Constitutive activation of Beta-catenin in uterine stroma and smooth muscle leads to the development of mesenchymal tumors in mice

Abstract

Leiomyomas and other mesenchymally derived tumors are the most common neoplasms of the female reproductive tract. Presently, very little is known about the etiology and progression of these tumors, which are the primary indication for hysterectomies. Dysregulated WNT signaling through beta-catenin is a well-established mechanism for tumorigenesis. We have developed a mouse model that expresses constitutively activated beta-catenin in uterine mesenchyme driven by the expression of Cre recombinase knocked into the Müllerian-inhibiting substance type II receptor promoter locus to investigate its effects on uterine endometrial stroma and myometrium. These mice show myometrial hyperplasia and develop mesenchymal tumors with 100% penetrance that exhibit histological and molecular characteristics of human leiomyomas and endometrial stromal sarcomas. By immunohistochemistry, we also show that both transforming growth factor beta and the mammalian target of rapamycin are induced by constitutive activation of beta-catenin. The prevalence of the tumors was greater in multiparous mice, suggesting that their development may be a hormonally driven process or that changes in uterine morphology during pregnancy and after parturition induce injury and repair mechanisms that stimulate tumorigenesis from stem/progenitor cells, which normally do not express constitutively activated beta-catenin. Additionally, adenomyosis and endometrial gland hyperplasia were occasionally observed in some mice. These results show evidence suggesting that dysregulated, stromal, and myometrial WNT/beta-catenin signaling has pleiotropic effects on uterine function and tumorigenesis.

FIG. 1.

Constitutive activation of β-catenin in uterine mesenchyme. A) Gross uterus from Amhr2^tm3(cre)Bhr;Ctnnb1^tm1Mmt/+ nulliparous mouse showing abnormal growth (black and red arrowheads) on the anti-mesometrial side of the uterus. Nodules were also observed on the surface of the uterus (black arrowheads). B) Gross uterus from multiparous adult Amhr2^tm3(cre)Bhr;Ctnnb1^tm1Mmt/+ mouse showing tumorous growth (black arrowhead) and multiple hemorrhagic sites (black arrows). C) Normal uterus from a control Amhr2^tm3(cre)Bhr/+ mouse. H&E-stained section of 4-wk-old mutant uterus (D) showing polyp-like growth protruding from the myometrium on the anti-mesometrial surface of the uterus (red arrowhead) and ESS-like lesions in the endometrial stroma (black arrowhead) and control uterus (E). ACTA2-immunostained longitudinal section of a nulliparous mutant uterus (F) with positively stained polyps (white arrowhead) and control (G). H) In situ hybridization in the E13.5 urogenital ridge shows that the Amhr2 mRNA is expressed in the coelomic epithelium and subjacent mesenchyme of the Müllerian duct (outlined in red with the Wolffian duct). CE, coelomic epithelium; MD, Müllerian duct; WD, Wolffian duct; O, ovary; LE, luminal epithelium; ES, endometrial stroma; M, myometrium. Bars = 2 mm (A–C), 50 μm (D–H).

FIG. 2.

Amhr2^tm3(cre)Bhr;Ctnnb1^tm1Mmt/+ mutant mice develop smooth muscle tumors of uterine myometrium. H&E-stained cross (A) and longitudinal (B) sections of mutant uteri showing the smooth muscle tumor (outlined with white line) in the myometrium. H&E-stained section of mutant uterus (C) at higher magnification showing tumorous growth (outlined in white in B) and human leiomyoma (D). Serial sections of mutant uteri in A and B are shown stained with ACTA2 antibody in E and F. G) Higher magnification image of neoplastic area from F showing that most of the tumorous cells are positive for ACTA2. H) Representative section of a human uterine smooth muscle tumor showing the typical staining pattern with the ACTA2 antibody. Phosphohistone staining of tumor area in a mutant uterus is shown in I and at higher magnification with white arrows in J. K and L) TGFB3 immunostaining in the central area of a mutant smooth muscle tumor and a human leiomyoma, respectively. Nuclei are stained with DAPI or hematoxylin. LE, luminal epithelium; ES, endometrial stroma; M, myometrium. Bar = 50 μm.

FIG. 3.

FRAP1 expression in Amhr2^tm3(cre)Bhr;Ctnnb1^tm1Mmt uteri. A) FRAP1 expression was detected by immunohistochemistry at high levels in the central areas of a leiomyoma (outlined in white) in a mutant uterus, the epithelial cells, the ES as well as in the circular myometrial layer (MC) and longitudinal myometrial layer (ML). B) A higher magnification view of FRAP1 expression in the MC and ML of a mutant mouse. C) In control uteri, strong FRAP1 expression is found in the EG, LE, and ES. D) Expression in the control myometrium appears limited to the endothelial cells. LE, luminal epithelium; ES, endometrial stroma; M, myometrium; EG, endometrial glands. Bars = 50 μm (A and C), 100 μm (B and D).

FIG. 4.

Constitutive activation of β-catenin causes development of ESS-like tumors in mouse uteri. A) H&E-stained section of mutant uterus. Neoplastic lesions are seen in endometrial stroma (outlined in white). B) ESS-like neoplastic growths are ACTA2-negative. The dotted lines indicate the location of ESS lesions detected on a serial section by H&E. C) Both cytoplasmic and nuclear staining for β-catenin were observed in tumorous lesions (outlined by white dotted line) and normal-looking myometrium of mutant uterus. Membranous staining for β-catenin is present in the LE and EG of mutant uteri and in control mice (D). E) The ESS-like tumors were vimentin-positive but in a pattern that was different from that observed normally in stromal areas of mutants and in control stroma (F). Nuclei are stained with DAPI. LE, luminal epithelium; ES, endometrial stroma; M, myometrium. Bar = 50 μm.

FIG. 5.

Sections of the mutant uteri with ESS-like tumors, outlined in a white dashed line in all the panels, were analyzed by immunofluorescence for various markers. The tumors were ESR1-positive (A), KIT-positive (B), and were negative for the pan-hematopoietic marker ITGAM (CD11B) and the endothelial marker PECAM1 (C). The ESS-like lesions were also positive for HMGA2 (D), and negative for E-cadherin (CDH1) (E) and PDGFRB (CD140B) (F). Nuclei are shown stained with DAPI. LE, Luminal epithelium; ES, Endometrial stroma; M, Myometrium. Bars = 50 μm.

FIG. 6.

A) H&E-stained section of mutant uterus showing hyperplasia and pseudostratification of epithelium of EGs. B) The longitudinal section of mutant uterus depicting the presence of EG in the myometrium stained with CDH1 (green) and ACTA2 (red) antibody, and counterstained with DAPI (blue). The arrowheads indicate ectopic endometrial tissue within the myometrium. LE, luminal epithelium; ES, endometrial stroma; EG, endometrial gland; M, myometrium. Bar = 50 μm.