Hierarchical clustering of immunohistochemical analysis of the activated ErbB/PI3K/Akt/NF-kappaB signalling pathway and prognostic significance in prostate cancer

Abstract

Background: The PI3K/Akt signalling pathway, induced by epidermal growth factor receptor (EGFR) and Her-2, is involved in the constitutive activation of NF-kappaB in prostate cancer cell lines. In this study, we extended the in vitro observation using an ex vivo model of prostate cancer tissues and assessed the prognostic significance of the PI3K/Ak/NF-kappaB signalling determinants.

Methods: We analysed a prostate cancer tissue microarray of 63 patients for the expression of total and activated EGFR, Her-2 receptors and the signalling molecules PTEN, phospho-PTEN, Akt, phospho-Akt and the NF-kappaB subunit p65. Data were analysed using Spearman's rho test, Kaplan-Meier curves and multivariate Cox regression analysis. In addition, a non-supervised hierarchical clustering analysis was applied to stratify patients according to prognostic groups in terms of risk of recurrence.

Results: The concomitant overexpression of activated EGFR and Her-2 was correlated with the nuclear expression of NF-kappaB. EGFR, phospho-EGFR, phospho-Her-2, ErbB3 and nuclear NF-kappaB were associated with the overall biochemical recurrence (BCR) of patients. The non-supervised hierarchical clustering analysis resulted in the separation of patients into five groups according to BCR.

Conclusions: These results validate the previous in vitro data on ErbB involvement in NF-kappaB activation and shows evidence for a significant role of ErbB/PI3K/Akt/NF-kappaB signalling in the progression of prostate cancer.

Figure 1

Representative staining for immunohistochemistry of EGFR, Her-2 and ErbB3 in prostate cancer tissues. (A, C) Low and moderate EGFR and phospho-EGFR cytoplasmic staining, respectively. (B, D) EGFR and phospho-EGFR strong cytoplasmic immunostaining, respectively. (E, G) Low and moderate staining for Her-2 and phospho-Her-2, respectively. (F) Strong cytoplasmic Her-2 staining. (H) Strong cytoplasmic staining with nuclear staining for phospho-Her-2. (I) Moderate cytoplasmic ErbB3 staining. (J) Strong cytoplasmic and moderate nuclear ErbB3 staining. Each individual marker is indicated. Magnification × 20.

Figure 2

Representative staining for immunohistochemistry of PI3K/Akt/PTEN signalling pathway markers in prostate cancer tissues. (A, B) Low and strong cytoplasmic PI3K immunostaining. (C) Low cytoplasmic staining for PTEN. (D) Strong cytoplasmic staining and nuclear staining of PTEN. (E) Phospho-PTEN low cytoplasmic staining. (F) Strong cytoplasmic staining with high nuclear staining of phospho-PTEN. (G) Moderate cytoplasmic p-Akt staining. (H) Strong cytoplasmic staining with nuclear p-Akt (top right corner). Each individual marker is indicated. Magnification × 20.

Figure 3

Representative staining for immunohistochemistry of p65/RelA in prostate cancer tissues. (A) Strong cytoplasmic staining with no nuclear staining of NF-κB p65 subunit. (B) Moderate and strong cytoplasmic staining with nuclear NF-κB p65 subunit. (C) Low and moderate phospho-NF-κB p65 subunit. (D) Moderate and strong phospho-NF-κB p65 subunit cytoplasmic staining with nuclear staining. Each individual marker is indicated. Magnification × 20.

Figure 4

Kaplan–Meier analysis of the ErbB/PI3K/Akt/NF-κB signalling pathway. Kaplan–Meier curves of BCR-free survival in 63 patients with prostate cancer. Each individual marker is indicated in the graph. Significance (P) is indicated by log rank.

Figure 5

EGFR, Her-2 and PI3K/Akt/NF-κB signalling pathways in an unsupervised hierarchical analysis and association with outcome. (A) Unsupervised hierarchical clustering. Each column represents a patient. Each row represents a marker staining as indicated on the left side. Grey colour represents weak marker staining, black represents strong marker staining. (B) Kaplan–Meier curve analysis. The five groups of patients identified by unsupervised clustering (clusters 1, 2, 3, 4 and 5 in Figure 4A) were analysed using Kaplan–Meier analysis for biochemical recurrence-free survival. P=0.003, significance (P) is indicated by log rank.