EZH2-shRNA-mediated upregulation of p21waf1/cip1 and its transcriptional enhancers with concomitant downmodulation of mutant p53 in pancreatic ductal adenocarcinoma.
OBJECTIVE
Enhancer 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, p21(waf1/cip1), is also observed frequently. We sought to investigate the role of the p53-p21(waf1/cip1) pathway in relation to EZH2-mediated inhibition of PDAC.


METHODS
The PANC-1 cell line was utilized in chromatin immunoprecipitation, gene profiling, Western blot, cell invasion, cell proliferation, and tumor xenograft assays.


RESULTS
Western 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 p21(waf1/cip1). Gene expression profile analysis indicated significantly enhanced transcripts of transcriptional inducers of p21(waf1/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.


CONCLUSIONS
Downregulation of mutant p53 with concomitant enhanced expression of p21(waf1/cip1) and its transcriptional trans-activators may contribute toward EZH2-mediated suppression of PDAC.
Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI; John D. Dingell VA Medical Center, Wayne State University, Detroit, MI. Electronic address: rbatchu@med.wayne.edu.
Ramesh B
RB
Batchu
Aamer M
AM
Qazi
Oksana V
OV
Gruzdyn
Assaad
A
Semaan
Shelly M
SM
Seward
Sreedhar
S
Chamala
Vasu B
VB
Dhulipala
David L
DL
Bouwman
Donald W
DW
Weaver
Scott A
SA
Gruber
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000818:Animals; D017209:Apoptosis; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D000071221:Enhancer of Zeste Homolog 2 Protein; D016158:Genes, p53 / Q000502:physiology*; D006801:Humans; D051379:Mice; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D063151:Polycomb Repressive Complex 2 / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2