Mammary Epithelial Stem Cells Research Articles

On Murine Mammary Epithelial Stem Cells: Discovery, Function, and Current Status

Cell-surface markers expressed on mammary stem cells and progenitors have helped to establish a preliminary mammary cell lineage hierarchy. Further characterization of these cells depends on overcoming several technical obstacles.

On Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

Epigenetic programs have been extensively studied in embryonic stem cells. However, epigenetic controls in mammary gland development and in the differentiation of mammary epithelial stem cells have not been defined.

Neoplastic transformation of mammary epithelial stem cells: Are they linked to the development of age-dependent breast cancer

Neoplastic transformation of mammary epithelial stem cells: Are they linked to the development of age-dependent breast cancer

Abstract A45: Targeting breast cancer stem cells using cancer preventive rexinoids

Abstract Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesize that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. Results: Our results showed that the rexionids LG100268 and bexarotene inhibit the growth of breast cell lines only at a high concentrations (the IC50 (concentration that reaches a 50% growth inhibiton) in most of the tested cell lines exceeded 10uM). Results of in vitro mammosphere assays showed that 100nM LG100268 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A45.

Dot Bennett

Dot Bennett

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs

Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.

Wnt signalling in murine postnatal mammary gland development

The mammary gland undergoes numerous developmental processes postnatally, from the elongation of the ductal tree-like structure to pregnancy-induced lobulo-alveolar development. Mammary epithelial stem cells have been suggested to be central to the control of enormous tissue expansion and remodelling during phases of mammary development. The Wnt signalling pathway plays a critical role in these biological steps and is suggested to be involved in the maintenance of the stem cell population. This review provides insight into recent findings on the activity of Wnt signalling during ductal and lobular mammary development and discusses the potential interplay between Wnt signals and mammary stem cells in mice.

G9a‐induced epigenetic silencing of maspin in human claudin‐low breast tumor initiating cells

Mammary Serine Protease Inhibitor (maspin) is a tumor suppressor gene that is silenced by epigenetic mechanisms in most breast cancer cell lines. Down-regulation of maspin contributes to metastatic progression and is associated with poor prognosis in breast cancer. Breast cancer can be classified into distinct tumor subtypes and the claudin-low subtype most closely resembles the mammary epithelial stem cell. We have generated a model human claudin-low breast tumor initiating cell line by exogenous expression of the OCT4 transcription factor that displays down-regulation of a panel of tumor suppressor genes, including maspin. In order to better understand the epigenetic mechanism by which maspin is silenced in these cells, we utilized siRNA to knock down the histone H3 lysine 9 and histone H3 lysine 27 methyltransferases (G9a and EZH2) that are responsible for gene repression. Knockdown of G9a results in increased maspin expression. However, knockdown of EZH2 did not change maspin expression. These results suggest that G9a targets maspin in claudin-low tumor initiating cells to induce gene repression through histone H3 lysine 9 methylation and subsequent DNA methylation. Thus, modulation of this epigenetic enzyme or the histone H3 lysine 9 methylation mark in the maspin promoter could potentially restore proper gene expression of the maspin breast tumor suppressor gene.

Abstract P2-07-04: Relevance of Mutated PIK3CA to Normal Mammary Gland Development and Malignancy

