 1,000 nmol/L) for 5, 15, 30, and 60
minutes at 37jC. As a positive control, PC3 cells were treated with 1
Ag/mL anisomycin, a potent MAPK pathway stimulating agent (34).
All cells were harvested in lysis buffer [20 mmol/L Tris-HCl (pH 7.5),
150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L
EGTA, 1 mmol/L Na3VO4, 50 mmol/L NaF, 1 mmol/L phenylmethyl-
sulfonyl fluoride, 50 mmol/L h-glycerophosphate] and 1 Complete
PC3 and LNCaP
Human Cancer Biology
www.aacrjournals.org Clin Cancer Res 2005;11(23) December1, 20058296
Ghrelin Activates MAPKin Prostate Cancer
www.aacrjournals.org Clin Cancer Res 2005;11(23) December1, 20058303
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