Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression

Abstract

IntroductionEffective in vivo models of breast cancer are crucial for studying the development and progression of the disease in humans. We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC).MethodsMIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes.ResultsOf the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a T50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies.ConclusionsWe have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway.

HMGB2 regulates satellite-cell-mediated skeletal muscle regeneration through IGF2BP2

“…Previous studies have demonstrated that all of them are validated for use (Aguilera et al, 2011;Averous et al, 2012;Della Pietra et al, 2015;Dixon et al, 2011;Gong et al, 2015;Kim et al, 2011;Li et al, 2012;Murphy et al, 2011;Nissar et al, 2012;Rao et al, 2014;Sarkar and Zohn, 2012;Takeda et al, 2014;Tan et al, 2011;Taniguchi et al, 2011;Wu et al, 2010;Zhang et al, 2011).…”

Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma

“…To further understand the spatiotemporal events leading to loss of apical-basal polarity, we examined tumors from mice expressing the polyoma virus middle-T antigen (PyMT) under the mouse mammary tumor virus (MMTV) promoter/enhancer (Lin et al 2003) or as a doxycycline-inducible system with a cross of two mouse strains: the PyMT-IRES-CRE (MIC) and the MMTV-reverse tetracycline transactivator (rtTA) (MTB) (Rao et al 2014). These mice form multifocal HR + luminal-type tumors with high penetrance (>75%) that progress through stages, including hyperplasia, mammary intraductal neoplasia (MIN), and adenocarcinoma, that are morphologically similar to human preinvasive breast lesions (Guy et al 1992;Lin et al 2003;Herschkowitz et al 2007;Rao et al 2014).…”

“…These mice form multifocal HR + luminal-type tumors with high penetrance (>75%) that progress through stages, including hyperplasia, mammary intraductal neoplasia (MIN), and adenocarcinoma, that are morphologically similar to human preinvasive breast lesions (Guy et al 1992;Lin et al 2003;Herschkowitz et al 2007;Rao et al 2014). To determine whether this model resembles human cancer in regard to progressive loss of apical-basal polarity and tissue organization, we immunostained tissue sections from different stages with a panel of apical-basal polarity markers, including ZO1, Par6, aPKC, Ezrin, Ecadherin, and Dlg1 ( In hyperplasia, we observed an increase in the number and size of ducts, suggesting that branching morphogenesis is increased at the earliest steps.…”

“…To further examine key steps in breast carcinoma progression, we established three-dimensional (3D) organotypic cultures from mammary epithelial cells from MMTV-rtTA-PyMT mice, which drive PyMT oncogene expression through a doxycycline-dependent inducible promoter (Rao et al 2014). Cells from noninduced mice were cultured to establish polarized structures with lumina, and then doxycycline was added to the culture medium to induce PyMT expression (Fig.…”

“…The PyMT model is a widely used mouse model of breast cancer that recapitulates signaling features of luminal breast cancer and progresses through preinvasive stages that are histologically and molecularly similar to human breast cancer progression (Maglione et al 2001;Lin et al 2003;Herschkowitz et al 2007;Fluck and Schaffhausen 2009;Rao et al 2014). Human cancers are thought to initiate from alterations in individual cells, and although this initiating event may not be recapitulated in transgenic models that have uniform expression of an oncogene, the PyMT model is a useful tool to study progression because of its high penetrance, short latency, and highly reproducible progression to carcinoma (Guy et al 1992;Rao et al 2014).…”