BACKGROUND: E- and P-cadherin are expressed in normal bladder mucosa where E-cadherin is recorded throughout all bladder epithelial cell layers with P-cadherin localized to the basal cell layer. Loss or reduction of E-cadherin expression in bladder tumorigenesis is linked to the acquisition of invasive potential and has been reported to have prognostic significance in bladder cancer. In contrast, little is known of the expression profile or possible role of P-cadherin in bladder tumor progression. In this study we have investigated the function and role of P-cadherin in bladder tumorigenesis.
METHODS: Forced expression of P-cadherin in bladder carcinoma cell lines, EJ and UM-UC-3, was achieved using lipofection. Immunoprecipitation of P-cadherin with the cadherin complex was performed following immunoprecipitation of β-catenin followed by probing for P-cadherin in western blot analysis. Knockdown of P-cadherin expression in RT112 and CUBIII bladder carcinoma cells was performed using targeted siRNA with oligofectamine, using a scrambled siRNA sequence as a control. Changes in the expression profiles of E-, N-cadherin, α, β, γ, and p120 catenins, focal adhesion kinase (FAK) and signaling molecules including MAPK, AKT and STAT’s, were determined using Western blot analysis and immunocytochemistry (ICC). Alterations in migration or invasive potential were established using standard in vitro assays.
RESULTS: Novel expression of P-cadherin in EJ and UM-UC-3 cells was confirmed in western blot analysis showing concomitant up-regulation of β-catenin and plakoglobin in EJ/P-cadherin transfectants, with no change in N-cadherin, α-catenin, p120^ctn, or the expression or phosphorylation status of AKT, MAPK, FAK, STAT1 and 3. In UM-UC-3/P-cadherin transfectants α-catenin was recorded up-regulated accompanied by reduced expression of FAK. ICC demonstrated mainly cytoplasmic localization of P-cadherin in transfectants with limited evidence of membranous P-cadherin. However, P-cadherin co-precipitated with β-catenin in immunoprecipitation. An assessment of the migration and invasive potential of transfectanted cells revealed enhanced migration in EJ and UM-UC-3/P-cadherin transfectants (>300%) with no detectable change in invasion. SiRNA knockdown of P-cadherin in the same transfectants reduced the migration potential to control levels, demonstrating a direct cause and effect. Use of siRNA to knockdown constitutively expressed P-cadherin in bladder carcinoma cell lines co-expressing E-cadherin (RT112, CUBIII) resulted in no change in expression of alternative elements of the cadherin complex. However, cells showing reduced expression of P-cadherin following siRNA knockdown displayed enhanced migratory potential in in vitro assays.
CONCLUSIONS: In this study we have shown that P-cadherin plays a role in the control of migratory but not invasive behavior of bladder carcinoma cells. Interestingly, enhanced migration was recorded in cells representative of invasive tumors following the introduction of P-cadherin and in cells representative of superficial lesions following knockdown of constitutively expressed P-cadherin. Although the mechanism by which the same migrational behavioral change was recorded in these two models with opposite effects on P-cadherin expression is not known, it is clear from these experiments that P-cadherin is a primary modulator of migration in bladder epithelial cells. P-cadherin may be important in the migration events associated with differentiation in normal bladder mucosa and in late-stage events in bladder tumorigenesis.