PPM1D as a Novel Biomarker for Prostate Cancer After Radical Prostatectomy

Materials and Methods

Patients and prostate specimens. A total of 234 PCa samples were collected from patients with an average age of 64.5 years (50-86 years) at the Department of Urology, Changhai Hospital, Second Military Medical University, China. All patients underwent radical prostatectomy from January 1997 to January 2011. The control group with BPH comprised of 80 age-matched patients, with an average age of 66.4 years (58-78 years), who were recruited during the same period. All slices from tumor samples were collected from areas of invasive adenocarcinoma and pathologically-identified according to hematoxylin and eosin staining. The tumor grade and clinical stage of these samples were assessed based on the 2002 TNM classification and Gleason system.

Follow-up. All patients were scheduled to have their serum prostate-specific antigen (PSA) level evaluated postoperatively every three months for the first year, every six months from the second to the fifth year and annually thereafter. Follow-up data were obtained by consulting the hospital medical records and the PCa database of our Department and by contacting the patients or their family members. Biochemical recurrence was defined as the sustained elevation of the serum total PSA level above 0.2 ng/ml on two or more occasions, and the date of biochemical recurrence was assigned as the date of the first value greater than 0.2 ng/ml. Follow-up time ranged from 3 to 176 months. This study was approved by the Second Military Medical University Ethical Committee (EC1008-32).

Immunohistochemistry. Tissue slices (4 μm) were de-paraffinized and incubated in water with H₂O₂ for 30 min to remove endogenous peroxidase activity. The sections were then immersed in 10 mM citrate buffer (pH=6) and heated in a microwave oven for 30 min. Sections were blocked with 5% normal goat serum in phosphate-buffered saline for 1 h and then incubated overnight with antibody to PPM1D (Santa Cruz, CA, USA) at 4°C. For the negative control, 5% normal goat serum was used to replace the primary antibody. Staining was detected using a standard avidin–biotin–peroxidase complex followed by hematoxylin counterstaining. Positive staining was defined as the presence of brown oxidized 3,3’-diaminobenzidine (DAB) in the given cellular compartments without background signal. The immunostaining scores of PPM1D were evaluated under a light microscope by two pathologists who were unaware of patients' clinical information and determined using a combined scoring system based on the sum of the staining intensity and the percentage of positively stained cells. Scores from 0-3 were given in terms of the staining intensity and the percentage of positively stained cells as follows: score 0, no staining or fewer than 10% of the tumor cells; score 1+, weak staining in 10% or more of the tumor cells; score 2+, moderate staining in 10% or more of the tumor cells; and score 3+, strong staining in 10% or more of the tumor cells. Scores 0 and 1+ were considered negative for PPM1D expression, while scores 2+ and 3+ were considered positive for PPM1D expression.

Cell culture and siRNA transfection. Human PCa cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection (ATCC) repository (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone, USA) at 37°C in an incubator with 5% CO₂. PPM1D-specific siRNA and non-specific control siRNA (both from Invitrogen, USA) were transfected into PC-3 and LNCaP cells with Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions.

Western blot. Total protein was isolated from PCa cells. Equal amounts of protein samples (60 μg) were subjected to a 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, USA). The membranes were blocked in 5% non-fat milk and then were incubated with primary antibody (anti-PPM1D, 1:1000; Abcam, CA, USA) or anti-GAPDH (1:2000; Santa Cruz, CA, USA) at 4°C overnight. The membranes were washed and incubated with specific peroxidase-conjugated secondary antibodies. Proteins were detected using the ECL system (Santa Cruz, CA, USA).

Cell vitality, invasion and migration assay. Cells were seeded into a 96-well plate at a density of 4000 cells per well and cultured for 5 days. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (Sigma-Aldrich, IL, USA) according to the manufacturer's protocol. Cell migration and invasion ability were measured by transwell assay. Subsequently, 1×10⁴ transfected cells were plated in the upper chambers of the transwell (Santa Cruz, CA, USA) in fetal bovine serum-free medium and 10% fetal bovine serum-containing medium was deposited in the lower chambers. For cell invasion assays, transwell membranes were coated with matrigel (BD Biosciences, NJ, USA) before plating cells. After 24 h, cell were removed from the upper chambers, whereas cells on the lower chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Five independent fields for each well were counted and photographed by microscope.

Statistical analysis. Pearson's χ² test was used to analyze the association between PPM1D expression and clinicopathological parameters. Survival and PSA recurrence curves were evaluated using the Kaplan–Meier method, and the log-rank test. All clinical parameters were analyzed with univariate and multivariate Cox proportional hazards models. Data were analyzed with analysis of variance among three groups. All statistical analyses were performed using the SPSS16.0. A value of p<0.05 was considered significant.