The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

<div><p>Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.</p> </div

PG promotes cell-cell adhesion in prostate cancer cell lines.

<p>A. Representative image of a dispase assay in PC3-M cells after transduction with PG OE adenovirus or control GFP adenovirus. B. Quantitations of dispase assays performed in triplicate; LNCaP, C4-2B, or PC3-M cells were plated in 6-well plates, and allowed to attach and spread. Cultures were transduced with GFP-containing adenovirus (GFP) or PG-containing adenovirus (PG OE), and after 24 hours the dispase assay was performed. Overexpression of PG in all 3 cell lines strengthens cell-cell adhesion. C. LNCaP cells were plated in 6-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool. The dispase assay was performed 96 hours after transfection. Suppression of PG expression results in a weakening of cell-cell adhesion in LNCaP cells. D. Representative image of a hanging drop assay in DU145 cells after transfection with control or PG siRNA pool. E–F. Quantitation of hanging drop assays performed in triplicate; C4-2B and PC3-M cells were transduced with GFP- or PG-containing adenoviruses (E), ARCaP E, LNCaP, DU145 and PC3 cells were transfected with control siRNA or PG siRNA pool (F). Aggregation assays were done 24 h (E), or 96 h (F) after the treatment. Graphs represent averages +/− SEM. *P<0.06; **P<0.005; ***P<0.0007, by paired Student t test.</p

PG-dependent Src activity is regulated by VN.

<p>A. Confocal images showing PG and pSrc expression in normal and malignant prostate tissue (using 68 cores from the prostate TMA). PG is in green and pSrc is red. Bar, 50 µm. B. Scatterplot demonstrating the inverse correlation between pSrc expression and PG expression in prostate tissue (using 68 cores). C. Confocal images showing PG and VN expression in normal and malignant tissue (T2 samples were shown for both pSrc and VN as representative of average expression levels in all malignant tissue samples). PG is in green and VN is red. Bar, 50 µm. D. Scatterplot showing the inverse correlation between VN expression and PG expression in prostate tissue (using 68 cores). E. Scatterplot showing the direct correlation between VN expression and pSrc in prostate tissue (using 68 cores). F. Western blot showing PG, pSrc, and Src expression in LNCaP cells transfected with control or PG siRNA plated on uncoated or VN-coated plastic. VN rescues Src activation in the absence of PG.</p

Model of the role of PG in prostate cancer adhesion and motility.

<p>Loss of PG results in a double negative hit on cell-cell junctions, while orchestrating alterations in extracellular matrix that promote motility. PG levels correspond directly to the strength of cell-cell adhesion in PCa cells. Down-regulation of PG leads to loss of cell-cell adhesion strength. Concomitantly, loss of PG causes up-regulation of VN and activation of Src. Together, these cell-cell and cell-ECM dependent effects of PG down-regulation lead to an increase in PCa cell motility.</p

Src regulates motility and PG down-regulates Src activity in prostate cancer.

<p>A–B. PC3-M, C4-2B (A), and LNCaP cells (B) were plated in 6 well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA (a pool of 4 sequences). PC3-M, C4-2B, and LNCaP cells were treated with medium containing PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the dispase assay. Inhibition of Src strengthens cell-cell adhesion in PCa cells in general and is able to rescue cell-cell adhesion in PG-deficient cells. C. PC3-M and ARCaP_M cells were plated in 24-well plates and allowed to attach and spread. The cells were treated with medium containing the selective Src- family kinase inhibitor PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the scratch wound assay. D. ARCaP_E cells were plated in 24-well plates and allowed to attach and spread. The cells were transduced with GFP-containing adenovirus or caSrc-containing adenovirus, and after 24 hours a scratch wound was made. Activity of Src is directly correlated with motility of PCa cells. E. ARCaP_M, PC3-M, and LNCaP cells were plated in 6-well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA pool and after 96 hours, the cells were lysed. ARCAP_M and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours the cells were lysed. The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against PG, pSrc, Src, and GAPDH. Levels of pSrc were determined by normalizing to Src levels for each cell line using Image J. Phosphorylation (activation) of Src is inversely correlated with PG levels. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized pSrc/Src ratio for the blot shown. The average ratio and standard deviation of pSrc/Src normalized for loading by GAPDH is as follows: ARCaP_M 0.6+/−0.2, LNCaP 1.7+/−0.2, PC3M 0.7+/−0.1. Graphs represent averages +/− SEM. *P<0.04; **P<003; ****P<0.0001, by paired Student t test.</p

Expression levels and localization of PG in prostate cancer cell lines.

