Prostate specific G protein coupled receptor is associated with prostate cancer prognosis and affects cancer cell proliferation and invasion

Patients’ recruitment and tissue samples

The pathology database at the University of Rochester Medical Center (URMC) from 2004
to 2005 was searched for radical prostatectomy and prostatic adenocarcinoma. 150 cases
were identified and retrieved for histologic review. The diagnosis of prostatic adenocarcinoma
for each case was confirmed by one of the two pathologists (FL and WQC). Clinical
and pathological information including age, stage, Gleason score, lymphovascular invasion,
extraprostatic extension, and survival were collected from the clinical notes and
pathology reports. Human tissue specimens obtained and processed at URMC were collected
under the protocol “Studies of biomarker expression in human tumors” (RSRB00020130)
approved by the University of Rochester Research Subjects Review Board (RSRB). The
institutional review board RSRB approved this retrospective study and waived the need
for consent. Patient records/information was anonymized and de-identified prior to
analysis.

Tissue microarrays

Archival, formalin-fixed, paraffin-embedded tissue blocks from the selected patients
were procured from the Department of Pathology and Laboratory Medicine at URMC. Tissue
microarrays (TMAs) were constructed from the selected paraffin blocks of 150 cases.
For each case, areas containing benign prostatic tissue, high grade prostatic intraepithelial
neoplasia, or prostatic adenocarcinoma were first marked by a pathologist for sampling.
Two 3 mm core samples were then retrieved from each selected area in the donor paraffin
blocks, and transferred to a TMA paraffin block. Six cores were taken from each individual
case. A TMA grid map was made to keep track of cores from the same prostatectomy specimen.
The Gleason grade for each PCa core was the same as that in the final pathology report.
Many tissue cores contained two of the three tissue types (normal and PIN, PIN and
PCa). Each TMA block also included tissue cores (normal liver, placenta, thyroid tissue,
or small bowel) as controls.

Reagents

?-ionone and ?-ionone were purchased from Sigma (St Louis, MO or NU-chek prep, INC.
Elysian, MO), dissolved in DMSO and stored at ?80 °C. Specific antibodies against
AR, PSGR, Phosphorylated P70 S6 kinase, phosphorylated mTOR, and phosphorylated 4EBP1
were obtained from Abcam (Cambridge, MA). The antibody against PSGR for immunohistochemistry
was from Novus Biologicals (Littleton, CO).

Immunohistochemistry

Tissue microarray sections were immunohistochemically stained with PSGR antibody as
previously described 11]. Briefly, TMA sections were de-paraffinized and rehydrated through graded alcohols.
Antigen retrieval was performed by boiling in an EDTA buffer (pH 9.0) at 98 °C for
20 min. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide. The
slides were incubated with antibody against PSGR (NLS6332, rabbit polyclonal, 1: 100)
at room temperature for one hour and were subsequently incubated for 30 min with EnVision?+?System
horseradish peroxidase-labeled polymer conjugated with biotinylated anti-rabbit secondary
antibody and 3,3?-diaminobenzidine substrate. All sections were counterstained with
Mayer’s hematoxylin. Normal prostate and testes were used as positive and negative
control for PSGR. Negative controls were also established by the replacement of primary
antibodies with normal serum.

Evaluation of immunohistochemical staining

Membranous and cytoplasmic PSGR staining was observed in the epithelial cell of normal
prostate, PIN and PCa (Fig. 1a). To more precisely represent the expression level of PSGR, the H-score system was
employed in this study. Two independent researchers (JZY and WQC) evaluated the stained
slides as described previously 12]. The staining intensity for each protein was scored as 0, no staining; 1+, weak;
2+, moderate; and 3+, strong staining. The percentage of tumor cells that stained
positive was also estimated (0–100 %). A H score (range 0–300) was generated by multiplying
the staining intensity score and the percentage of positively stained tumor cells
(H-Score?=?3× percentage of cells with strong staining?+?2× percentage of cells with
moderate staining?+?1× percentage of cells with weak staining?+?0× percentage of cells
with no staining). The Kappa value for inter-observer agreement between the two researchers
was 0.85.

