Mammary Growth Research Articles

Energy and lysine requirements and balances of sows during transition and lactation: A factorial approach

This study aimed to quantify daily requirements for metabolizable energy (ME) and standard ileal digestible (SID) lysine in late gestating and lactating sows using a factorial approach. Metabolizable energy and SID lysine required for fetal and mammary growth, colostrum and milk production, uterine components (including uterus wall, placenta and membrane fluids) and maintenance were estimated. It was estimated that maintenance, additional heat loss, colostrum production, fetal growth, mammary growth and uterine components accounted for 66.8%, 19.3%, 7.2%, 5.0%, 1.3% and 0.5% of total ME requirements, respectively, in the last 12 days of gestation. Oxidation/transamination, fetal growth, mammary growth, colostrum production, maintenance and uterine components were estimated to account for 29.5%, 22.7%, 16.8%, 16.1%, 10.4% and 4.5% of total SID lysine requirements, respectively, in the last 12 days of gestation. After parturition, ME and SID lysine requirements increased daily until peak lactation (day 17). At peak lactation, 95% and 72% of total required SID lysine and ME, respectively, were associated with milk production (including oxidation). Relative to day 104 of gestation, ME and SID lysine requirements increased by 60% and 149% at day 115 of gestation, and by 228%, and 338% at peak lactation, respectively. A dietary SID lysine: ME ratio of 0.55 was estimated as the ideal ratio to optimize milk production at peak lactation. It was estimated that the regressing uterus released about 30g SID lysine and 14MJ ME into the endogenous plasma pool during lactation. It was concluded that dramatic changes in energy and lysine requirements and balances occur during transition and lactation; that sows with high milk yield and /or low live weight require high SID lysine: ME ratio and that it would be beneficial to feed sows with two components at each meal to match the daily requirements for maintenance and production.

In Vitro Supplementation of Leucine Increases Murine HC11 Cell Proliferation

Mammary tissue undergoes multiples cycles of cell growth and proliferation during the lifetime of the female to meet the nutrient demand of the suckling neonate. On the basis of studies in rodents, pigs and humans demonstrating the anabolic effects of leucine (Leu) through the activation of mammalian target of rapamycin (mTOR) signaling pathway, there has been recent interest in the potential of supplementary Leu to improve skeletal muscle protein synthesis and overall nitrogen retention. To determine whether Leu supplementation also upregulates mTOR pathway in mammary tissue and increases mammary cell proliferation, a murine mammary cell line (HC11) was cultured in different concentrations of Leu (0, 1, and 2 mM). Cell proliferation was measured using flow cytometry at 0, 8, and 16 days. Reverse transcription quantitative PCR was performed to measure mRNA abundance of mammary Leu transporter LAT1 (SLC7A5), the intracellular Leu sensor for the mTOR signaling pathway leucyl‐tRNA synthetase (LARS2), mTOR and its downstream effector ribosomal protein S6 kinase (RPS6K1), and cyclin D (CCND1), a marker for cell proliferation. Guanine phosphoribosyl transferase (HPRT), RNA polymerase 3 (POL3), and ribosomal protein L3a (RPL3a) were used as reference genes. Statistical analysis was performed by ANOVA using a linear mixed model to account for treatment and replication effects. After 16 d cell proliferation increased in response to 1 mM Leu compared to 0 mM (P < 0.05), and increased further (P < 0.05) in cells supplemented with 2 mM Leu. Gene expression of SLC7A5, LARS2, mTOR, RPS6K1, and CCND1 did not differ between groups. Taken together, these results suggest that Leu supplementation increases mammary cell proliferation in vitro, but this effect is not associated with an increase in mRNA abundance of aforementioned genes. Further work is needed to elucidate the mechanisms by which dietary Leu supplementation increases mammary cell proliferation, as it may hold the potential to enhance mammary growth and subsequent milk production in animals.Support or Funding InformationARI 58982

