Citation:

Suen, A., W. Jefferson, AND C. Wood. SIX1 Oncoprotein Promotes Abnormal Uterine Basal Cell Development in Mice Exposed Neonatally to Diethylstilbestrol. Gordon Research Conference on Cellular and Molecular Mechanisms of Toxicity, Andover, NH, August 13 - 18, 2017.

Impact/Purpose:

Early-life exposure to estrogenic chemicals in the environment has been associated with increased susceptibility to cancer, infertility, and other adverse reproductive health outcomes later in life. Biological pathways driving these effects are still largely unknown. This case study investigated the role of a developmental protein called SIX1 as a molecular driver and biomarker of latent estrogenic effects in a mouse model of early-life estrogen exposure. The ultimate goal of this work is to enable more rapid identification of AOPs for cancer incorporating life stage susceptibility.

Description:

In a classical model of latent hormonal carcinogenesis, exposing mice on neonatal days 1-5 to the synthetic estrogen diethylstilbestrol (DES; 1 mg/kg/day) results in high incidence of uterine carcinoma. However, the biological mechanisms driving DES-induced carcinogenesis remain unclear. We previously showed that the sine oculis homeobox homolog 1 (SIX1) oncoprotein was aberrantly expressed in the uteri of mice exposed neonatally to DES. Abnormal SIX1 expression persisted with age and localized to abnormal uterine basal cells within all neoplastic lesions. In the current study, we investigated whether SIX1 is necessary for uterine basal cell differentiation and carcinogenesis in mice exposed neonatally to DES. We used a conditional knockout mouse model in which floxed SIX1 is excised specifically in the uterus using the PgR-cre transgene. We confirmed that SIX1 is effectively deleted in this model and is not induced by neonatal DES exposure by real time RT-PCR and immunoblotting immediately following treatment (day 5) and in adults (6 months). As expected, development of the female reproductive tract was normal following uterine deletion of SIX1. At 6 months of age, uterine carcinoma was observed in 4/10 (40%) of DES-exposed wildtype (Six1+/+) mice and 7/9 (78%) of DES-exposed SIX1 conditional knockout (Six1d/d) mice but not in either of the control groups. The presence of basal cell and squamous cell metaplasia of endometrial glands, atypical hyperplasia, and carcinomas in the absence of SIX1 indicates that SIX1 is not required for DES-induced carcinogenesis in the uterus. However, we found two distinct differences between DES-exposed Six1+/+ and Six1d/d mice. Abnormal basal cells expressing the basal cell marker Keratin 14 (K14) were present throughout the entire uterus (body and horns) of Six1+/+ mice, while K14-positive basal cells were restricted to the uterine body of the Six1d/d mice and not found in the uterine horns. Additionally, basal cell and squamous metaplasia was significantly reduced in DES-exposed Six1d/d mice, with >10-fold fewer K14 positive basal cells in endometrial glands of Six1d/d mice, suggesting SIX1 is necessary for differentiation of abnormal basal cells. Luminal epithelial cell number was not different between DES-exposed Six1+/+ and Six1d/d mice, as indicated by the K18 glandular epithelial cell marker. To further characterize this model, we performed a microarray analysis of all four groups at 6 months of age. There were 635 significantly different genes (>1.5 fold) between DES-exposed uteri from Six1+/+ and Six1d/d mice, suggesting aberrant SIX1 expression acts as a transcription factor in this model and contributes to altered gene expression. Taken together, these data indicate that SIX1 expression is not required for DES-induced carcinogenesis in the uterus but rather serves as a key differentiation factor for aberrant basal-type progenitor cells. This abstract does not reflect U.S. EPA policy.

Record Details: