We sought to develop a

We sought to develop a mouse model for breast and extra-uterine Müllerian cancer predisposition based on conditional inactivation of Brca1 not only in these organs, but also in hormone producing cells that regulate the menstrual cycle, including ovarian granulosa cells and the anterior pituitary gland, in order to mimic both the genetic background and the cell-nonautonomous conditions associated with strong predisposition to these cancers in humans. Transgenic constructs driving expression of Cre recombinase under the control of a combination of cell-specific promoters active in the various tissues of interest were introduced in mice carrying floxed egfr inhibitors not only in Brca1, but also in p53, a gene mutated in almost all human cancers associated with the BRCA1 mutation carrier state (Ahmed et al., 2010). The cell-specific promoters used included a previously characterized truncated form of the Follicle stimulating hormone receptor (Fshr) promoter (Griswold et al., 1995; Chodankar et al., 2005) and the Müllerian inhibiting substance receptor type 2 (Mis2r) promoter (Josso et al., 2001; Connolly et al., 2003). The latter is expressed in the Müllerian ducts during embryological development, which later differentiate into internal reproductive organs including fallopian tubes, uterus, cervix, and a portion of the vagina, as well as other extra-uterine structures carrying increased cancer risk in BRCA1 mutation carriers such as endosalpingiosis. Mis2r promoter is also active in mammary epithelium (Segev et al., 2001).
Materials and Methods
Results
Discussion We took advantage of 2 promoters with largely overlapping, but slightly different cell specificity to introduce conditional Brca1 and p53 double knockouts targeting the mammary gland, reproductive organs, and also organs playing a central role in controlling the estrous cycle, which is equivalent to the human menstrual cycle. The important drivers of estrous cycle activity targeted in this experimental model include ovarian granulosa cells and a subset of cells within the anterior pituitary. We had previously reported that Brca1 inactivation in ovarian granulosa cells leads to changes in estrous cycle dynamics including prolongation of the pre-ovulatory phase and increased circulating levels of sex steroid hormones (Hong et al., 2010) and also reported on evidence that alterations in these hormonal levels are also present in human BRCA1 mutation carriers (Widschwendter et al., 2013). Given the well-established importance of menstrual cycle activity as a risk modulator for breast and extra-uterine Müllerian carcinomas, our intention was to generate a mouse model suitable to investigate the interplay between genetic and hormonal factors of predisposition to both of these cancers. The truncated form of the Fshr promoter that we used in our studies is active in ovarian granulosa cells and in the anterior pituitary, both of which influence Müllerian tumorigenesis in a cell-nonautonomous manner, but it is not expressed in Müllerian epithelium (Chodankar et al., 2005). Thus, any contribution of genetic alterations driven by this promoter to Müllerian carcinogenesis can only be mediated through a cell-nonautonomous mechanism, hence its importance in our overall strategy. Several transgenic or knockout models for extra-uterine Müllerian/ovarian and for mammary carcinomas have been developed over the last 2 decades (see (Pfefferle et al., 2013; Hollern and Andrechek, 2014; Hasan et al., 2015) for reviews). In some cases, specific pathways, including Her-2/neu in mammary epithelium, Pten in Müllerian epithelium, and others have been targeted while others have focused on Brca1/2 inactivation in tissues corresponding to those with an elevated cancer risk in human BRCA1/2 mutation carriers. These contributions led to significant progress in our understanding of the cell-autonomous role of specific pathways in the development of these cancers. The model described here is associated with tumors that are morphologically similar to the human tumors associated with the BRCA1/2 mutation carrier state, as evidenced by the high-grade papillary serous appearance of Müllerian tumors and the basal appearance and triple negative nature of at least some of the mammary tumors that we observed. This model is not based on targeting any specific signaling pathway, but on the inactivation of cell cycle regulators associated with human mammary and extra-uterine Müllerian cancer predisposition. It is also distinguished from existing models based on the following features: (1) it not only targets tissues similar to those at elevated risk of cancer in human BRCA1/2 mutation carriers, but also organs that influence cancer predisposition from a distance via cell-nonautonomous mechanisms, closely mimicking the conditions associated with cancer predisposition in human BRCA1 mutation carriers; (2) the fact that a significant proportion of mice heterozygous for Brca1 and p53 mutations develop tumors further increases similarities to the genetic background associated with human familial breast and extra-uterine Müllerian cancer predisposition; (3) the possibility of generating tumors in mice that are heterozygous for a Brca1 mutation avoids confounders due to developmental defects associated with homozygous deletions of different splice forms of this gene, which have been reported both in the mammary gland and in the reproductive tract (Xu et al., 1999; Kim et al., 2006); (4) differences in the tissue specificity of the various promoters used to drive tissue-specific mutations, plus the possibility of using surgical manipulations entailing ovarian transplantation between mutant and wild type donors, make it possible to study cell-autonomous and the cell-nonautonomous mechanisms of cancer predisposition independently of each other and, therefore, to distinguish their respective contributions. Cell-nonautonomous mechanisms, which are mediated by circulating factors as opposed to intra-cellular changes, should be readily targetable pharmacologically, hence the importance of understanding their exact mechanisms.