EZH2 Regulates Growth of Estrogen Receptor Negative Breast Carcinomas by Regulating BRCA1 Localization
ME Gonzalez, X Li, K Toy, M DuPrie, CG Kleer. University of Michigan
Background: EZH2 is an oncogene essential to promote proliferation and capable to trigger neoplastic conversion in the breast. EZH2 is overexpressed in breast carcinomas with high propensity to metastasize. We have reported that EZH2 promotes proliferation of breast cancer through downregulation of BRCA1 protein, but the mechanism is unknown. BRCA1 shuttles between the cytoplasm and nucleus to maintain genomic stability. AKT1 up regulation disrupts BRCA1 function by altering its nuclear localization and inducing genomic instability. We hypothesize that EZH2 regulates nuclear-cytoplasmic shuttling of BRCA1 in breast cells through AKT1.
Design: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells, and a lentivirus-mediated shRNA interference to inhibit EZH2 in two ER negative breast cancer cells, CAL51 and MDA-MB-231. The effects of EZH2 on in vitro and in vivo tumorigenicity were determined by Western blots, immunofluorescence, proliferation, FACS, and xenograft and transgenic mouse models.
Results: EZH2 knockdown in breast cancer cells decreased their in vivo tumorigenicity. EZH2 shRNA knockdown in CAL51 cells increased BRCA1 nuclear localization in early S phase, decreased cell growth by delaying cell cycle progression, and decreased tetraploidy. EZH2 overepression in MCF10A cells decreased BRCA1 nuclear localization, increased proliferation and caused mitotic spindle defects and tetraploidy. EZH2 overepression increased total and phoshporylated AKT1 at serine 473. Specific pharmacologic inhibition of AKT reverted the effect of EZH2 on BRCA1 nuclear localization, proliferation, mitotic defects and tetraploidy. We also found that EZH2 directly interacts with AKT1 protein. Mammary glands of EZH2 trasnsgenic mice show up-regulation of AKT1 and a decreased in nuclear BRCA1 protein compared to wild type mice.
Conclusions: EZH2 inhibition decreases growth of ER negative breast cancer in mice. EZH2 regulates the nuclear-cytoplasmic shuttling of BRCA1 protein and influences BRCA1 functions on proliferation, 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.
Tuesday, March 23, 2010 1:30 PM
Platform Session: Section H, Tuesday Afternoon