Mammary Adipocytes Research Articles

Abstract P1-03-02: Estrogens contribute to cytokine upregulation and cancer stem cell recruitment upon breast cancer contact with mature human mammary adipocytes: Effects of estrogen type and adipocyte donor weight

Abstract Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. While obesity confers increased cancer risk and worse outcome, mechanisms thereof are not fully known. We show prolonged co-culture of fat cells (human adipocyte stem cells, differentiated adipocytes or mature adipocytes) from breast tissue together with breast cancer lines or cultured primary dissociated human breast tumor cells increases secretion of six different pro-inflammatory cytokines, each of which contributes to tumor progression through cancer stem cell recruitment. Prolonged exposure to fat cells or to each cytokine increases the proportion of cells that form mammosphere and express ALDH1 activity in vitro and that can initiate primary orthotopic tumors and metastasis in vivo. Adipocyte and cytokine exposures activate Src, and Src family kinase activity leads to induction of embryonic transcription factors that upregulate miR302b. miR302b induction is Sox2-dependent, promotes cytokine-driven sphere formation, and in turn, stimulates cMYC and SOX2 expression. Src is not only activated by adipocyte or cytokine exposures, it is also required to sustain cytokine induction, since Src inhibitors decrease cytokine production after co-culture. Cytokine upregulation was much greater after co-culture of ER+ breast cancer cells with mature, aromatase positive, adipocytes than with adipocyte stem cells. Cytokine induction was estrogen regulated. The mechanisms of cytokine induction, ER-coactivation and effects of different estrogenic ligands will be presented. Present data illuminate the increased risk of breast cancer after menopause, particularly in obese women and the increased breast cancer mortality with obesity: cancer cell invasion into local fat, in the presence of high local aromatase and intracellular estrogen would establish feed-forward loops to activate Src, maintain pro-inflammatory cytokine production and increase tumor initiating cell abundance, tumor growth and metastasis. These data link obesity related pro-inflammatory cytokines to Src activation and cancer initiating cell abundance, and provide a novel rationale for Src inhibitors together with endocrine therapy for breast cancer. Citation Format: Slingerland J, Picon-Ruiz M, Jang K, Morata-Tarifa C, Pan C, Besser A, Kim M, Ince TA, Howard GA, El-Ashry D. Estrogens contribute to cytokine upregulation and cancer stem cell recruitment upon breast cancer contact with mature human mammary adipocytes: Effects of estrogen type and adipocyte donor weight. [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 P1-03-02.

Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration.

Mammary branching morphogenesis is regulated by receptor tyrosine kinases (RTKs). We sought to determine how these RTK signals alter proliferation and migration to accomplish tube elongation in mouse. Both behaviors occur but it has been difficult to determine their relative contribution to elongation in vivo, as mammary adipocytes scatter light and limit the depth of optical imaging. Accordingly, we utilized 3D culture to study elongation in an experimentally accessible setting. We first used antibodies to localize RTK signals and discovered that phosphorylated ERK1/2 (pERK) was spatially enriched in cells near the front of elongating ducts, whereas phosphorylated AKT was ubiquitous. We next observed a gradient of cell migration speeds from rear to front of elongating ducts, with the front characterized by both high pERK and the fastest cells. Furthermore, cells within elongating ducts oriented both their protrusions and their migration in the direction of tube elongation. By contrast, cells within the organoid body were isotropically protrusive. We next tested the requirement for proliferation and migration. Early inhibition of proliferation blocked the creation of migratory cells, whereas late inhibition of proliferation did not block continued duct elongation. By contrast, pharmacological inhibition of either MEK or Rac1 signaling acutely blocked both cell migration and duct elongation. Finally, conditional induction of MEK activity was sufficient to induce collective cell migration and ductal elongation. Our data suggest a model for ductal elongation in which RTK-dependent proliferation creates motile cells with high pERK, the collective migration of which acutely requires both MEK and Rac1 signaling.

Molecular aspects of adipoepithelial transdifferentiation in mouse mammary gland.