Effects of Anethum graveolens L. (dill) Seed and Leaf Aqueous Extracts on the Growth of Mammary Gland Alveolar Buds of Rat

The effects of dill seed aqueous extract on the sex hormones have already been reported in our previous studies. In this study, we examined the oral administration of dill leaf and seed extracts on the mammary gland tissue that were exposed to these hormones. Adult female rats were divided into 3 groups of control, dill leaf and seed aqueous extracts (0.5 g/kg) treated groups (n = 7). After 25 days, the rats that were in estrus phase were dissected, and their pelvic mammary glands were spread on slides and stained by carmine. Then, the alveolar buds 1 and 2 (AB1 and AB2) and lobules of the whole mount mammary glands were counted and scored in tip, central and distal parts of mammary gland, then the ratio of mammary gland differentiation 1 and 2 (Dif1 and Dif2) was calculated. Estradiol concentration was measured via radioimmunoassay method and Progesterone, FSH and LH concentrations were measured using immunoradiometric assay. The data were analyzed by One-Way ANOVA test. The results showed that AB1 decreased but AB2 increased in total mammary gland of the experimental groups; however, these changes were not statistically significant. The numbers of lobules and Dif2 in the mammary gland were significantly increased in dill leaf and seed extracts treated groups. Progesterone concentration increased in dill seed extract-treated group. The results indicated that oral administration of dill seed and leaf aqueous extracts increases the mammary glands growth; therefore, dill may be considered as lactiferous herb.

Abstract P6-08-08: GATA3 inhibits breast basal-like tumorigenesis

Abstract BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation.BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation. Citation Format: Pei X-H, Chan HL, Liu S, Scott A, Pimentel E, Slingerland J, Robbins D, Capobianco A, Bai F. GATA3 inhibits breast basal-like tumorigenesis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-08.

In vivo reprogramming of non-mammary cells to an epithelial cell fate is independent of amphiregulin signaling.

Amphiregulin (AREG)-/- mice demonstrate impaired mammary development and form only rudimentary ductal epithelial trees; however, AREG-/- glands are still capable of undergoing alveologenesis and lactogenesis during pregnancy. Transplantation of AREG-/- mammary epithelial cells into cleared mouse mammary fat pads results in a diminished capacity for epithelial growth (∼15%) as compared to that of wild-type mammary epithelial cells. To determine whether estrogen receptor α (ERα, also known as ESR1) and/or AREG signaling were necessary for non-mammary cell redirection, we inoculated either ERα-/- or AREG-/- mammary cells with non-mammary progenitor cells (WAP-Cre/Rosa26LacZ+ male testicular cells or GFP-positive embryonic neuronal stem cells). ERα-/- cells possessed a limited ability to grow or reprogram non-mammary cells in transplanted mammary fat pads. AREG-/- mammary cells were capable of redirecting both types of non-mammary cell populations to mammary phenotypes in regenerating mammary outgrowths. Transplantation of fragments from AREG-reprogrammed chimeric outgrowths resulted in secondary outgrowths in six out of ten fat pads, demonstrating the self-renewing capacity of the redirected non-mammary cells to contribute new progeny to chimeric outgrowths. Nestin was detected at the leading edges of developing alveoli, suggesting that its expression may be essential for lobular expansion.

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted invivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation invitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Abstract C28: Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment

