Characterization of a novel doxycycline inducible model of EZH2 overexpression in mammary glands

Background: Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase and catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In human breast cancer, EZH2 overexpression is an independent prognostic marker and is significantly associated with negative ER and PR expression. However, the timing and functional significance of EZH2 overexpression in breast cancer development is still unclear. Towards this, we generated a conditional model of EZH2 overexpression to the mammary gland in FVB mice. Methods: We generated a doxycycline-inducible transgenic mouse model MMTV-rtTA;TetO-EZH2 and appropriate controls in an FVB background to enable targeted overexpression of EZH2 within mammary epithelial cells. We administered doxycycline (2 mg/mL) in the drinking water to 10-week-old female mice for 96 hours and in chow for 6 months, to induce EZH2 overexpression. At this age, the mammary glands are mature. At study endpoints, mammary glands were resected and analyzed using whole-mount carmine alum staining to assess ductal architecture. Mammary glands were embedded in paraffin and studied by histopathology and immunostaining using anti-EZH2 antibodies. Results: Induction of EZH2 expression in adult mice for 96 hours led to increased numbers of ductal branches compared to induced and uninduced controls. Long-term EZH2 induction also resulted in a hyperbranching phenotype and development of mammary epithelial nodules observed in carmine alum stains of whole glands, which were not present in the controls. Additional studies, including histopathological evaluation, immunostaining, and spatial analyses are underway to further characterize the biological and molecular consequences of EZH2 induction. Conclusions: We present the development and initial characterization of a novel inducible EZH2 overexpression model in the mammary glands of adult mice. Preliminary studies show that EZH2 overexpression results in intraductal epithelial hyperplasia and ductal hyperbranching recapitulating human preneoplastic lesions. Future studies will combine this novel model with other breast cancer mouse models to explore the consequences of EZH2 overexpression on tumor initiation and progression.