Enhancer of Zeste homologue 2 (EZH2) regulates BRCA1 transiently nuclear export during the cell cycle, and induces tetraploidy of breast cells through AKT1.

MARIA E GONZALEZ; XIN LI; KATHY TOY; MATTEW DU PRIE; ALEJANDRA C VENTURA; HEATHER KRUGER; CELINA KLEER

Conferencia; 2010 AACR Annual Meeting; 2010

EZH2 is overexpressed in breast carcinomas with high propensity to metastasize. EZH2 is an oncogene essential to promoteproliferation and capable to trigger neoplastic conversion in the breast. We have reported that EZH2 promotes proliferationof breast cancer through downregulation of BRCA1 protein, but the mechanism is unknown. BRCA1 shuttles between thecytoplasm and nucleus to maintain genomic stability. AKT1 up regulation disrupts BRCA1 function by altering its nuclearlocalization and inducing genomic instability. We hypothesize that EZH2 regulates nuclear-cytoplasmic shuttling of BRCA1in breast cells through AKT1.Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammarycells and a lentivirus-mediated shRNA interference targeting EZH2 model in CAL51 breast cancer cells (tetraploid,hormone receptor negative, and with intact p53 and BRCA1 genes). Our recently developed EZH2 transgenic mouse modelwas analyzed to determine the in vivo relevance of our in vitro data obtained by Western blots, immunofluorescence,proliferation, and FACS.Results: EZH2 shRNA knock-down in CAL51 cells increased BRCA1 nuclear localization in early S phase, decreased cellgrowth by delaying cell cycle progression, and tetraploidy. EZH2 overexpression in MCF10A cells decreased BRCA1nuclear localization, increased proliferation and caused mitotic spindle defects and tetraploidy. EZH2 overexpressionincreased total and phoshporylated AKT1 at serine 473. Specific pharmacologic inhibition of AKT reverted the effect ofEZH2 on BRCA1 nuclear localization, proliferation, mitotic defects and tetraploidy. We also found that EZH2 directlyinteracts with AKT1 protein. Mammary glands of EZH2 transgenic mice show up-regulation of AKT1 and a decreased innuclear BRCA1 protein compared to wild type mice.Conclusion: EZH2 regulates the nuclear-cytoplasmic shuttling of BRCA1 protein and influences BRCA1 functions onproliferation, mitotic spindle control, and maintenance of genomic stability in benign breast cells and in breast cancer cells.The regulation of BRCA1 localization and function by EZH2 requires AKT.