Abstract A role for the tumor microenvironment (TME) in cancer progression is irrefutable and our laboratory has been at the forefront of this field providing evidence for both tumor suppressive and oncogenic roles of the TME. The PDGF pathway is an exemplar for the study of tumor-stroma interaction as PDGF receptors (PDGFR) are frequently expressed in the fibroblasts and pericytes within the tumor-associated stroma of epithelial tumors including breast cancer. In contrast, PDGF ligands are expressed by the epithelial tumor cells themselves. However, beyond a few descriptive studies, the role of interactive PDGFRβ signaling in the TME during breast cancer initiation, progression and metastases is not understood. This can be attributed in part to limited in vivo models to study the complex TME, especially for breast cancer associated metastases. To overcome this limitation, we have established a transgenic knock-in mouse model that expresses constitutively active PDGFRβ in the stroma of the mammary gland as well in the lung and the brain, two common sites of metastatic breast cancer dissemination. We have found that these mice develop mammary gland hyperplasia highlighting the importance of PDGFRβ in the TME in driving mammary epithelial cell growth. To test whether activation of mutant PDGFRβ in either the lung or the brain increases metastatic growth at either site, two experimental metastases assays were performed: (1) tail vein and (2) intracranial injections to test for lung and brain metastatic outgrowth, respectively. Tail vein injection of the non-metastatic murine mammary cancer cell line DB7 cells led to pronounced lung metastases in PDGFRβ knock-in mice in less than 4 weeks. No macrometastases were seen in the control at this same time point. Similar to the surge in lung metastasis, intracranial injection of DB7 cells led to an increase in tumor growth in brains of the mutant versus wild type controls, revealing an important role for PDGFRβ signaling in the breast cancer metastatic microenvironment. In addition, knockdown of PDGF-B in mammary cancer cells represses intracranial growth in wild type animals. Combined these data support a role for PDGFRβ signaling in the breast cancer metastatic microenvironment. Ongoing investigation is aimed to delineate how activated PDGF-B to PDGFRβ signaling primes the metastatic niche. Citation Format: Gina Sizemore, Anisha Mathur, Katie Thies, Chelsea Bolyard, Steven Sizemore, Raleigh Kladney, Anthony Trimboli, Balveen Kaur, Gustavo Leone, Michael Ostrowski. Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C28.

Telomerase and estrogen-sensing activities are essential for continued mammary growth in vivo but dispensable for "reprogramming" neural stem cells.

It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland in vivo, we serially transplanted mammary fragments from wild type (TER+/+), heterozygous (TER+/−), and homozygous (TER−/−) mammary tissues into the cleared mammary fat pads of immune-compromised nude mice. Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/− cells were able to direct neural stem cells to mammary cell fates.

Abstract 3681: Nischarin disruption promotes breast tumor development in mouse models

Abstract Nischarin is a novel tumor suppressor that was first discovered and characterized in our laboratory. The accumulating evidence suggests that Nischarin is a critical regulator of cell migration and breast tumor growth, metastasis and invasion. However, little is known about the exact function of Nischarin in in vivo physiological conditions, probably due to lack of any transgenic Nischarin animals. We recently generated Nischarin knockout mouse. This Nischarin knockout mouse has a dramatic unusual phenotype of mammary development and overall growth defect. Nischarin knockout mice do not spontaneously develop breast tumors. To determine the importance of Nischarin in regulation of breast cancer development, we bred Nischarin knockout virgin mice with PyMT (polyoma middle T antigen) and Neu background mice and the progeny produced tumors exclusively in the mammary gland. We found Nischarin knockout virgins with PyMT accelerated breast cancer onset and progression, which further suggests Nischarin inhibits tumor growth. Mammary epithelial cells (MECs) are surrounded by basement membrane, which mainly consists of extracellular matrix. We noted that the basement membrane of Nischarin knockout virgins contain much more collagen and fibronectin than those of wild type virgins. In addition, we observed that Nischarin disruption significantly changed extracellular acidification rates (ECAR) in mouse primary breast tumour cells. We suspected that this changed acidic microenvironment might facilitate breast tumor progression. Recent studies indicated that clinical anti-diabetic drug Metformin, an AMPK activator, inhibits breast cancer cell growth. Our data show that Het-PyMT and Null-PyMT primary cells were much more sensitive for metformin treatment. Thus we examined the importance of AMPK signaling in our mouse models. Interestingly, both tumor tissues and primary cells derived from Null-PyMT breast tumors had lower AMPK activity. Together, our data strongly support that Nischarin disruption promotes breast tumor development and AMPK signaling is important for Nischarin-mediated suppression of breast tumor. Citation Format: Shengli Dong, Suresh K. Alahari. Nischarin disruption promotes breast tumor development in mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3681.

Consumption of endophyte-infected fescue seed during the dry period does not decrease milk production in the following lactation

Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90±4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (−60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation.

Multiple Endocrine Disrupting Effects in Rats Perinatally Exposed to Butylparaben.

Parabens comprise a group of preservatives commonly added to cosmetics, lotions, and other consumer products. Butylparaben has estrogenic and antiandrogenic properties and is known to reduce sperm counts in rats following perinatal exposure. Whether butylparaben exposure can affect other endocrine sensitive endpoints, however, remains largely unknown. In this study, time-mated Wistar rats (n = 18) were orally exposed to 0, 10, 100, or 500 mg/kg bw/d of butylparaben from gestation day 7 to pup day 22. Several endocrine-sensitive endpoints were adversely affected. In the 2 highest dose groups, the anogenital distance of newborn male and female offspring was significantly reduced, and in prepubertal females, ovary weights were reduced and mammary gland outgrowth was increased. In male offspring, sperm count was significantly reduced at all doses from 10 mg/kg bw/d. Testicular CYP19a1 (aromatase) expression was reduced in prepubertal, but not adult animals exposed to butylparaben. In adult testes, Nr5a1 expression was reduced at all doses, indicating persistent disruption of steroidogenesis. Prostate histology was altered at prepuberty and adult prostate weights were reduced in the high dose group. Thus, butylparaben exerted endocrine disrupting effects on both male and female offspring. The observed adverse developmental effect on sperm count at the lowest dose is highly relevant to risk assessment, as this is the lowest observed adverse effect level in a study on perinatal exposure to butylparaben.

Low-dose effects of bisphenol A on mammary gland development in rats.

Bisphenol A (BPA) is widely used in food contact materials, toys, and other products. Several studies have indicated that effects observed at doses near human exposure levels may not be observed at higher doses. Many studies have shown effects on mammary glands at low doses of BPA, however, because of small number of animals or few doses investigated these data have not been used by EFSA as point of departure for the newly assessed tolerable daily intake (TDI). We performed a study with perinatal exposure to BPA (0, 0.025, 0.25, 5, and 50mg/kg bw/day) in rats (n=22 mated/group). One of the aims was to perform a study robust enough to contribute to the risk assessment of BPA and to elucidate possible biphasic dose-response relationships. We investigated mammary gland effects in the offspring at 22, 100, and 400days of age. Male offspring showed increased mammary outgrowth on pup day (PD) 22 at 0.025mg/kg BPA, indicating an increased mammary development at this low dose only. Increased prevalence of intraductal hyperplasia was observed in BPA females exposed to 0.25mg/kg at PD 400, but not at PD 100, and not at higher or lower doses. The present findings support data from the published literature showing that perinatal exposure to BPA can induce increased mammary growth and proliferative lesions in rodents. Our results indicate that low-dose exposure to BPA can affect mammary gland development in male and female rats, although higher doses show a different pattern of effects. The observed intraductal hyperplasia in female rats could be associated with an increased risk for developing hyperplastic lesions, which are parallels to early signs of breast neoplasia in women. Collectively, current knowledge on effects of BPA on mammary gland at low doses indicates that highly exposed humans may not be sufficiently protected.

Mammary tumor growth and metastasis are reduced in c-Kit mutant Sash mice.