Fig. 1. Expression of PSGR protein in normal prostate (N), prostatic intraepithelial neoplasia
(PIN) and prostate cancer (PCa) by immunohistochemistry. A Low (a-c) and high magnification (d-f) images of PSGR staining in N (a, d), PIN (b, e) and PCa (c, f); B Low (a and b) and high (c and d) magnification images of PSGR in N and adjacent PIN (a, c), and PIN and adjacent PCa (b, d) (original magnification x 10 and x 20 for low and high respectively)

Cell culture and invasion assay

LNCaP, PC3, and C4-2 human PCa cell lines were obtained from the American Type Culture
Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented
with 10 % of FBS and antibiotics in 100 mm cell culture dishes or culture plates.
12 h prior to experimentation, cells were cultured in Dulbecco’s modified Eagle’s
medium with 5 % charcoal treated FBS and antibiotics.

To measure cell invasion, an insert with 8.0 ?m pores (Corning) was coated with matrigel
(BD Biosciences) and 5?×?10
⁵
C4-2 cells were seeded. After 3 days with ?- or ?-ionone treatment, the cells remaining
in the upper compartment of insert were removed by cotton swabs, and those invaded
through the matrix were stained with 0.1 % crystal violet and counted under a light
microscope at five individual fields per insert. Results were presented as an average
of triplicate experiments.

Cell proliferation and growth assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay and anchorage-independent
growth assay were performed to measure cell proliferation following previously published
protocols 13]. Briefly, for MTT assay, cells were seeded in 24-well plates. After 3 days culture,
the medium was removed and the cells were washed once with PBS. 300 ?l of serum-free
medium and 30 ?l of MTT reagent (5 mg/ml) were added, and incubated for one and a
half hours in a humidified 5 % CO2 incubator at 37 °C. The absorbance was measured
using a BIORAD Microplate reader at wavelength of 570 and 620 nm. The different absorbance
values at 570 and 620 nm wavelength represented the direct correlation with number
of viable cells per well. For anchorage-independent growth assay: soft agar plates
were prepared in six-well plates with a bottom layer of 0.8 % Noble agar in serum-free
DMEM 2?×?10
⁴
cells mixed with 0.8 % Noble agar in 10 % fetal calf serum-supplemented DMEM were
seeded as the top agar layer onto the agar plates. Colonies were visualized after
six weeks culture by staining with 0.005 % crystal violet. Two wells were prepared
for each treatment and the experiments were repeated twice.

Western blot

Cells were lysed in a 500 ?l lysis buffer (PBS, 0.5 % Triton X-100, 5 mM EDTA, and
protease inhibitors) on ice for 30 min. The lysate was collected and cleared by centrifuging
at 12,000?g for 20 min at 4 °C. Protein concentration of supernatants was determined by the Bradford’s
method (Bio-Rad Laboratories protein assay, Hercules, CA, USA). 10 ?g of the sample
was separated by SDS-polyacrylamide gel electrophoresis and blotted as previously
described 14]. Specific antibodies against AR, PSGR, Phosphorylated P70 S6 kinase, phosphorylated
mTOR, and phosphorylated 4EBP1 were used for western blots (1:1000).

Statistical analysis

Statistical analysis was performed using the Prism 5 statistical package from GraphPad
Software, Inc. (La Jolla, CA). Data was analyzed and expressed as mean?±?S.E. Overall
survival (OS) was defined as the time from the date of diagnosis to the date of death
or final clinical follow-up. The Kaplan-Meier method was used to estimate survival
probabilities in patient subgroups. Significant difference of PSGR expression among/between
groups was determined by one-way analysis of variance or Student’s t-test. A p value less than 0.05 was considered statistically significant.