EZH2 Expands Breast Stem Cells by Activating Notch, Acting To Accelerate the Onset of Breast Cancer
Maria E Gonzalez, Xin Li, Heather M Moore, Matthew L DuPrie, Kathy A Toy, Celina G Kleer. University of Michigan, Ann Arbor, MI
Background: EZH2 is a critical epigenetic regulator of cell type identity and has oncogenic functions in breast and other cancers, acting primarily as a transcriptional repressor through trimethylation of histone 3 at lysine 27 (H3K27me3). EZH2 upregulation signals increased risk for breast cancer when detected in benign breast biopsies. We hypothesized that EZH2 may promote breast cancer initiation through regulation of mammary stem cells.
Design: EZH2 was overexpressed in nontumorigenic breast cells using a Doxycycline-regulated or an adenoviral system. EZH2 was knocked down in breast cancer cell lines and cells derived from patients' tumors using specific shRNAs. The effect of EZH2 levels on mammary stem cells was investigated using mammosphere assay as well as flow cytometry (ALDH1+ and CD44+/CD24- assays). To study the effect of EZH2 on NOTCH1 transcriptional activity and promoter binding we employed reporter assays and chromatin immunoprecipitation (ChIP assays). The effect of EZH2 in tumor initiation was investigated using xenografts and genetic mammary specific EZH2 overexpression in MMTV-neu mice.
Results: EZH2 overexpression increased, while EZH2 knockdown (KD) decreased the number of mammospheres and the percentage of ALDH1+ and CD44+/CD24- cells in nontumorigenic and in breast cancer cells, respectively. NOTCH1 was one of the most significantly downregulated genes upon EZH2 KD in breast cancer cells. In nontumorigenic breast cells, EZH2 overexpression led to NOTCH1 mRNA and protein upregulation, and an increase in NOTCH1 transcriptional activity. Mechanistically, EZH2 binds to NOTCH1 promoter (-1.2kb) independenly of the SET domain, but requires the HII domain. Consistent with EZH2-induced NOTCH1 activation, the binding of EZH2 to NOTCH1 promoter was associated with increased H3K4me3 activating mark and with binding of RNA polymerase. Blockade of NOTCH signaling with GSI or NOTCH1 downregulation using siRNAs rescued the EZH2-mediated stem cell expansion in breast cells. Genetic EZH2 overexpression in MMTV-neu mice led to accelerated tumor initiation (log rank p<0.005).
Conclusions: EZH2 overexpression promotes breast cancer initiation in vivo by expanding the pool of functional breast stem cells via upregulation of NOTCH1. We uncover a novel role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to NOTCH1 promoter through the HII EZH2 domain. NOTCH1 is required for EZH2-mediated stem cell expansion in breast cancer. The new mechanism described herein may shed light into the tumor promoting effects of EZH2 in multiple solid tumors.
Monday, March 4, 2013 11:00 AM
Proffered Papers: Section G2, Monday Morning