Targeted Overexpression of EZH2 to the Mammary Gland Accelerates ErbB2-Driven Tumorigenesis
Xin Li, Maria E Gonzalez, Matthew L DuPrie, Kathy A Toy, Celina G Kleer. University of Michigan, Ann Arbor
Background: EZH2 protein is overexpressed in estrogen receptor negative invasive breast carcinomas with concomitant HER-2/neu overexpression and is a predictor of metastasis. We have demonstrated that mammary specific EZH2 overexpression causes intraductal hyperplasia but is not sufficient to induce invasive carcinomas. To address the biological impact of EZH2 overexpression in mammary tumorigenesis, we generated a novel mouse model of ErbB-2 and EZH2 overexpression in the mammary epithelium.
Design: Mammary specific EZH2 overexpressing transgenic mice were developed in our laboratory. MMTV-neu mice were purchased (Jackson laboratories, FVB/N-TgN(MMTVneu)202Mul/J). EZH2+;neu and EZH2 wt;neu mice were obtained by synchronized breeding of male heterozygous EZH2 transgenics and female MMTV-neu mice. Xenotransplants were carried out to determine the effect of EZH2 overexpression in the stem cell population. Flow cytometry, real-time PCR and immunoblots were performed in mouse mammary glands and cell lines. EZH2, Ki-67, Stat5, and Notch1 were determined by IHC.
Results: EZH2 overexpression enhanced tumor initiation in MMTV-neu mice. EZH2+neu mice (n=30) showed accelerated tumor development (median time to tumor initiation=234 days) compared to EZH2 wt;neu controls (n=25)(median time to tumor initiation=295 days) (Log-rank p < 0.0001). Despite no differences in tumor size or histology, flow cytometry revealed that EZH2+;neu mice had an expansion of the luminal progenitors (CD24+CD29loCD61+) in the mammary glands at 8 weeks of age, preceding tumor development. Transplantation of mammary stem cells derived from 8-week old EZH2+neu and EZH2 wt;neu controls into FVB mice showed that EZH2 overexpression induced stem cell proliferation, intraductal hyperplasia and the formation of solid nests composed of primitive cells. The function of EZH2 in mammary stem cells was further validated in breast cell lines by showing that EZH2 overexpression increased the number of stem cells and mammospheres. EZH2 increased the levels of stem cell regulators NOTCH1 and STAT5a tested by RT-PCR and immunoblots.
Conclusions: EZH2 overexpression accelerates the initiation of ErbB2-induced mammary tumors in mice. EZH2 overexpressing mammary glands exhibited an increase in the progenitor cell population able to recapitulate the cellular heterogeneity of the mammary gland. EZH2 overexpression deregulated several stem cell pathways, including NOTCH1 and STAT5, which is under investigation. We provide first in vivo evidence that EZH2 cooperates with ErbB2 in breast cancer initiation and increases the stem cell population in the mammary gland.
Tuesday, March 20, 2012 11:30 AM
Platform Session: Section B, Tuesday Morning