Increased levels of EZH2, a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER negative breast cancer progression, EZH2 expression was inhibited in ER negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G2/M cell cycle transition, while not affecting apoptosis. In vivo, EZH2 down-regulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown up regulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 down-regulation in proliferation, G2/M arrest, and on the levels of hyperphosphorlated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER negative breast carcinomas show significant overexpression of EZH2 and down-regulation of BRCA1 proteins. Taken together, we show that EZH2 plays a role in ER negative breast cancer progression in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER negative breast cancer progression.

Increased levels of EZH2, a critical regulator of cellular memory, signal the presence of metastasis and poor outcome in breast cancer patients. High levels of EZH2 are associated with nuclear pleomorphism, lack of estrogen receptor expression, and decreased nuclear levels of BRCA1 tumor suppressor protein in invasive breast carcinomas. The mechanism by which EZH2 overexpression promotes the growth of poorly differentiated invasive carcinomas remains to be defined. Here, we show that EZH2 controls the intracellular localization of BRCA1 protein. Conditional doxycycline-induced upregulation of EZH2 in benign mammary epithelial cells results in nuclear export of BRCA1 protein, aberrant mitoses with extra centrosomes, and genomic instability. EZH2 inhibition in CAL51 breast cancer cells induces BRCA1 nuclear localization and rescues defects in ploidy and mitosis. Mechanistically, EZH2 overexpression is sufficient for activation of the phosphoinositide 3-kinase/ Akt (PI3K/Akt) pathway specifically through activation of Akt isoform 1. EZH2-induced BRCA1 nuclear export, aneuploidy, and mitotic defects were prevented by treatment with the PI3K inhibitors LY294002 or wortmannin. Targeted inhibition of Akt-1, Akt-2, and Akt-3 isoforms revealed that the EZH2-induced phenotype requires specific activation of Akt-1. The relevance of our studies to human breast cancer is highlighted by the finding that high EZH2 protein levels are associated with upregulated expression of phospho-Akt-1 (Ser473) and decreased nuclear expression of phospho-BRCA1 (Ser1423) in 39% of invasive breast carcinomas. These results enable us to pinpoint one mechanism by which EZH2 regulates BRCA1 expression and genomic stability mediated by the PI3K/Akt-1 pathway. Cancer Res; 71(6); 2360-70. Ó2011 AACR.

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif ( Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae . These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

The endosomal innate receptor CD158d (KIR2DL4) induces cellular senescence in human NK cells in response to soluble ligand (HLA-G or agonist antibody). These senescent NK cells display a senescence-associated secretory phenotype (SASP) and their secretome promotes vascular remodeling and angiogenesis. To understand how CD158d initiates signaling for a senescence response, we mapped the region in its cytoplasmic tail that controls signaling. We identified a conserved TRAF6 binding motif, which was required for CD158d-induced NF-κB activation and IL-8 secretion, for TRAF6 association with CD158d, and for TRAF6 recruitment to CD158d+ endosomes in transfected cells. The adaptor TRAF6 is known to couple proximal signals from receptors such as endosomal TLRs and CD40 through the kinase TAK1 for NF-κB-dependent pro-inflammatory responses. siRNA-mediated silencing of TRAF6 and TAK1, and inhibition of TAK1 blocked CD158d-dependent IL-8 secretion. Stimulation of primary, resting NK cells with soluble Ab to CD158d induced TRAF6 association with CD158d, induced TAK1 phosphorylation, and inhibition of TAK1 blocked the CD158d-dependent reprogramming of NK cells that produces the SASP signature. Our results reveal that a prototypic TLR and TNF-receptor signaling pathway is used by a killer cell immunoglobulin-like receptor that promotes secretion of pro-inflammatory and pro-angiogenic mediators as part of a unique senescence phenotype in NK cells.

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Supplementary Methods from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer

Supplementary Figures 1-5 from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer

Supplementary Figures 1-5 from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer