The p63 Protein Isoform ΔNp63α Inhibits Epithelial-Mesenchymal Transition in Human Bladder Cancer Cells: ROLE OF MIR-205

Mai N Tran,Woonyoung Choi,Matthew F Wszolek,Neema Navai,I-Ling C Lee,Giovanni Nitti,Sijin Wen,Elsa R Flores,Arlene Siefker-Radtke,Bogdan Czerniak,Colin Dinney,Michelle Barton,David J Mcconkey

doi:10.1074/jbc.m112.408104

Abstract

Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, "stemness," and drug resistance. EMT is typically characterized by the loss of the epithelial marker E-cadherin and increased expression of EMT-associated transcriptional repressors, including ZEB1 and ZEB2. The miR-200 family and miR-205 prevent EMT through suppression of ZEB1/2. p53 has been implicated in the regulation of miR-200c, but the mechanisms controlling miR-205 expression remain elusive. Here we report that the p53 family member and p63 isoform, ΔNp63α, promotes miR-205 transcription and controls EMT in human bladder cancer cells. ΔNp63α, E-cadherin and miR-205 were coexpressed in a panel of bladder cancer cell lines (n = 28) and a cohort of primary bladder tumors (n = 98). Stable knockdown of ΔNp63α in the "epithelial" bladder cancer cell line UM-UC6 decreased the expression of miR-205 and induced the expression of ZEB1/2, effects that were reversed by expression of exogenous miR-205. Conversely, overexpression of ΔNp63α in the "mesenchymal" bladder cancer cell line UM-UC3 induced miR-205 and suppressed ZEB1/2. ΔNp63α knockdown reduced the expression of the primary and mature forms of miR-205 and the miR-205 "host" gene (miR-205HG) and decreased binding of RNA Pol II to the miR-205HG promoter, inhibiting miR-205HG transcription. Finally, high miR-205 expression was associated with adverse clinical outcomes in bladder cancer patients. Together, our data demonstrate that ΔNp63α-mediated expression of miR-205 contributes to the regulation of EMT in bladder cancer cells and identify miR-205 as a molecular marker of the lethal subset of human bladder cancers.

Highlights

Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, “stemness,” and drug resistance
We report that the p53 family member and p63 isoform, ⌬Np63␣, promotes miR-205 transcription and controls EMT in human bladder cancer cells. ⌬Np63␣, E-cadherin and miR-205 were coexpressed in a panel of bladder cancer cell lines (n ‫ ؍‬28) and a cohort of primary bladder tumors (n ‫ ؍‬98)
Our data implicate ⌬Np63␣, the most abundant isoform of p63 expressed in Bladder cancer (BC), in the control of EMT

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cell lines were obtained from the MD Anderson Bladder SPORE Tissue Bank and cultured as in Ref. 19. EX-Z5740-M02) were transfected into cells using Lipofectamine 2000 SiRNAs were transfected into cells using Lipofectamine RNAiMAX RNA Isolation and Real-time Reverse Transcription PCR (qRT-PCR) Analysis—RNA was isolated from cells using the mirVanaTM miRNA isolation kit (Ambion/Life Techonologies). The AgPath-ID One-Step RT-PCR kit (Applied Biosystems/ Life Technology) was used for real-time reverse transcription PCR. Indirect staining was performed for P-cadherin using a polyclonal rabbit anti-P-cadherin antibody Precipitated DNA and the DNA input were amplified and analyzed by quantitative real-time PCR with SYBR Green qPCR Master Mix (Applied Biosystems/Life Techonologies) using the primers listed in supplemental Table 1. Statistical analyses were carried out using Splus 7 (Insightful Corp., Seattle, WA)

RESULTS

Findings

DISCUSSION