<p>A. Prostate cancer cell lines were lysed and subjected to SDS-PAGE, followed by immunoblotting with antibodies against PG and GAPDH. PG levels were determined by normalizing to GAPDH levels for each cell line using Image J. The immunoblot shows that the expression of PG in prostate cancer cell lines inversely correlates with the degree of aggressiveness of the cell line. B. Prostate cancer cell lines were plated onto coverslips and allowed to attach and spread. Immunofluorescence was performed using antibodies against PG to demonstrate the localization of PG in ARCaP_E, ARCaP_M, LNCaP, C4-2B, DU145, PC-3, and PC3-M cell lines. The immunofluorescence shows weaker staining and a loss of cell-cell border localization of PG in the more aggressive cell lines (ARCaP_M, C4-2B, and PC3-M). Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG protein levels for the blot shown. All the PG protein levels were normalized to ARCaP_E levels within each blot. The average level and standard deviation of PG protein levels in panel A is as follows: ARCaP_E 1.0, ARCaP_M 0.3+/−0.2, LNCaP 1.5+/−0.5, C4-2B 0.5+/−0.2, DU145 3.4+/−1.2, PC-3 1.0+/−0.4, PC3-M 0.5+/−0.2.</p

PG alters the profile of expression of ECM molecules in PCa cell lines.

<p>A–E. ARCaP_E, ARCaP_M, LNCaP, C4-2B, and PC3-M cells were plated in 6-well plates and allowed to attach and spread. The cells were either transduced with GFP- or PG-containing adenovirus and were lysed after 24 hours (PC3-M, C4-2B, ARCaP_M); or the cells were transfected with control siRNA or PG siRNA pool and after 96 hours lysed (ARCaP_E, LNCaP, C4-2B). The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against VN (A), FN (B), LN (C), Col. I (D), Col IV (E) and GAPDH. Levels of ECM proteins were determined by normalizing to GAPDH levels for each cell line using Image J. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG siRNA/Con siRNA or PG OE/GFP ratio for the blot shown. The average ratio and standard deviation of GAPDH normalized PG siRNA/Con siRNA and PG OE/GFP is as follows: (A) ARCaP_E 3.1+/−1.3, LNCaP 2.0+/−0.9, PC3-M 0.3+/−0.1; (B) LNCaP 0.7+/−0.05, C4-2B KD 0.8+/−0.1, C4-2B OE 1.2+/−0.2, PC3-M 1.5+/−0.05; (C) ARCaP_M 0.6+/−0.1, PC3-M 0.4+/−0.3; (D) 0.3+/−0.1; (E) 0.4+/−0.2.</p

PG inhibits motility in prostate cancer cell lines.

<p>A. PC3-M and ARCaP_M were plated in 24-well plates and allowed to attach and spread. The cells were then transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours a scratch wound was made. Images were taken at 0 and 24 hours and the % wound closure was calculated by comparing the size of the wound at 24 hours to the 0 hour time point for each condition. Overexpression of PG suppresses motility in these two cell lines. B. ARCaP_E and C4-2B cells were plated in 24-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, the scratch wounds were made. Suppression of PG leads to an increase in motility in ARCaP_E and C4-2B cells. Scratch wound assay done in triplicate in prostate cancer cell lines after overexpression or knockdown of PG. C. ARCaP_M, C4-2B, DU145 and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours plated onto Matrigel coated transwell membranes for an invasion assay. Overexpression of PG suppresses invasion in these four cell lines. D. ARCaPE, LNCaP, C4-2B, DU145, PC-3 and PC3-M cells were transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, plated onto Matrigel coated transwell membranes for an invasion assay. Suppression of PG leads to an increase in invasion in these six cell lines. Invasion assays were done in triplicate. Average of the total number of invading cells is presented. Graphs represent averages +/− SEM. *P<0.1; **P<0.05; ***P<0.001, by paired Student t test.</p