Evaluation of Kallikrein-related Peptidase 5 Expression and its Significance for Breast Cancer Patients: Association with Kallikrein-related Peptidase 7 Expression

Patients and Methods

Study population. The study group consisted of 74 patients with breast malignancies who underwent surgery for primary breast cancer at Saint Savvas Oncologic Hospital of Athens and 6 normal breast tissues. No chemotherapy or radiotherapy had been administered before surgery. Histological diagnoses and grading of the tumors were made based on the revised World Health Organization (WHO) classification for breast tumors. Cancer and normal tissues were evaluated by eosin-hematoxylin staining of paraffin sections. Patients' mean age was 64.2±1.6, with a range of 35-88 years. Sixteen patients (22%) received no adjuvant treatment, 22 (30%) received tamoxifen and 18 (24%) received chemotherapy with or without tamoxifen. Estrogen (ER) and progesterone receptor (PR) status was established as previously described (27). Clinical and pathological information documented at the time of surgery included stage and grade of the disease, histological type, size and nodal status of the tumors and the existence of ER and PR. The staging of the tumors followed the TNM system. The agreement of the Institute's Ethics Committee for the scientific analysis of tumor tissues was available, as well as patients' written informed consent. Investigations were carried out in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983.

Cell lines and culture conditions. Human breast cancer cell lines MCF-7, MDA-MB-435, BT-20, the ovarian cancer cell line CaOV-3 and the prostate cancer cell line PC-3 were obtained from the American Tissue Culture Collection and the breast cancer cell line MCF-10A was provided by the Department of Pharmacology, Wayne State University, Detroit, USA. All the culture media were obtained from Invitrogen (Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum, as well as 40 mg/l gentamicin sulfate. The media for MCF-7 were additionally supplemented with 10 ng/ml insulin whereas the media for MCF-10A were additionally supplemented with 10 ng/ml insulin, 20 ng/ml EGF, and 1.3 nM hydrocortisone. All the cells were grown at 37°C in an atmosphere of 5% CO₂.

RNA isolation – semi-quantitative RT-PCR. Upon collection, the breast tissues were snap frozen in liquid nitrogen and subsequently kept at −80°C until required. The tissues were pulverized and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The purity and concentration of RNA were determined by spectrophotometric methods. Two micrograms of total RNA were reverse-transcribed into first-strand cDNA using Superscript™ pre-amplification system (Invitrogen), following the manufacturer's instructions. The integrity of cDNA was examined by PCR amplification of GAPDH housekeeping gene, as previously described (27). In order to optimize PCR conditions, different quantities of cDNA (0.005-2 μl) from the CaOV-3 ovarian cancer cell line were amplified under exponential, non-saturating conditions, for 27, 31, 35, 37 and 40 cycles to confirm that amplification was in the linear range and to determine the appropriate cycle number for semi-quantitative PCR as described previously (28). For the amplification of KLK5, the forward primer anneals to exon 3 and the reverse primer anneals to exon 5 (forward 5’-CCA CTA CTC CCT GTC ACC AG-3’; reverse 5’-GTA ATC TCC CCA GGA CAC GA-3’), detecting the classical KLK5 form as well as splice variants 1 and 2 and giving an amplicon of 435 bp. For the amplification of GAPDH (amplicon 233 bp) the primers used were: forward 5’-ATG GGG AAG GTG AAG GTC G-3’; reverse 5’-GGG TCA TTG ATG GCA ACA ATA TC-3’. After optimization, PCR was carried out in a 20 μl reaction mixture containing 0.8 μl of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 mM dNTPs (deoxynucleoside 5’-triphosphate), 5 μM of each primer and 2.5 units of Taq DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) on a thermal cycler (MJ Research, Waltham, Massachusetts, USA). The cycling conditions for KLK5 were: a denaturation step at 94°C for 5 min, followed by 36 cycles of 94°C for 30 sec, an annealing step at 68°C for 50 s and 72°C for 45 s and a final extension step at 72°C for 10 min. The cycling conditions for GAPDH were: a denaturation step at 95°C for 10 min, followed by 30 cycles of 94°C for 30 s, an annealing step at 60°C for 1 min and 72°C for 1 min and a final extension step at 72°C for 8 min. Equal quantities of PCR products for the KLK5 and GAPDH genes were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. The primers spanned more than two exons to avoid contamination by genomic DNA. For the study of KLK7 expression the same primers and conditions as those we reported previously were used (25).

Following densitometric measurements of the band intensities using a Gel Logic 100 Imaging System and 1D Image Analysis Software, version 3.6 (Eastman Kodak Company, Rochester, NY, USA), the ratio of KLK5 and KLK7 to GAPDH band intensity was calculated. Based on this ratio, KLK5 and KLK7 expression was characterized as low or high compared to the ratio for non-cancer samples. A cut-off value of the mean ratio for non-cancer samples +2SD was calculated. KLK5 and KLK7 expression in samples with ratios higher than this value was considered high, whereas KLK5 and KLK7 expression in samples with ratios less than or equal to this value was characterized as low, as previously described (28). Expression analysis was performed twice for each sample. The identity of PCR products was verified by sequencing with an automated DNA sequencer.

Steroid hormone receptor analysis. Steroid hormone receptors were quantified as described elsewhere (29). The results of the dual ligand binding assay, in which dextran-coated charcoal was used to separate bound ligand from free ligand, were interpreted by Scatchard analysis. Tumors with ER and PR concentrations of ≤10 fmol/mg protein were characterized as negative, whereas concentrations of 10-300 fmol/mg protein were characterized as positive.

Statistical analysis. Associations between KLK5 status and qualitative variables were analyzed using the Chi-square test or the Fisher's exact test, where appropriate. A Cox proportional hazard regression model was developed to evaluate the association (i.e. hazard ratio and its confidence interval) between the potential prognostic marker and disease-free survival (DFS) or overall survival (OS). This analysis was conducted at both univariate and multivariate levels. Survival analysis was performed by constructing Kaplan-Meier DFS and OS curves for patients with KLK5 status low or high and the Wilcoxon signed ranks test was used to compare survival between subgroups of patients. DFS was defined as the time between the date of surgical removal of the primary tumor and the date of the first documented evidence of relapse. OS was defined as the time interval between the date of surgery and the date of death, or the date of last follow-up for those who were alive at the end of the study. Associations between KLK5 expression status and hormone receptor status were evaluated by the use of Mann-Whitney test. Finally, association between KLK5 and KLK7 expression was studied by the use of Fisher's exact test.