Figure 5.

ATDC overexpression results in epigenetic silencing of PTEN expression by up-regulation of DNMT3A. (A) Immunohistochemical staining reveals loss of PTEN expression in CAG-ATDC bladder tumors. Bar, 100 μm. (B) Western blotting of total protein extracts from normal bladder tissues (N1, N2) from control littermates or invasive bladder tumors (T1, T2, T3) from CAG-ATDC transgenic mice confirms loss of PTEN expression in ATDC-expressing tumors. (C) Methylation-specific PCR assay (MSP) analysis of PTEN promoter from genomic DNA derived from CAG-ATDC invasive bladder tumors demonstrates PTEN promoter methylation in tumors. N1 and N2: Normal bladder from control littermates; T1, T2 and T3: invasive bladder tumors from CAG-ATDC mice. (D) Western blotting for ATDC, PTEN and DNMT3A in lysates derived from UC9 cells transfected with control or ATDC shRNA demonstrates decreased DNMT3A and increased PTEN expression. β-actin serves as a loading control. (E) Overexpression of ATDC in the immortalized urothelial SV-HUC1 cells increases DNMT3A and decreases PTEN expression (n=4). (F) Knockdown of DNMT3A in UC9 cells increases PTEN expression. (F–H) Knockdown of DNMT3A in UC9 cells reduces cellular invasion and proliferation (n = 3, mean±SE, *p < 0.05 vs control shRNA). (I) Western blotting of lysates from UC9 cells transfected with either control, ATDC or PTEN targeting shRNA or PTEN expression vector demonstrates silencing of ATDC and/or PTEN by shRNA and recovery of PTEN expression by ectopic expression. (J) Silencing of ATDC expression reduces invasion which is restored by silencing of PTEN. Ectopic of expression of PTEN decreases invasion. (n =3, mean±SE, *p < 0.05 for groups indicated with asterisks when compared to control shRNA group). (K) Representative images depicting ATDC, DNMT3A and PTEN expression in normal human urothelium and invasive human bladder cancer. (n=120, ATDC-DNMT3A p value = 0.03, ATDC-PTEN p value = 0.07).