The mouse mammary gland is a complex tissue, which is continually undergoing changes in structure and function. Embryonically, the gland begins with invasion of the underlying fat pad by a rudimentary ductal structure. Postnatal growth occurs in two phases: ductal growth and early alveolar development during estrous cycles, and cycles of proliferation, differentiation, and death that occur with each pregnancy, lactation, and involution. The variety of epithelial structures and stromal changes throughout the life of a mammary gland makes it a challenge to study. The purpose of this histological review is to give a brief representation of the morphological changes that occur throughout the cycle of mouse mammary gland development so that developmental changes observed in mouse models of mammary development can be appreciated.

Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.

The goals of this study were to determine the cellular sites of insulin-like growth factor (IGF) and IGF type-I receptor (IGF-IR) expression and to begin to elucidate functional roles for the IGFs during postnatal development of the murine mammary gland. Using in situ hybridization analyses, we determined that IGF-I, IGF-II, and IGF-IR messenger RNAs were expressed in the highly proliferative terminal end buds during pubertal ductal growth. Consistent with these data, IGF-I (in combination with mammogenic hormones) promoted ductal growth in pubertal stage mammary glands cultured in vitro. During postpubertal and pregnancy stages, IGF-II and IGF-IR continued to be expressed in ductal epithelium. Expression of IGF-II in ductal and alveolar epithelium correlated with the pattern of rapidly proliferating cells, as determined by incorporation of 5-Bromo-2'-deoxyuridine, suggesting a potential autocrine or paracrine role for IGF-II as a mitogen for ductal epithelial cells. IGF-I expression was reinitiated in mammary epithelium in the differentiated alveoli at the end of pregnancy, suggesting an additional role for this factor in maintenance of the alveoli during lactation. Taken together, these data support an in vivo role for locally-produced IGFs in promoting ductal growth during puberty and suggest that IGF-I and IGF-II may have distinct functions during pregnancy-induced alveolar development.

Background: Mammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.

IntroductionPolyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis.α-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells.MethodsMDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection.ResultsDFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35–40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm2 vs 4.51 mm2, P < 0.05).ConclusionTaken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early.

The insulin-like growth factors are mitogens and survival factors for normal mammary epithelial cells in vitro. Data reviewed here demonstrate that mRNAs for IGF-I and IGF-II, the IGF type I receptor and the IGFBPs are expressed locally in mammary tissue during pubertal and pregnancy-induced growth and differentiation of murine mammary glands. IGF-I, IGF-II and the IGF-IR were expressed in terminal end buds (TEBs) in virgin glands during ductal growth. In addition, IGF-II and IGF-IR mRNAs were expressed in ductal and alveolar epithelium in glands throughout postnatal development. Consistent with these results, IGF-I promoted ductal growth and proliferation in mouse mammary glands in organ culture. In addition to endogenous expression of the IGFs and IGF-IR, the IGFBPs showed a varied pattern of expression in mammary tissue during postnatal development. For example, IGFBP-3 and -5 mRNAs were expressed in TEBs and ducts while IGFBP-2 and -4 mRNAs were expressed in stromal cells immediately surrounding the epithelium. These results support a role for the IGFs and IGFBPs as local mediators of postnatal mammary gland growth and differentiation.

Abstract. Metastasis occurs, in part, due to tumor cell responses to chemokine secretion by ectopic organs or tissues. SDF-1 is constitutively expressed in tissues where metastases frequently develop while breast carcinoma cells express the receptor for SDF-1, CXCR4, which is correlated with increased bone metastasis and poor overall survival. We hypothesized that treatment with a CXCR4 antagonist, CTCE-9908, would decrease incidence of bone and lung metastasis. Treatment with CTCE-9908 (25 mg/kg) began the day prior to or the day of intravenous or intracardiac tumor cell inoculation of MDA-MB-231 human breast carcinoma cells expressing enhanced green fluorescent protein (GFP) into athymic mice. After 5 or 8 weeks (i.c. and i.v. injections, respectively), the presence of fluorescent foci at metastatic sites was assessed. Somewhat surprisingly, CTCE-9908 treatment did not decrease incidence of metastasis as hypothesized. However, CTCE-9908 did decrease metastatic burden (i.e., size of metastases) in all organs examined (lungs, bone, heart, liver, kidneys, pancreas and spleen). Based upon this and other studies, the use of CTCE-9908 is promising as an adjuvant therapy for metastatic disease.

Induction of cyclin proteins is required for progression of cells through the G(1)-S and G(2)-M cell cycle checkpoints and is a primary mechanism by which mitogens regulate cell cycle progression. IGF-I and the epidermal growth factor (EGF)-related ligands are mitogens for mammary epithelial cells in vitro and are essential for growth of the mammary epithelium during development. We report here that IGF-I in combination with EGF or TGFalpha is synergistic in promoting DNA synthesis in mammary epithelial cells in the intact mammary gland cultured in vitro. We further investigated the role of IGF-I and EGF in cyclin expression and cell cycle progression in the mammary gland and demonstrate that IGF-I and EGF induce expression of early G(1) cyclins. However, we show that IGF-I, but not EGF, induces late G(1) and G(2) cyclins and is required for mammary epithelial cells to overcome the G(1)-S checkpoint. These data demonstrate that IGF-I is essential for cell cycle progression in mammary epithelial cells and that it is required for EGF-mediated progression past the G(1)-S checkpoint in these cells.