Prostate Apoptosis Response-4 (PAR4) Suppresses Growth and Invasion of Breast Cancer Cells and Is Positively Associated with Patient Survival

Materials and Methods

Materials. Human breast cancer cell lines MDA-MB-231, MCF7 and ZR-75-1 were purchased from the European Collection of Cell Cultures (Salisbury, UK). The cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 10% foetal calf serum and antibiotics. Monoclonal mouse anti-PAR4 was obtained from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Unless otherwise stated, other materials and reagents were purchased from Sigma-Aldrich Ltd (Poole, Dorset, UK).

Collection of breast tissue samples. Breast cancer tissues (n=127) and normal background breast tissues (n=34) were collected during surgery and stored at −80°C until use. All patients were followed-up clinically after surgery, with a median follow-up period of 120 months (June 2004). Histopathological details were obtained from pathological reports and confirmed by a consultant pathologist (Table I). All protocols were reviewed and approved by the Bro Taf Health Authority local Research Ethics Committee and patients participated with informed consent.

RNA extraction, reverse transcription polymerase chain reaction (RT-PCR) and quantitative PCR. Total RNA was extracted from frozen tissues and cultured cells using TRI regent® (Sigma-Aldrich Ltd). The concentration of RNA was determined using a UV spectrophotometer (WPA UV 1101; Biotech Photometer, Cambridge, UK).

Reverse transcription was carried out using an RT kit with an oligo (dT) primer supplied by ABgene® (Life Technologies Ltd., Paisley, UK) using 0.5 μg of total RNA for each 20 μl RT reaction. The quality of cDNA was verified by examining a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Conventional PCR was performed with specific primers for PAR4. The conditions for the PCR were: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a final extension for 7 min at 72°C. The products were visualized on a 1.5% agarose gel after staining with ethidium bromide. PAR4 transcript levels in the breast tissue specimens were determined using real-time qPCR, based on the Amplifluor™ technology, as reported previously (21). Briefly, pairs of qPCR primers were similarly designed using the Beacon Designer™ software (PREMIER Biosoft, Palo Alto, CA, USA), but an additional sequence was added to the antisense primer. This is known as the Z sequence (5’-actgaacctgaccgtaca-3’), which is complementary to the universal Z probe (Intergen Inc., Oxford, UK). The reaction was carried out using iCycler iQ™ (Bio-Rad, Hemel Hempstead, Hertfordshire, UK), which is equipped with an optical unit that allows real-time detection of 96 reactions. The levels of the PAR4 transcript are reported here as the number of transcript copies per 50 ng RNA, generated from an internal standard, cytokeratin 19 (CK19) that was simultaneously amplified during the same qPCR. The sequences of the primers used in the current study are listed in Table II.

Immunohistochemical staining of PAR4. Frozen sections of breast tumours and background breast tissue were cut at a thickness of 6 μm. The sections were air-dried and fixed in a mixture of 50% acetone and 50% methanol. The sections were then placed in Tris-buffered saline buffer for 20 min to rehydrate. Sections were then incubated for 20 min in a 0.6% bovine serum albumin blocking solution and probed with the primary antibody to PAR4 (1:100 dilution) for 1 h at room temperature. Following extensive washing, sections were incubated for 30 min in the secondary biotinylated antibody (Multi Link Swine anti-goat/mouse/rabbit immunoglobulin; Dako Inc., Ely, Cambridgeshire, UK). Avidin/Biotin Complex (Vector Laboratories Ltd, Peterborough, UK) was then applied to the sections, followed by washing. The sections were subsequently incubated with the chromogen 3,3’-diaminobenzidine (Vector Laboratories Ltd) in the dark for 5 min, and were then counterstained in Gill's haematoxylin. The IHC stained sections were assessed by two researchers independently, the description of the PAR4 staining was agreed by both researchers with a final validation by a third researcher.

Western blot analysis. Cells were lysed in HCMF buffer containing 1% Triton, 0.1% sodium dodecyl sulfate, 2 mM CaCl₂, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin and 1 μg/ml aprotinin for 30 min before centrifuging at 13,000 × g for 10 min. Protein concentrations were quantified using a spectrophotometer (BioTek™ ELx800™; BioTek, Swindon, UK). Equal amounts of each protein sample (20 μg/lane) were separated in a 10% polyacrylamide gel. After electrophoresis, proteins were blotted onto nitrocellulose sheets. After blocking in 10% skimmed milk for 60 min, the blots were probed with the antibody against PAR4 and peroxidase-conjugated secondary antibody. Protein bands were developed using the Supersignal™ West Dura system (Pierce Biotechnology, Inc., Rockford, IL, USA), and photographed with an UVITech imager (UVITech, Inc., Cambridge, UK).

Overexpression of PAR4 in MCF-7 and MDA-MB-231 cells. The PAR4 overexpression vectors were constructed using a pEF6/V5/His vector and used to transfect MCF-7 and MDA-MB-231 cells. Following transfection, cells were selected using blasticidin (5 μg/ml). MCF-7PAR4exp, MDA-MB-231PAR4exp that overexpressed PAR4 and respective controls (MCF-7pEF and MDA-MB-231pEF), which carried empty plasmid vectors, were established. MCF-7WT and MDA-MB-231WT were designated for the wild-type cells.

In vitro cell growth assay. A standard procedure was used as previously described (20, 21). Cells were plated into a 96-well plate (2,500 cells/well). Cell growth was assessed after 1, 3 and 5 days. Crystal violet was used to stain cells, and absorbance was determined at a wavelength of 540 nm using a spectrophotometer (BioTek™ ELx800™; BioTek).

In vitro invasion assay. According to a standard procedure, transwell inserts with 8 μm pore size were coated with 50 μg Matrigel (BD Matrigel™ Basement Membrane Matrix; BD Bioscience, Oxford, UK) and air-dried. After rehydration, 20,000 cells were added to each well. After 96 hours, cells that had migrated through the matrix to the other side of the insert were fixed, stained and then counted under a microscope.

Cell-matrix adhesion assay. As previously described (22), 40,000 cells were added to each well of a 96-well plate which was pre-coated with Matrigel™ (5 μg/well). After 40 min of incubation, non-adherent cells were washed-off using balanced salt solution buffer. The remaining adhered cells were fixed, stained and then counted.

Tumour growth in an athymic mouse model. Female athymic nude mice (4-8 week old CD-1® Nude mice; Charles River Laboratories, Oxford, UK) were subcutaneously injected with MCF-7 breast cancer cells (1.0×10⁶ cells) in Matrigel™ (2.5 mg/ml). The mice were kept in sterilised, filtered cages under 12-h dark/12-h light standardized environmental conditions. They were weighed twice weekly in accordance with Home Office regulations. Tumour size was measured twice a week using digital callipers and calculated as volume (mm³)=0.512 × width² × length. This study was performed in strict accordance with the recommendations in the “Research and Testing Using Animals” guidance published by the UK Government Home Office (19). The study protocol and procedure (project licence no. 30/2591) were approved by the UK Government Home Office.

Statistical analysis. Statistical analyses were performed using Minitab® version 14 and SPSS version 18 statistical software packages (SPSS Inc., Chicago, IL, USA). Non-normally distributed data were assessed using the Mann–Whitney test, while the two sample t-test was used for normally distributed data. Kaplan–Meier survival analysis, Cox proportional hazards model and Spearman rank correlation coefficient were used to analyse data using SPSS. Differences were considered to be statistically significant at p<0.05.