Pregnancy-associated breast cancers (PABCs) are tumors diagnosed during pregnancy or up to 5 years following parturition, and are usually high-grade, connective tissue-rich, and estrogen receptor (ER)/progesterone receptor-negative. Little is known about the cellular origin of PABCs or the mechanisms by which PABCs are initiated. Using the RCAS retrovirus to deliver the ErbB2 oncogene into the mammary epithelium of our previously reported MMTV-tva transgenic mice, we detected high-grade, poorly differentiated, stroma-rich and ER-negative tumors during pregnancy and lactation. These high-grade and stroma-rich tumors were less frequent in involuted mice or in age-matched nulliparous mice. More importantly, by generating a WAP-tva transgenic line for expression of ErbB2 selectively in WAP(+) mammary alveolar cells, we found that tumors had similar morphological phenotypes (high grade, poorly differentiated, stroma-rich and ER-negative), irrespective of the time since pregnancy and even in the absence of pregnancy. These data suggest that PABCs arise preferentially from an alveolar cell population that expands during pregnancy and lactation. This somatic mouse model may also be useful for preclinical testing of new prophylactic and therapeutic strategies against PABC.

Background

The mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.

Methods

We utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice.

Results

We demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.

Conclusions

Herein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.

The phosphoinositide 3-kinase (PI3K) signaling cascade is a key mediator of cellular growth, survival and metabolism and is frequently subverted in human cancer. The gene encoding for the alpha catalytic subunit of PI3K (PIK3CA) is mutated and/or amplified in ∼30% of breast cancers. Mutations in either the kinase domain (H1047R) or the helical domain (E545K) are most common and result in a constitutively active enzyme with oncogenic capacity. PIK3CA(H1047R) was previously demonstrated to induce tumors in transgenic mouse models; however, it was not known whether overexpression of PIK3CA(E545K) is sufficient to induce mammary tumors and whether tumor initiation by these two types of mutants differs. Here, we demonstrate that expression of PIK3CA(E545K) in the mouse mammary gland induces heterogenous mammary carcinomas but with a longer latency than PIK3CA(H1047R)-expressing mice. Our results suggest that the helical domain mutant PIK3CA(E545K) is a less potent inducer of mammary tumors due to less efficient activation of downstream Akt signaling.

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

Amplification and overexpression of erbB2/neu proto-oncogene is observed in 20-30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.