Besides its well‐known function in allergic response, mast cell, one of the key immune cells present in tumor microenvironment, plays important roles in cancer progression. However, the functional role of mast cells in breast cancer development and metastasis is not well understood. To test the involvement of mast cells in breast cancer, we examined the effects of loss of mast cells on mammary tumor development by crossing the well‐known mast cell deficient mouse strain sash (KitW‐sh/W‐sh) with the mammary tumor transgenic mouse strain MMTV‐Polyoma Middle T antigen (PyMT). Although mammary tumor onset was not affected in the absence of mast cells, mammary growth and metastasis were reduced in PyMT/KitW‐sh/W‐sh mice compared with PyMT/wild‐type mice (WT). Histological and immunofluorescent analyses showed that tumors from PyMT/KitW‐sh/W‐sh mice showed largely differentiated morphology with reduced angiogenesis compared with MMTV‐PyMT/WT mice. Our results suggest that mast cells may promote breast cancer growth and metastasis. Agents that can block mast cells growth are potential new therapies to treat metastatic breast cancer.

Abstract P3-04-02: Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are estrogen receptor (ER)-positive, however, endocrine response and estrogen signaling are not well understood in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Based on these observations, we hypothesize that ER regulates unique signaling pathways in ILC cells that control growth and endocrine response. To identify putative targets that regulate endocrine response in ILC, we assessed genome-wide ER-mediated gene expression and ER genomic binding in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44). Among ILC-specific estrogen-regulated genes, the most strongly induced was the secreted ligand WNT4. Additionally, we identified an ILC-specific ER binding site (ERBS) at WNT4, suggesting that WNT4 is directly controlled by ER in ILC cells. Direct ER-regulation of WNT4 is in contrast to control of WNT4 by progesterone receptor (PR) during mammary gland development; however, further ER ChIP experiments suggest that ILC cells place WNT4 under ER control via unique use of the WNT4 ERBS. Thus, ILC cells may hijack a tightly regulated developmental program to drive estrogen-regulated cell phenotypes. Based on the role of WNT4 in mammary gland growth and expansion, we hypothesized that WNT4 is required for estrogen-induced growth in ILC cells. To test this, we used siRNA to knock down WNT4 in ILC and IDC cell lines. Using either of two siRNAs, WNT4 knockdown completely blocked estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells, suggesting that these genes are downstream of WNT4 signaling. Wnt signaling typically acts via the canonical, β-catenin-dependent pathway; however, we observed that β-catenin is dysfunctional in ILC cells, and WNT4 cannot activate β-catenin signaling in cancer cell lines. This suggests that WNT4 regulates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Clinical observations suggest that ER regulates unique downstream pathways in ILC. We identified WNT4 as a putative downstream effector of ER signaling in ILC, as WNT4 is strongly-induced and directly regulated by ER specifically in ILC cells. Further, WNT4 is necessary for estrogen-induced growth in ILC cells, and likely signals via a non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on characterizing the signaling pathway controlled by WNT4 in order to identify putative therapeutic targets. Citation Format: Sikora MJ, Oesterreich S. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-02.

Abstract P5-06-01: The role of thyroid hormones in breast tumorigenesis: A translational study utilizing mouse models, cell culture and patient data