Abstract In the current study we generated transgenic mice with mammary gland specific-overexpression of PI3KCA wild type and mutant protein with the aim to characterize the effects of activation of PI3K signaling on normal mammary gland and tumor development. PIK3CA encodes p110α, the predominant isoform of the catalytic subunit of class 1A phosphatidylinositol 3-kinase (PI3K), a lipid phosphokinase. The family of PI3Ks provides signaling for diverse cellular functions, including proliferation, metabolism, migration, translation, apoptosis avoidance, and angiogenesis. Disruption of this tightly regulated pathway by gene loss (PTEN), mutation (PIK3CA, AKT1, or less commonly PIK3R1), or amplification (PIK3CA), is one of the most common alterations in human cancers. PIK3CA mutations result in constitutive activation of p110α , increasing lipid kinase activity and resulting in an increase in activated AKT. The majority of mutations in breast cancer occur at three hotspots: E542K and E545K at exon 9, which encodes the helical domain, and H1047R at exon 20, which encodes the kinase domain. Previously, it was described that human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. In a mouse model in which PI3K is activated by forced recruitment of p110α to the membrane, increased ductal branching, alveolar hyperplasia, intraductal neoplasia and a low frequency of mammary tumors were observed. In breast carcinoma, mutations in PIK3CA and loss of PTEN function are almost always mutually exclusive. However, mutations in PIK3CA are often correlated with overexpression of Her2/neu (48%) in primary breast carcinomas, indicating that gain of function in these two signaling components could have a synergistic effect in counteracting PTEN. To date, no in vivo studies have been reported to determine if PIK3CA mutations play a causative role in breast tumorigenesis. In the current study we generated transgenic mice with mammary gland specific-overexpression of PIK3CA wild type and mutant protein and analyzed the effects of activation of PI3K signaling on normal mammary gland and tumor development. Mice harboring Exon 9 and Exon 20 mutations showed increased ductal branching at 8 weeks of age. Two mice harboring low and high copy numbers of Exon 20 mutation developed mammary tumors at 14 and 16 months of age, respectively. Histological analysis revealed that both were carcinomas with tubular, papillary and comedo features. PI3K activation increases the mammary epithelial stem cell population (CD24med/CD49f hi) in the normal gland. This population, which was characterized both by in vitro and in vivo assays, is highly expanded in the tumor cells. Tumor formation and mammary gland development is being observed in PIK3CA mutants crossed with MMTV-Her2neu mice. In conclusion, PI3K signaling activation appears to alter normal mammary gland development and initiate mammary tumor formation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-04.

Abstract P4-06-24: KLF8 Induction of Mammary Epithelial Stem Cell Formation and Tumorigenesis by Regulating EMT-Associated MicroRNAs

Abstract Although it is well established that the epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells, the molecularmechanisms responsible for this change remain largely undefined. We set out to identify the mechanisms by examining the expression of stem cell markers as well as microRNAs in MCF-10A cells during EMT induction. By immunostaining and FACS analysis, we found that during EMT induction by either TGF-β treatment or overexpression of KLF8, both the expression of CD44high/CD24low and the activity of aldehyde dehydrogenase (ALDH) were dramatically increased. Using microRNA PCR array, we identified a microRNA signature associated with the EMT. Using microRNA inhibitor, We further demonstrated that one of the microRNAs, miR-146a, is upregulated and plays an important role in keeping the stem cell percentage during EMT induced by both TGF-β and KLF8, as revealed by manipulation of miR-146a expression, analysis of the stem cell markers and mammosphere growth. Taken together, these results highlight a novel role for miR-146a in regulating the stem-like cell induction from MCF-10A cells by EMT. Experiments are in progress to investigate the contribution of KLF8-regulated expression of miR146a and its targets to the stem cell induction and transformation. Supported by: NIH, ACS, Komen and NYSTEM grants to J.Z. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-24.

Estrogen expands breast cancer stem-like cells through paracrine FGF/Tbx3 signaling

Many tumors contain heterogeneous populations of cells, only some of which exhibit increased tumorigenicity and resistance to anticancer therapies. Evidence suggests that these aggressive cancer cells, often termed "cancer stem cells" or "cancer stem-like cells" (CSCs), rely upon developmental signaling pathways that are important for survival and expansion of normal stem cells. Here we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast cancer cell lines and freshly isolated breast cancer cells, whereas cotreatment of cells with tamoxifen or a small molecule inhibitor of FGFR signaling was sufficient to prevent the estrogen-induced expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene expression was able to abrogate tumorsphere formation, whereas ectopic Tbx3 expression increased tumor seeding potential by 100-fold. These findings demonstrate that breast CSCs are stimulated by estrogen through a signaling pathway that similarly controls normal mammary epithelial stem cell biology.

Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

BackgroundIn the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer.Methods and FindingsWe show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.ConclusionsThese findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

Murine Mammary Epithelial Stem Cells: Discovery, Function, and Current Status

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.