The circular, reversible conversion of the mammary gland during pregnancy and involution is a paradigm of physiological tissue plasticity. The two most prominent cell types in mammary gland, adipocytes and epithelial cells, interact in an orchestrated way to coordinate this process. Previously, we showed that this conversion is at least partly achieved by reciprocal transdifferentiation between mammary adipocytes and lobulo-alveolar epithelial cells. Here, we aim to shed more light on the regulators of mammary transdifferentiation. Using immunohistochemistry with cell type-specific lipid droplet-coating markers (Perilipin1 and 2), we show that cells with an intermediate adipoepithelial phenotype exist during and after pregnancy. Nuclei of cells with similar transitional structural characteristics are highly positive for Elf5, a master regulator of alveologenesis. In cultured adipocytes, we could show that transient and stable ectopic expression of Elf5 induces expression of the milk component whey acidic protein, although the general adipocyte phenotype is not affected suggesting that additional pioneering factors are necessary. Furthermore, the lack of transdifferentiation of adipocytes during pregnancy after clearing of the epithelial compartment indicates that transdifferentiation signals must emanate from the epithelial part. To explore candidate genes potentially involved in the transdifferentiation process, we devised a high-throughput gene expression study to compare cleared mammary fat pads with developing, contralateral controls at several time points during pregnancy. Incorporation of bioinformatic predictions of secretory proteins provides new insights into possible paracrine signaling pathways and downstream transdifferentiation factors. We discuss a potential role for osteopontin (secreted phosphoprotein 1 [Spp1]) signaling through integrins to induce adipoepithelial transdifferentiation.

Abstract B17: Social isolation stress promotes obesity and mammary tumorigenesis and increases both Mdm2 and p53 expression and autophagy in the mammary gland

Abstract Background: Social isolation is a potent psychosocial stressor and is associated with increase breast cancer mortality in human and animal studies. The effects of social isolation are unlikely to be mediated through an increase in stress hormone levels or macrophage-mediated immune responses, as these changes are transient and normalize during chronic social isolation. A recent study reported that an exposure to a persistent social isolation induced metabolic reprogramming in mammary gland adipocytes. We therefore tested whether the effects of social isolation on mammary cancer are potentiated in mice fed obesity-inducing high fat (HF) diet. Female C57BL/6 mice were group housed or isolated at weaning (3 weeks of age) and fed either AIN93G -based control or HF diet. Mammary tumors were induced by priming mammary glands with progestin (MPA) and then gavaging mice with DMBA weekly for 4 weeks. Results: Mice that were isolated and fed HF diet, became obese and developed insulin resistance. Mammary tumorigenesis was highest in socially-isolated obese mice, and also higher in group-housed, obese mice and isolated, normal weight mice, when compared to group-housed mice fed a control diet (p<0.001). Serum leptin levels were not affected by stress, but adiponectin levels were reduced in mice housed in social isolation (p=0.042), compared to group-housed mice. We found no evidence of inflammatory changes (expression of Tnf-α, Il1b or Il6, or macrophage number) in the mammary glands of the socially-isolated mice, and neither was the expression of tumor suppressor genes p16, p21 or Sirt1 altered by stress or diet. Since social isolation reduces mammary tumorigenesis in heterozygous p53 knockout mice, and p53 is known to respond to metabolic changes and to influence metabolic pathways, we studied whether its expression was altered by social stress. The results indicated that social isolation (p<0.001) (but not diet) significantly increased p53 mRNA expression. Apoptosis in Tunel assay was not altered, but cell proliferation, measured by Ki67 staining, was significantly increased in socially-isolated mice (p=0.002). The latter is consistent with a significant upregulation of cyclin D1 in the stressed mice (p<0.001). As the expression of Jnk1 (p=0.003) and Dram1 (p=0.003) were elevated in the mammary glands of stressed mice, p53-induced autophagy may have been increased. Finally, Mdm2 was upregulated in these mice (p=0.001), indicating that the negative feedback between this gene and p53 was disrupted by social isolation. All the observed changes were most apparent in HF fed, socially-isolated mice, but also seen in control diet fed mice exposed to social isolation stress. Conclusion: Our data suggest that HF-induced obesity potentiates the effects of social isolation on mammary carcinogenesis, and together, perhaps by a disruption of the Mdm2–p53 interaction and induction of genes that stimulate autophagy, increase mammary tumorigenesis in mice. Citation Format: Allison Sumis, Fabia O. Andrade, Leena A. Hilakivi-Clarke. Social isolation stress promotes obesity and mammary tumorigenesis and increases both Mdm2 and p53 expression and autophagy in the mammary gland. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B17.

Chronic Social Isolation Is Associated with Metabolic Gene Expression Changes Specific to Mammary Adipose Tissue

Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of "triple-negative" breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e., during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2, and Acly was found in the adipocyte, rather than the epithelial fraction. Surprisingly, metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected, increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin secretion from this adipose depot. Furthermore, application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed-conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent preinvasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer.

Regulation of apoptosis in adipocytes and breast cancer cells by 1,25-dihydroxyvitamin D3: a link between obesity and breast cancer.

Modulation of apoptosis is emerging as a promising strategy for prevention and treatment of breast cancer and obesity because removal of mammary cancer cells and mature adipocytes through this process will result in decreasing tumor size and produce long-term reduction in adipose tissue mass. The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) triggers apoptosis in breast cancer cells and adipocytes via the induction of the apoptotic Ca2+ signal - a sustained increase in concentration of intracellular Ca2+. This signal acts as an apoptotic initiator that directly recruits Ca2+-dependent apoptotic effectors, calpain and caspase 12, in breast cancer cells and adipocytes. Normal mammary epithelial cells are resistant to 1,25(OH)2D3-induced, Ca2+-mediated apoptosis because the mechanisms regulating Ca2+ in these cells do not sustain Ca2+ increase at the apoptosis-inducing level. Induction of apoptosis with 1,25(OH)2D3 in adipose tissue, particularly in the tumor-surrounding adipose tissue involved in tumor progression, can contribute to the anticancer effects of the hormone. The 1,25(OH)2D3-Ca2+ link between obesity and breast cancer supports the rationale to include Ca2+-dependent apoptotic proteases as molecular targets for the discovery of new therapeutic and preventive agents for breast cancer and obesity; it also supports the recommendation to maintain adequate or increased vitamin D and calcium intakes as one of the possible ways to protect against breast cancer and decrease adiposity.

Abstract 1354: Chronic social stress in a mouse model of triple-negative breast cancer is associated with oncogenic changes in mammary fat.

Abstract Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen (Tag) FVB/N mouse model of “triple-negative” breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e. during the in situ carcinoma stage). To further understand how underlying accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2 and Acly was found primarily in the adipocyte, rather than epithelial fraction. Increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin expression. Furthermore, culture media from isolated mouse mammary adipose tissue resulted in relatively increased proliferation and decreased apoptosis of mammary epithelial cancer cells derived from the same mouse strain. Additional gene expression analyses identified significant upregulation of the novel carbohydrate response element binding protein beta isoform (CHREBP-β) in socially isolated mammary fat. We hypothesize that the response to social isolation induces metabolic changes in mammary fat that are coordinated through CHREBP-β and are pro-oncogenic. We are currently performing global lipidomic and metabolomic analyses of mammary adipose tissue and secreted culture media to identify bioactive molecules involved in tumor growth. Citation Format: Paul A. Volden, Erin Wonder, Maxwell Skor, Feenalie Patel, Martha McClintock, Matthew Brady, Suzanne Conzen. Chronic social stress in a mouse model of triple-negative breast cancer is associated with oncogenic changes in mammary fat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2013-1354