Abstract Breast cancer and thyroid hormone signaling have been linked since the 1960s. Breast cancer patients have a higher incidence of thyroid cancer, and thyroid cancer patients have a higher incidence of breast cancer than would be predicted by chance alone, supporting a link between thyroid hormone signaling and breast malignancy. Despite many correlative studies, the role and mechanism of thyroid hormone signaling in mammary tumorigenesis has not been elucidated. Past studies have not comprehensively evaluated thyroid hormones (T3, T4) and thyroid stimulating hormone (TSH) levels with breast cancer status in the same individual. The results are further confounded by issues of temporality; studies have either assessed thyroid hormone levels before or after diagnosis, prohibiting conclusions regarding whether thyroid disruption is a cause or effect of breast cancer. In vitro models demonstrate that mammary cells have thyroid hormone receptors and respond to thyroid hormones; however these studies have not been extended to in vivo models. In the current study, we took a translational approach by combining data from cell culture, mouse models and breast cancer patients from the City of Hope Cancer Registry (CHCR). We used the murine MMTV-PyMT model of breast cancer and treated mice with the anti-thyroid drug PTU; lowering T4 levels and increasing TSH. These mice developed significantly larger mammary tumors than untreated animals or those treated with T4 (p= 0.0012 and p=0.0183, respectively). Next, we showed that MCF10a cells in vitro are sensitive to both T4 and TSH added to the culture medium. We hypothesize that even subtle perturbations in normal thyroid hormone levels can stimulate mammary cell growth, increasing risk of transformation. We extended these findings to a small pilot study of 879 invasive and 136 in situ female breast cancer patients with comprehensive thyroid hormone lab analyses in the CHCR (total=984). Pre-treatment results were available for 44 women. TSH was significantly elevated for invasive versus in situ patients (Pt-test =0.016), regardless of the timing of the test. Pre-treatment TSH levels were significantly increased as the severity of disease increased (Pt-test =0.026). Women diagnosed stage 1 disease with no recurrence had a mean TSH level of 1.68 ± 1.87 (Standard Deviation: SD), whereas women diagnosed with stage 1 disease who returned with metastasis had a mean of 2.64 ± 1.72 SD. In the same women, free T4 and total T4 levels were lower and T3 levels were higher (Pt-test< 0.05) in women with invasive versus in situ disease. These studies support the hypothesis that even minimally dysregulated thyroid hormone levels may increase the risk of breast cancer development and progression to aggressive disease. Many women over the age of 50 suffer from sub-clinical hypothyroidism, and our results suggest that sub-clinical hypothyroidism increases breast cancer risk and disease progression. Interestingly, our data indicate that as disease progresses, dependence on TSH lessens, and the most striking differences may be seen in early and low grade disease. Collectively, our data highlight a need to further investigate the role of sub-clinical hypothyroidism in breast cancer. Citation Format: Franco A, Jolly LA, Russell S, Goldstein L, DeHart J. The role of thyroid hormones in breast tumorigenesis: A translational study utilizing mouse models, cell culture and patient data. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-01.

Abstract IA13: Clonal dynamics of normal and malignant human mammary cell growth in xenografts

Abstract Most human breast cancers have diversified genomically and biologically by the time they become clinically evident and little is known about their origin from normal human mammary cells, or the cellular and molecular mechanisms that lead to their genesis and evolution. We have developed methods to quantify, purify and characterize different subsets of normal human mammary cells and have used these to identify properties that may influence their propensity for transformation. We have also developed methods for inducing the rapid transformation in vivo of these purified subsets following their transplantation into immunodeficient mice. The results demonstrate the ability of a single oncogene (KRASG12D) to induce the formation of serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. Both primary and secondary tumors are phenotypically heterogeneous and transcriptome analyses of primary tumors assign them to a “normal-like” category. DNA barcoding of the cells at the time of their initial transduction with KRASG12D has revealed a dramatic change in the numbers and sizes of clones they generate after 2 weeks in vivo. DNA barcoding also showed the unexpected appearance of many “new” clones in tumors generated upon passage into secondary recipients, thus recapitulating some features of in vivo passaged human breast cancer cell lines and patients’ tumor xenografts. This system challenges previous concepts about the process of human mammary oncogenesis and provides a new system for analyzing factors that can influence its speed, efficiency and heterogeneity of outcomes. Citation Format: Connie J. Eaves, Long Nguyen, Davide Pellacani, Nagarajan Kannan, Sylvan Lefort, Sneha Balani, Claire Cox, Tomo Osako, Samuel Aparicio, Martin Hirst. Clonal dynamics of normal and malignant human mammary cell growth in xenografts. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA13.