Expression Pattern of Selected Cell Adhesion Molecules in Mammary Duct Repopulating Cells Sorted From The Mouse Mammary Anlagen*

Mammary stem cells (MaSCs) is an ideal model for studies of organogenesis, cell proliferation, differentiation, survival and apoptosis. Recent researches showed that many adult stem cell surface markers belonged to cell adhesion molecule (CAM) family. Therefore, analysis of relationship between mammary stem/progenitor cells and the expression pattern of CAM have directive meanings for identifying embryonic mammary stem/progenitor cells and understanding their properties. Here, cells in E14 mouse mammary anlagen were purified using the adult mouse mammary epithelial stem cell (MaESC) markers of CD24 and CD49f. It was found that CD24 and CD49f double-positive cells contain two distinct cell populations: CD24hiCD49f + and CD24medCD49f +, and their percentages in total mammary anlagen cells were 16% and 47%, respectively. In the following monolayer culture and in vivo transplantation tests, it was found that the CD24medCD49f + cells could attach plate and regenerate mammary ductal units; correspondingly, CD24hiCD49f + cells did not possess of these capabilities. These results indicate that the two mammary anlagen cell populations represent different cell types, and CD24medCD49f+ population may contain the self-renewal mammary anlagen stem/progenitor cells. Afterwards, the differences in the expressions of 19 mammary-related CAM transcripts in the two cell populations were validated by quantitative real-time PCR analysis. The data show that the CAM gene expressions of mammary repopulating-CD24medCD49f + cells are dramatically different from that of CD24hiCD49f + cell population. The expressions of several adult stem cell markers in CD24medCD49f+ cell population are remarkable higher than those in CD24hiCD49f+ cell population.

Abstract 5267: Mammary stromal fibroblast activity is modulated by HoxC6: impacts on growth and metastatic potential

Abstract Crosstalk between mammary epithelium and stromal cell types, including fibroblasts and adipocytes is 1) essential for mammary gland development 2) likely impacts breast cancer potential and 3) significantly impacts the growth, invasion and metastasis of tumor cells. Although well recognized, molecular mechanisms important in stromal-epithelial crosstalk are still poorly understood. The purpose of this study is to better elucidate one potentially significant mechanism involving stromal fibroblast to epithelial paracrine signaling in the mammary gland. We previously demonstrated that HoxC6 was required for mammary epithelial cell growth and ductal branching morphogenesis using mouse models and 3-dimensional in vitro culture of human mammary epithelial cell lines. We also demonstrated that constitutive ErbB2/Her2/Neu receptor activation in epithelium of transgenics is sufficient to rescue HoxC6 deficiency in epithelial growth but not branching. Significantly, this model also shows that HoxC6 null stroma facilitates tumor metastasis. Here, we focus attention on stromal fibroblasts (vimentin positive cells) from wild type and HoxC6 null prepubertal mouse thoracic mammary glands (TMGs). When cultured in vitro, HoxC6 null TMG fibroblasts display multiple filopodia and extended cellular processes that could indicate an activated myofibroblast phenotype. In contrast, cultured wild type TMG fibroblasts appear quite similar to mouse embryonic fibroblasts. Quantitative comparisons of gene expression patterns in isolated fibroblasts indicate that HoxC6 null fibroblasts are more activated than wild type fibroblasts. Activation markers studied include alpha smooth muscle actin (ACTA2/SMA), S100A4 (Fsp1) and Wnt1-induced secreted protein 1 (Wisp1). Since estrogens are know to repress HoxC6 expression and are implicated in modulating paracrine stromal-to-epithelial interactions, stromal fibroblasts were isolated and cultured under estrogen-free conditions and treated with 17beta-estradiol, bisphenol A and vitamin D. In addition, stromal fibroblasts were co-cultured with mammary epithelial stem cells and stem-like mammary tumor cells to study the impact of stromal fibroblast HoxC6 status on epithelial cell activities. The results obtained will be discussed as they pertain to stromal-to-epithelial signaling in mammary gland development and in the growth, invasion and metastasis of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5267.

