Activation of nuclear factor-kappaB in human prostate carcinogenesis and association to biochemical relapse

Abstract

Nuclear factor (NF)-kappaB/p65 regulates the transcription of a wide variety of genes involved in cell survival, invasion and metastasis. We characterised by immunohistochemistry the expression of NF-kappaB/p65 protein in six histologically normal prostate, 13 high-grade prostatic intraepithelial neoplasia (PIN) and 86 prostate adenocarcinoma specimens. Nuclear localisation of p65 was used as a measure of NF-kappaB active state. Nuclear localisation of NF-kappaB was only seen in scattered basal cells in normal prostate glands. Prostatic intraepithelial neoplasias exhibited diffuse and strong cytoplasmic staining but no nuclear staining. In prostate adenocarcinomas, cytoplasmic NF-kappaB was detected in 57 (66.3%) specimens, and nuclear NF-kappaB (activated) in 47 (54.7%). Nuclear and cytoplasmic NF-kappaB staining was not correlated (P=0.19). By univariate analysis, nuclear localisation of NF-kappaB was associated with biochemical relapse (P=0.0009; log-rank test) while cytoplasmic expression did not. On multivariate analysis, serum preoperative prostate specific antigen (P=0.02), Gleason score (P=0.03) and nuclear NF-kappaB (P=0.002) were independent predictors of biochemical relapse. These results provide novel evidence for NF-kappaB/p65 nuclear translocation in the transition from PIN to prostate cancer. Our findings also indicate that nuclear localisation of NF-kappaB is an independent prognostic factor of biochemical relapse in prostate cancer.

Figure 1

Upper panels. Immnunohistochemical stain for p65 in prostate cancer tissues. (A) Prostate cancer staining with the antibody to NF-κB/p65 (C-20, sc-372, Santa Cruz) dilution 1 : 180, magnification × 400. (B) Immunoreactivity was blocked by preabsortion of the primary antibody with the antigen peptide (sc-372 P, Santa Cruz), magnification × 400. Lower panels. Immunofluorescence stain for p65 prostate cancer cells (PC-3). (C) Inmunofluorescence staining with the antibody to NF-κB p65 (C-20, Santa Cruz, dilution 1 : 180) in nonstimulated human prostate cancer cells (PC-3) shows only cytoplasmic staining. (D) PC-3 cells stimulated with TNF-α 100 ng ml⁻¹ for 10 min exhibit nuclear staining.

Figure 2

Patterns of NF-κB immunoreactivity in prostate tissues (diaminobenzidine with hematoxylin counterstaining; magnification, × 400). (A) Cells of normal prostate glands with weak cytoplasmic NF-κB staining while nuclear NF-κB staining was only seen in scattered basal cells (arrows). (B) High-grade prostate intraepithelial neoplasia with intense cytoplasmic NF-κB staining. No nuclear immunoreactivity was seen in cells of PIN lesions. (C) Prostate cancer specimen with diffuse cytoplasmic NF-κB staining but no nuclear immunoreactivity. (D) Prostate cancer specimen with both cytoplasmic and nuclear NF-κB staining in tumour cells.

Figure 3

Upper panels, Kaplan–Meier survival curves for cytoplasmic and nuclear NF-κB expression in prostate adenocarcinomas. (A) No association between NF-κB cytoplasmic expression and biochemical disease recurrence was detected (P=0.74). (B) Nuclear expression of NF-κB was associated to the chance of biochemical disease-free survival. Patients with nuclear NF-κB had a higher risk of biochemical disease recurrence compared with those whose primary tumours did not have nuclear NF-κB (P=0.0009). Lower panels, Kaplan–Meier survival curves for NF-κB nuclear expression combined with presurgical serum prostate specific antigen (PSA) levels and Gleason score. (C) Patients with positive nuclear NF-κB expression and both (PSA⩾10 ng ml⁻¹ and gleason ⩾7) adverse prognostic factors had a greater chance of biochemical disease recurrence than those who presented one (PSA⩾10 ng ml⁻¹ or gleason ⩾7) or no (PSA⩽10 ng ml⁻¹ and gleason⩽6) adverse independent prognostic factors (P=0.0032). (D) Patients with negative nuclear NF-κB expression, PSA<10 ng ml⁻¹ and Gleason⩽6 presented had a better prognosis compared to patients with negative nuclear NF-κB expression and one (PSA⩾10 ng ml⁻¹ or gleason⩾7) or both (PSA⩾10 ng ml⁻¹ and gleason⩾7) adverse independent prognostic factors (P=0.046).