Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential

Abstract

The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with < 1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.

Figure 1

Western blot analysis showing endogenous levels of E-cadherin, N-cadherin, plakoglobin and β-actin expression in a representative panel of bladder carcinoma cell lines. Lane 1, RT4; lane 2, EJ; lane 3, 5637, lane 4, RT112; lane 5, HT1376; lane 6, HU456; lane 7, KK47; lane 8, UM-UC-3; lane 9, SCaBER; lane 10, J82; lane 11, TCCSUP; lane 12, BC16. Note the cell lines TCCSUP, J82, UM-UC-3 and EJ express N-cadherin in the absence of E-cadherin and display correspondingly low levels of plakoglobin.

Figure 2

Western blot analysis showing EJ and J82 transfectants probed for expression of β-catenin, N-cadherin, plakoglobin and β-actin. Lanes 1–3 in each panel represent three clones expressing high levels of plakoglobin following transfection. Lanes 4–6 represent three control clones harboring the neomycin-resistance gene.

Figure 3

Parental cell lines (A) J82 and (B) EJ displayed no detectable staining of plakoglobin in immunohistochemistry. In J82 transfectants (C) cytoplasmic with limited membranous plakoglobin was observed. Less than 1% of transfectants displayed nuclear localization of plakoglobin. In EJ transfectants (D) cytoplasmic and membranous localization of plakoglobin was recorded with no detectable nuclear plakoglobin.

Figure 4

In vitro migration and invasion assay results showing decreased migration of plakoglobin transfectants with no significant alteration in the invasive capacity as compared to neomycin controls.

Figure 5

siRNA directed against plakoglobin downregulates protein expression. EJ and J82 cells were transfected with siRNA directed against plakoglobin or control transfected with a scrambled siRNA. Cells were lysed 48-h post-transfection and Western blots were probed with anti-plakoglobin or anti-β-actin antibody.

Figure 6

Migration assay of EJ and J82 cells transfected with plakoglobin siRNA or control transfected controls harbouring scrambled siRNA sequence. The cells transfected with siRNA directed against plakoglobin displayed a statistically significant increase in migration capacity compared to the mock-transfected controls. Numbers are expressed as percent control where mock-transfectants were considered 100%.

Figure 7

Western blot analysis showing desmocollin-2/3 and desmoglein-2 expression in a panel of bladder carcinoma cell lines. Note that cell lines expressing reduced levels of plakoglobin (Figure 1) also show reduced expression of desmoglein and desmocollin. Lane 1, EJ; lane 2, 5637; lane 3, J82, lane 4, TCCSUP; lane 5, T24; lane 6, SCaBER; lane 7, RT112; lane 8, RT4; lane 9, PSI; lane 10, BC16.1; lane 11, CUBIII; lane 12, EJ; lane 13, HT1197; lane 14, HT1376; lane 15, HU456; lane 16, UM-UC-3; lane 17, KK47; lane 18, MGHU-1. Cell lysate from the EJ cell line was included in all gels.

Figure 8

Western blot analysis showing total cell lysates from EJ plakoglobin transfectant clones (lanes 1–3) and neomycin control cell lysates (lanes 4–6) probed for desmoglein-2, plakoglobin and β-actin.