EZH2-shRNA–mediated upregulation of p21waf1/cip1 and its transcriptional enhancers with concomitant downmodulation of mutant p53 in pancreatic ductal adenocarcinoma

PurposeEnhancer of zeste homologue 2 (EZH2), a component of the chromatin modification protein complex, is upregulated in pancreatic ductal adenocarcinoma (PDAC), whereas loss of p53 and its downstream target, p21waf1/cip1, is also observed frequently. We sought to investigate the role of the p53-p21waf1/cip1 pathway in relation to EZH2-mediated inhibition of PDAC.MethodsThe PANC-1 cell line was utilized in chromatin immunoprecipitation, gene profiling, Western blot, cell invasion, cell proliferation, and tumor xenograft assays.ResultsWestern blot analysis with antibodies that recognize both wild-type and mutant p53 did not show any alterations in band intensity; however, antibody that detects only mutant p53 showed a band of significantly lesser intensity with EZH2 knockdown. Western blot analysis further revealed a significant upregulation of p21waf1/cip1. Gene expression profile analysis indicated significantly enhanced transcripts of transcriptional inducers of p21waf1/cip1, with downregulation of mutant p53 transcript, corroborating the Western blot analysis. PANC-1 cells expressing EZH2-short hairpin RNA displayed markedly attenuated growth in SCID mice.ConclusionDownregulation of mutant p53 with concomitant enhanced expression of p21waf1/cip1 and its transcriptional trans-activators may contribute toward EZH2-mediated suppression of PDAC.