Abstract PL01-04: Stress and the social environment: Impact on cancer risk

Abstract The breast cancer research community has long considered the impact of exogenous estrogen exposure, nutrition, alcohol consumption, and exercise on breast cancer biology. More recently, experimental studies of exposure to chronic stressors in the social environment and the resulting stress response have suggested that epinephrine- and glucocorticoid-mediated signaling pathways might potentiate breast cancer growth both at primary and metastatic tumor sites. To study this hypothesis in more detail, we initiated a transdisciplinary study of a well-established social stressor for female rodents- chronic social isolation. We found that exposing a young female rodent to chronic socially isolation for 12 weeks (compared to group living/social interaction with three other female mice) was associated with increased mammary tumor growth in genetically predisposed rodents. For example, in the C3(1)/SV40 T-antigen (Tag) FVB/N mouse model of “triple-negative” (ER,PR and ErbB-negative breast cancer) the heightened stress response elicited by chronic social isolation was associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e. during the in situ carcinoma stage when the animals are 15 weeks of age). This suggested that metabolic changes within the mammary gland could affect breast cancer biology. To further understand how accelerated mammary tumor growth may be associated with the physiological response to social isolation, we separated the mammary gland adipose tissue from its adjacent ductal epithelial cells and then analyzed individual cell types for changes in metabolic gene expression. We found that the major increase in expression of the key metabolic genes Acaca, Hk2 and Acly was primarily in the adipocyte (fat cell) fraction of the mammary gland. In contrast, metabolic gene expression was not significantly increased in visceral or gonadal adipose depots of socially isolated female mice, suggesting that the stress response affects individual fat depots differently in this model. We confirmed that the increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and local leptin production; interestingly, systemic leptin levels were not significantly altered. Furthermore, compared to the group housed mammary fat, application of media that had been cultured with isolated mouse mammary adipose tissue resulted in relatively increased proliferation of mammary cancer cells. These results suggest that the physiological response to a chronic social stressor (isolation) results in metabolic changes in mammary gland adipocytes that in turn contribute to increased proliferation of pre-invasive malignant epithelial cells. Adipocyte-secreted metabolites and/or tumor growth-promoting proteins could identify biomarkers and/or targets for preventive intervention in breast cancer. We are currently examining the mammary fat of the isolated versus group-housed mice to identify differential production of adipokines and lipid species that may influence early breast cancer growth in this model. Citation Format: Suzanne Conzen. Stress and the social environment: Impact on cancer risk. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PL01-04.

Adipocyte-released insulin-like growth factor-1 is regulated by glucose and fatty acids and controls breast cancer cell growth in vitro

Aims/hypothesisType 2 diabetes and obesity are associated with increased risk of site-specific cancers. We have investigated whether metabolic alterations at the level of adipose-derived differentiating cells may affect specific phenotypes of breast cancer cells.MethodsGrowth profiles of breast cancer cell lines were evaluated in co-cultures with differentiated adipocytes or their precursor cells and upon treatment with adipocyte conditioned media. Production and release of cytokines and growth factors were assessed by real-time RT-PCR and multiplex-based ELISA assays.ResultsCo-cultures with either differentiated mouse 3T3-L1 or human mammary adipocytes increased viability of MCF-7 cells to a greater extent, when compared with their undifferentiated precursors. Adipocytes cultured in 25 mmol/l glucose were twofold more effective in promoting cell growth, compared with those grown in 5.5 mmol/l glucose, and activated mitogenic pathways in MCF-7 cells. Growth-promoting action was also enhanced when adipocytes were incubated in the presence of palmitate or oleate. Interestingly, 3T3-L1 and human adipocytes released higher amounts of keratinocyte-derived chemokine/IL-8, the protein ‘regulated upon activation, normally T expressed, and secreted’ (RANTES), and IGF-1, compared with their precursor cells. Their levels were reduced upon incubation with low glucose and enhanced by fatty acids. Moreover, both undifferentiated cells and differentiated adipocytes from obese individuals displayed about twofold higher IGF-1 release and MCF-7 cell growth induction than lean individuals. Finally, inhibition of the IGF-1 pathway almost completely prevented the growth-promoting effect of adipocytes on breast cancer cells.Conclusions/interpretationIGF-1 release by adipocytes is regulated by glucose and fatty acids and may contribute to the control of cancer cell growth in obese individuals.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2629-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Abstract 4116: Mammary adipocyte-specific metabolic alterations are associated with paracrine effects on mammary tumorigenesis

