Desmosomal plakophilins in the prostate and prostatic adenocarcinomas: implications for diagnosis and tumor progression

Abstract

The plakophilins, members of the armadillo-repeat family, consist of three different proteins (PKP1-3) that are specifically recruited to desmosomal plaques in a highly cell type-specific manner. Using immunofluorescence, immunoelectron microscopy, and immunoblot, we found that all three plakophilins occurred in luminal and basal cells of the pseudostratified prostate epithelium. The analysis of 135 cases of prostatic adenocarcinomas grouped into tumors with low (Gleason score < or = 6), intermediate (Gleason score 7), and high Gleason score (8 < or = Gleason score < or = 10) showed that the expression of PKP1 was reduced or lost in adenocarcinomas with high Gleason scores. The expression of PKP2 was unchanged in all prostatic adenocarcinomas analyzed. In contrast, PKP3 expression was increased in carcinomas with high Gleason scores in comparison with carcinomas with low Gleason scores. In DU 145 cell lines with either overexpression or knockdown of PKP3, both imbalances resulted in fewer desmosomal cell contacts. In addition, overexpression of PKP3 in DU 145 cells led to an augmentation in proliferation rate. Our data imply that both loss of PKP1 and up-regulation of PKP3 expression are biologically important events in prostate cancer and are associated with a more aggressive phenotype.

Figure 1

Immunofluorescence results of double-labeling experiments on cryostat sections throughout the human prostate. The localization of plakophilins PKP1-3 (A, red, PKP2; D, red, PKP3; and G, red, PKP1) is compared with that of desmoplakin (B and E, green, DP) or desmoglein (H, green, DSG). The corresponding merged pictures are also shown (C, F, and I). Note the punctate staining and the pronounced localization at the apical portion of the basolateral membranes typical for desmosomes in polar epithelia. In higher magnifications (D–I), basal cells occur also positive for plakophilins. Abbreviation: L, lumen. Scale bars = 20 μm.

Figure 2

Detection of PKP1 on formaldehyde-fixed, paraffin-embedded tissues of tumor-free human prostate (A, D, and G) compared with the localization of keratin 14 (B and E, K14) and keratin 8 and keratin 18 (H, K8/18). In D–F, a higher magnification of the glandular epithelium is shown. The merged pictures are given in (C, F, and I). Note PKP1 is detectable in both types of cells, luminal and basal cells. Abbreviations: L, lumen; B, blood vessel. Scale bars: 20 μm (C and I); 10 μm (F).

Figure 3

Conventional electronmicroscopy of glandular epithelial cells of human prostate (A) and immunoelectron microscopic localization of plakophilins (immunogold reactions, followed by silver enhancement) to desmosomes using antibodies specific for PKP1 (B), PKP2 (C), or PKP3 (D). Note the prominent decoration of the electrodense cytoplasmic plaques of desmosomes with all three antibodies specific for PKP1-3. Abbreviations: D, desmosome; N, nucleus. Scale bars: 500 nm (A); 200 nm (B–D).

Figure 4

Immunoblot identification of plakophilins PKP1-3 in various cultured prostatic cell lines and human prostate tissue. The reactions are shown from following total lysates: human prostate (lane 1), BPH-1 (lane 2), DU 145 (lane 3), and LNCaP (lane 4). As a loading control, an immunoblot for β-actin (actin) was performed. Molecular weight markers are as indicated in kilodaltons in the left margin.

Figure 5

Occurrence of plakophilins (PKP1, PKP2, and PKP3) in prostatic adenocarcinoma in correlation with Gleason scores (low: Gleason score 3 to 6; intermediate: Gleason score 7; high: Gleason score 8 to 10). The percentages of negative (white color), inhomogeneous (light gray color), and positive (dark gray color) cases are given.

Figure 6

Detection of plakophilins on formaldehyde-fixed, paraffin-embedded tissue of prostatic adenocarcinoma grouped in low (A, D, G, and J), intermediate (B, E, H, and K), and high (C, F, I, and L) Gleason score by immunofluorescence. H&E is shown for a representative example of low (A, Gleason score 3 to 6), intermediate (B, Gleason score 7), and high (C, Gleason score 8 to 10) Gleason score. Immunolocalizations of PKP1 (D–F), PKP2 (G–I), and PKP3 (J–L) are presented. Note the heterogeneous detection of PKP1 in intermediate Gleason score and the loss of PKP1 in high Gleason score. Scale bars = 50 μm.

Figure 7

Detection of PKP1 on formaldehyde-fixed, paraffin-embedded tissue of an adenocarcinoma of the human prostate (A and C, PKP1) compared with the localization of keratin 8 and keratin 18 (B and D, K8/K18). Note the loss of PKP1 localization in adenocarcinoma of the prostate with high Gleason pattern. Scale bars = 50 μm.

Figure 8

Influence of overexpression (D–F, PKP3-EGFP) or knockdown (G–I, shPKP3-1350) of PKP3 on DU 145-cultured cells compared with untreated cells (A–C, DU 145). The localization of endogenous PKP3 (A and G, PKP3) using monoclonal antibody clone PKP3-270.6.2 or exogenous PKP3 (D, PKP3-EGFP) is compared with the desmosomal marker desmoglein (B, E, and H, DSG), using desmoglein-specific antibody rb5. The corresponding merged pictures are shown in C, F, and I. Diffuse cytoplamics staining in G arises from the exogenous EGFP expression introduced with the shPKP3-1350 cDNA construct. Note an imbalance of PKP3 expression in DU 145 cells results in fewer desmosomes. Scale bars = 20 μm.

Figure 9

Comparison of the cellular proliferation of untreated DU 145 cells (DU 145) and DU 145 cell lines with knockdown (shPKP3-1350) or overexpression (PKP3-EGFP) of PKP3 as measured by BrdU incorporation. For experimental details, see Materials and Methods. The data represent the mean cumulative values of three independent experiments including SD. The absorbance obtained for DU 145 cells was set as reference point 1.0. A significant difference (*P < 0.05) compared with DU 145 cells was observed for PKP3-overexpressing cells as assessed with Student’s t-test for unpaired experiments.