Endometrial hyperplasia with loss of APC in a novel population of Lyz2-expressing mouse endometrial epithelial cells

Abstract

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of β-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Graphical Abstract

Figure 1.

Loss of APC in mice LysM + cells results in uterine hypertrophy. Representative gross images of uteri from littermate control (A) and Apc^cKO (B) mice at 28 weeks postnatal. Uterine wet weight as a percentage of body weight for littermate control and Apc^cKO mice at the indicated ages are shown in (C). Data are represented as the mean ± SD. P-values were generated using a two-tailed, unpaired t-test.

Figure 2.

LysM is expressed in 1% of endometrial epithelial cells. Representative images of LysM IHC on 28-week-old control uterine sections showing LysM + luminal (A, B) and glandular epithelial cells (C, D). Boxed areas in A and C are shown at higher magnification in B and D. Arrowheads in A and C indicate prominently stained cells. EpCAM + endometrial epithelial cells from control and LysM-EGFP knock-in mice were analyzed by flow cytometry for GFP (E). Total GFP + endometrial epithelial cells from uteri of random cycling control and LysM-EGFP mice were quantitated by flow cytometry at the indicated ages (F). Data are represented as the mean ± SD.

Figure 3.

LysM + endometrial epithelial cells proliferate, and daughter cells can be LysM-. LysM-Cre mice were bred to mT/mG reporter mice to trace the lineage of uterine LysM + daughter cells. GFP + endometrial epithelial cells were observed by fluorescence microscopy in uterine sections from mice at 3 (A), 8 (B) and 28 (C) weeks of age. Inset in A is an image of section from a littermate control. Flow cytometry gating strategy to quantitate EpCam + endometrial epithelial cells that are GFP + in the LysM-Cre lineage (D). Flow cytometry analysis of EpCAM + cells in uteri from random cycling LysM-Cre;mTmG lineage mice at 3, 8 and 28 weeks of age, along with age-matched controls that lack LysM-Cre (E). Data are represented as mean ± SD.

Figure 4.

Loss of APC in LysM + endometrial epithelial cells results in endometrial hyperplasia. Representative H&E stained sections from 28-week-old control (A) and Apc^cKO mice demonstrating cystic endometrial hyperplasia (B), villoglandular or papillary hyperplasia (C), complex atypical hyperplasia or EIN (D), nuclear atypia (E) and deciduoma/endometrial stroma sarcoma (F) histology. Images of endometrium of 28-week-old LysM-Cre; mTmG as a control (G) and LysM-Cre; Apc^cKO; mTmG (H) mice. Flow cytometry analysis of GFP + cells in uteri from random cycling control (LysM-cre negative), LysM-cre; mTmG and LysM-cre; APC^cKO; mTmG mice at 28 weeks postnatal (I). Data are represented as the mean ± SD. Representative IHC with a β-catenin antibody was performed on endometrium from control mice at 20 weeks postnatal (J, K). Representative images of IHC with β-catenin (L, M) and cyclin D1 (N, O) antibodies on uterine serial sections from an 8-week-old Apc^cKO mouse. Panels K, M and O are higher magnification images of J, L and N, respectively. Epithelial cells with nuclear β-catenin show increased expression of cyclin D1.

Figure 5.

Endometrial hyperplasia in Apc^cKO mice is hormone dependent. Uterine wet weight as a percentage of total body weight for 28-week-old intact control and Apc^cKO mice compared to those from 28-week-old ovariectomized control and Apc^cKO mice as indicated (A). H&E stained endometrial sections from 28-week-old ovariectomized control (B) and Apc^cKO (C) mice showing similar pathology. Serum estradiol (D) levels are slightly elevated in the Apc^cKO mice compared to control mice. Progesterone (E) levels are not significantly different in control and Apc^cKO mice. Data are represented as the mean ± SD. P-values were generated using a two-tailed, unpaired t-test. (F) IHC with β-catenin and ER-α antibodies on serial sections from 20-week-old Apc^cKO endometrium shows that endometrial epithelial cells with increased β-catenin also have increased ER-α. Red arrowheads indicate prominent areas with excess β-catenin and ER-α expression.