Abstract In rodent models of breast cancer, chronic social stress from isolated housing is associated with increased mammary tumor growth. Interestingly, in several diverse mouse strains, social isolation is also associated with increased expression of the key metabolic genes Acaca, Hk2 and Acly in the adipocyte fraction of the mammary gland. We hypothesized that these gene expression changes would be associated with increased mammary fat glucose metabolism, lipid synthesis, and secretion of paracrine growth promoting adipokines. We found that increased metabolic gene expression in mammary fat did indeed increase both lipid metabolism and leptin secretion from the mammary adipocytes of isolated mice. Surprisingly, visceral fat metabolism and systemic metabolic parameters were unchanged in social isolates, suggesting an effect of social stress that is specific to the mammary fat depot. When mouse mammary cancer cells were treated with culture media derived from the incubation of isolated versus group-housed mouse mammary adipose tissue, the mammary fat secretome of social isolates resulted in significantly increased cancer cell proliferation compared to mammary fat from grouped mice. Current experiments are designed to elucidate the metabolic factors promoting tumor cell proliferation in the mammary fat derived from the stressed animals. We propose a model wherein social isolation and the ensuing heightened neuroendocrine response significantly alters mammary adipocyte metabolism and associated adipokine secretion, thereby potentiating estrogen-independent mammary tumor growth. These studies have implications for understanding the environmental, endocrine, and molecular risk factors associated with estrogen receptor-negative breast cancer. DOD W81XWH-11-1-014901, NIH/NIDDK T32 DK087703, NIH R01CA148814 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4116. doi:1538-7445.AM2012-4116

Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor, modulating mammary epithelial cell growth

The vitamin D(3) receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D(3) synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D(3) -induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α-hydroxylase, 1α-OHase), the enzyme that generates the biologically active VDR ligand, 1α,25-dihydroxyvitamin D(3) (1,25D(3) ), and CYP24 (24-hydroxylase, 24-OHase), a VDR-1,25D(3) induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25-hydroxyvitamin D(3) (25D(3) ) to the active ligand, 1,25D(3) , and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co-cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D(3) or 1,25D(3) compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D(3) to 1,25D(3) , signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D(3) -induced growth regulation of ductal epithelium.

The effect of feeding systems on the characteristics of products from small ruminants

Small ruminants are the most efficient transformers of low quality forage into high quality animal products with distinguished chemical composition and organoleptic characteristics. There is a wide range of sheep and goat farming systems from highly extensive, based on natural grasslands or rangelands, to very intensive ones, based on natural grazing and supplementary feeding. Usually, the systems which are under comparison are those based mainly on pasture vs. the indoor ones. The aim of this paper is to provide an integrated analysis of the major aspects of the nature and composition of small ruminants products, such as milk and meat, and then the effect of feeding systems on chemical composition and quality characteristics of those products (i.e. fatty acid profile, antioxidants, vitamins, muscle:fat ratio, flavour, taste, etc.), since milk and meat quality is constantly evolving, partly in response to the rising concerns of consumers in terms of safety, health, ethical aspects, origin etc. As small ruminants milk is mostly transformed into cheese and its yield depends on milk composition, the main objectives of the dairy sheep and goat breeders are to improve milk quality by increasing the total milk solids output and stabilizing the milk composition (fat and protein) through the appropriate level of nutrition. Factors such as forage:concentrate ratio, dietary fat supplements, pasture, etc. have essential effect on small ruminants milk yield, milk composition and fatty acid (FA) profile. A number of studies have shown that milk from sheep and goats in pasture is enriched in substances of natural origin like phenolic compounds, fat soluble vitamins, flavours terpenes, bioactive lipid components, unsaturated FA and CLA, in addition to being naturally high in medium-chain FA in comparison to those fed conventional concentrate-forage diets. However, there are species differences between sheep and goats as the dietary effects on those parameters concerns, which could be explained by the differences found in mRNA of stearoyl-CoA desaturase of their mammary adipocytes. The feeding system effects on meat quality is more difficult to be identified because lambs and kids of different breed, weaned at different age and live weight or raised on different types of pastures have different growth rate and carcass characteristics like level of fatness, FA profile, flavour, tenderness, taste, etc. It has been demonstrated that lambs and kids raised under a grazing system without any supplementation, present an inferior fatness degree and a higher meat fat concentration of n− 3 PUFA and CLA. Lamb meat has higher fat content, higher proportions of SFA and lower MUFA compared to goats, under similar dietary treatment, which make goat meat especially valuable nutritionally and for consumer health. In conclusion, the existing unfavourable properties of small ruminant products can be improved by nutritional intervention to modify their FA profile for the consumer's health benefit.