Abstract 3469: Effect of age and mutagen on the enriched murine mammary epithelial stem and progenitor cell population

Abstract Epidemiological studies indicated that aging is a significant risk factor for breast cancer. Yet, there is insufficient knowledge on the mechanisms affecting breast cancer initiation and promotion in older women. Recent studies indicated epithelial stem/progenitor cell population, responsible for lifelong tissue maintenance and regeneration, as a target of transformation. It has been reported earlier by serial transplantation of murine mammary tissues that functional mammary outgrowths indicating stem cell function is unaffected by the age of the donor. But it is unclear that whether spontaneous or induced mutagenesis in the aging stem/progenitor cell population has the potential to impair stem/progenitor functions and/or to initiate tumorigenesis. In this study, first we have isolated lineage negative (CD31−CD45−Ter119−) mammary epithelial cells (Lin−MEC) from the thoracic/inguinal mammary glands of young (6 months, n=7) and old (28 months, n=6) mice by serial enzymatic dissociation followed by magnetic nanoparticle separation method (StemCell Technology). These cells were utilized to form mammospheres, representing mammary stem/early progenitor cells. Although the frequency of mammosphere formation remained the same, the number of larger mammospheres (>60µm) from old mice were significantly lower than the young mice, probably indicating decreased stem cell activity in the older population. To study the effect of mutagen in vivo, we treated two young and two old mice with methyl nitrosourea (MNU, 25mg/kg body weight) once a week for three consecutive weeks. Animals were sacrificed one week after the last treatment. Both total and larger mammospheres (>60µm) from the isolated Lin−MEC of the treated older mice were observed in significantly higher number than the treated younger mice. In a recent clinical study larger mammospheres (>50µm) obtained from metastatic breast cancer cells has been correlated to their ability to induce tumors. These Lin−MEC cells were also analyzed and compared for DNA damage by alkaline comet assay and a higher percentage of cells (>10%) with irreversible DNA damage in the older population were observed compared to younger population (<2%) indicating probable lower DNA repair capacity of the former cells. Thus, our preliminary study indicated that enriched stem/progenitor cells in the old mice with decreased in vitro stem cell activity might have the potential to be vulnerable to DNA damage by mutagens and subsequent tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3469.

C-Jun Induces Mammary Epithelial Cellular Invasion and Breast Cancer Stem Cell Expansion

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

Effects of milk replacer formulation on measures of mammary growth and composition in Holstein heifers

Overfeeding prepubertal heifers may impair mammary parenchymal growth and reduce milk production, but evidence suggests that increased intake of a high-protein milk replacer before weaning may be beneficial. This study was designed to evaluate effects of milk replacer (MR) composition on mass and composition of mammary parenchyma and fat pad, growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. Specifically, we hypothesized that positive effects of faster rates of gain during the preweaning period alter the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed 1 of 4 MR diets (n=6/diet): control [20% crude protein (CP), 21% fat MR fed at 441g of dry matter (DM)/d], high protein, low fat (28% CP, 20% fat MR fed at 951g of DM/d), high protein, high fat (27% CP, 28% fat MR fed at 951g of DM/d), and high protein, high fat+ (27% CP, 28% fat MR fed at 1,431g of DM/d). Water and starter (20% CP, 1.43% fat) were offered ad libitum. Animals were killed on d 65 and mammary tissue was subjected to biochemical, molecular, and histological examination. No differences in mammary parenchymal mass or composition, with or without adjusting for empty body weight, were detected. Mass was increased and composition of the mammary fat pad was altered by nutrient intake. No diet differences in putative mammary epithelial stem cell abundance or abundance of transcripts for genes of the GH/IGF-I axis were detected. In this study, growth of the mammary epithelium, size of the mammary epithelial stem cell population, and components of the GH/IGF-I axis did not depend on diet. However, an underlying positive correlation between telomerase, a marker of mammary stem cells, and growth of the mammary parenchyma was detected. Implications of diet-induced effects on mammary fat pad and possible effects on subsequent development and function remain to be determined.

Efficacy, Safety, and Tolerability of Low-Dose Hormone Therapy in Managing Menopausal Symptoms

Use of the lowest clinically effective dose of postmenopausal hormone therapy conforms to current recommendations and good clinical practice. Although accumulating evidence demonstrates the efficacy and tolerability of low hormone therapy doses, data about their use are limited by a lack of long-term, randomized studies. This review evaluates current evidence on the efficacy, safety, and tolerability of these preparations and their role in menopausal management.