Effects of late-gestation heat stress on immunity and performance of calves

Lactating cows that experience heat stress will have reduced dry matter intake and milk yield and shift metabolism, which ultimately reduces the efficiency of milk production. Dry cows that are heat stressed similarly experience lower intake, reduced mammary growth, and compromised immune function that ultimately results in a poorer transition into lactation and lower milk yield in the next lactation. A recent focus in our laboratory is on the effects of late gestation, in utero heat stress on calf survival and performance. We have completed a series of studies to examine preweaning growth and health, and later reproductive and productive responses, in an attempt to quantify acute and persistent effects of in utero heat strain. Late gestation heat stress results in calves with lower body weight at birth, shorter stature at weaning, and failure to achieve the same weight or height at 12mo of age observed in calves from dams that are cooled when dry. A portion of the reduced growth may result from the lower immune status observed in calves heat stressed in utero, which begins with poorer apparent efficiency of immunoglobulin absorption and extends to lower survival rates through puberty. Heat-stressed calves, however, have permanent shifts in metabolism that are consistent with greater peripheral accumulation of energy and less lean growth relative to those from cooled dams. Comparing reproductive performance in calves heat stressed versus those cooled in utero, we observe that the cooled heifers require fewer services to attain pregnancy and become pregnant at an earlier age. Tracking the milk production in calves that were heat stressed in utero versus those cooled in late gestation revealed a significant reduction of yield in the first lactation, approximately 5kg/d through 35 wk of lactation, despite similar body weight and condition score at calving. These observations indicate that a relatively brief period of heat stress in late gestation dramatically alters the health, growth, and ultimate performance of dairy calves. Thus, it is critical to effectively manage heat stress of dry cows to avoid negative effects on the calf.

Abstract A74: Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions

Abstract Breast cancer is one of the frequently diagnosed and life-threatening cancer diseases in women worldwide, especially, the triple negative breast cancer (ER-/PR-/HER2- TNBC) is a clinically challenging breast cancer disease due to lack of efficient targeted therapeutics. Recently, the development of phytocompounds derived from traditional medicinal herbs into potential chemotherapeutic or chemopreventive agent for human cancer management has aroused a great interest. In our previous study, we identified deoxyelephantopin (DET), a major germacranolide sesquiterpene lactone from a traditional medicinal herb Elephantopus scaber L., which could significantly suppress TS/A (ER+) mammary cancer cell growth, motility and metastasis in vitro andin vivo; however, a relatively less suppressive effect was observed in human TNBC cell line, MDA-MB-231. We thus designed and created a DET derivative (designated DETD) by semi-organic synthesis, which exhibited a 4-fold less in IC50 value than DET in inhibiting MDA-MB-231 cell proliferation. To address the modes of action of both authentic DET and novel DETD compounds against TNBC, we established and used mass spectrometry (MS)-based quantitative proteomics, direct binding assay of DET-near infrared (NIR) fluorescent compound conjugate, and mammary tumor model to gain the mechanistic insight. Our data showed that DET and DETD can markedly induce the reactive oxygen species (ROS) production in early stage of treatment, that may further promote nonautophagic cell death through cytoplasmic vacuoles production. Furthermore, we observed the release of exosome vesicles from MDA-MB-231 cells was elevated by DET or DETD treatment compared to the vehicle control. Quantitative investigation of exosomal proteome showed that DET and DETD responsive exosomal proteins were involved in the regulation of EIF2 signaling, mTOR signaling, regulation of eIF4 and p70S6K signaling, CDK5 signaling, and inhibition of angiogenesis. Of note, the immunofluorescence cell staining showed that DET-NIR was co-localized with exosomes of MDA-MB-231 cells, on the other hand, several nuclear exosomal proteins of mouse TNBC 4T1 cells could interact directly with DET. Together results of this study suggest that DET and DETD compounds may serve as a good candidate in treatment of TNBC disease, through modulation of oxidative stress and specific exosomal proteins in cancer cells. Citation Format: Jeng-Yuan Shiau, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Wai-Leng Lee, Jacquelyn Gervay-Hague, Lie-Fen Shyur. Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A74.