SIX1 regulates aberrant endometrial epithelial cell differentiation and cancer latency following developmental estrogenic chemical exposure

Female mice exposed neonatally to the synthetic estrogen diethylstilbestrol (DES) develop metaplastic and neoplastic uterine changes as adults. Abnormal endometrial glands express the oncofetal protein sine oculis homeobox 1 (SIX1) and contain cells with basal (cytokeratin [CK]14+/18-) and poorly differentiated features (CK14+/18+), strongly associating SIX1 with aberrant differentiation and cancer. Here we tested whether SIX1 expression is necessary for abnormal endometrial differentiation and DES-induced carcinogenesis by using Pgr-cre to generate conditional knockout mice lacking uterine Six1 (Six1d/d). Interestingly, corn oil (CO) vehicle treated Six1d/d mice develop focal endometrial glandular dysplasia and features of carcinoma in situ as compared with CO wildtype Six1 (Six1+/+) mice. Furthermore, Six1d/d mice neonatally exposed to DES had a 42% higher incidence of endometrial cancer relative to DES Six1+/+ mice. While DES Six1d/d mice had >10-fold fewer CK14+/18- basal cells within the uterine horns as compared with DES Six1+/+ mice, the appearance of CK14+/18+ cells remained a feature of neoplastic lesions. We have employed whole genome microarray expression profiling as a discovery platform to identify genes associated with the phenotypic changes described above. Expression of three genes (Ihh, Krt14, and Trp63) were quantified by real-time PCR, confirming low variability between samples as well as the predicted response pattern. Gene expression analysis was conducted on uterine horn tissue from four independent biological replicates for each exposure group (CO and DES) and genotype (Six1+/+ or Six1d/d) at 6 months-of-age (mos).