Metabolic Syndrome and Breast Cancer Risk: Is There a Role for Metformin?

Obesity is one of the most important known preventable causes of cancer, accounting for up to 20% of cancer deaths in women. Obese women have increased risk of dying from breast cancer as well as an increased risk of distant metastasis. Metabolic Syndrome (MetSyn) is a group of metabolic conditions that include 1) abdominal obesity, 2) atherogenic dyslipidemia, 3) elevated blood pressure, and 4) insulin resistance. MetSyn is known to promote the development of cardiovascular disease and diabetes and may be associated with increased breast cancer risk. Emerging evidence supports an association between mammary adipocytes and their secreted adipocytokines and breast cancer initiation and progression. Metformin (1,1-dimethylbiguanide hydrochloride) is a drug used to treat type 2 diabetes and MetSyn. We review the potential association between MetSyn in promoting breast cancer and emerging evidence for the use of metformin in cancer prevention.

Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells

Previously, we have shown that Pck1 expression in mammary gland adipocytes and white adipose tissue maintains triglyceride stores through glyceroneogenesis, and these lipids were used for synthesis of milk triglycerides during lactation. Reduced milk triglycerides during lactation resulted in patterning of the newborn for insulin resistance. In this study, the role of Pck1 in mammary gland epithelial cells was analyzed. The developmental expression of Pck1 decreased in isolated mouse mammary gland epithelial cells through development and during lactation. Using HC11, a clonal mammary epithelial cell line, we found that both Janus kinase 2 signal transducers and activators of transcription 5 and the AKT pathways contributed to the repression of Pck1 mRNA by prolactin. These pathways necessitate three accessory factor regions of the Pck1 promoter for repression by prolactin. Using [U-(13)C(6)]glucose, [U-(13)C(3)]pyruvate, and [U-(13)C(3)]glycerol in HC11 cells, we determined that Pck1 functions in the pathway for the conversion of gluconeogenic precursors to glucose and contributes to glycerol-3-phosphate synthesis through glyceroneogenesis. Therefore, Pck1 plays an important role in both the mammary gland adipocytes and epithelial cells during lactation.

Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, β-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic β-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.

Abstract A57: Breast adipocytes bioactivate Vitamin D3 and regulate hormone-induced epithelial cell growth

Abstract Vitamin D3, a nutritionally modulated hormone, participates as a negative growth regulator of mammary gland development and transformation. However, the mechanism behind breast specific vitamin D3 synthesis, metabolism, and signaling to maintain glandular homeostasis and inhibit the onset of mammary transformation remains unknown. Our objective was to test the concept that vitamin D3 signaling in mammary adipose tissue contributes to the negative growth regulation of mammary epithelial cells. We hypothesized that the mammary adipose tissue bioactivates 25-hydroxyvitamin D3 [25(OH)D3] to the active metabolite, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], and induces adipocyte Vitamin D3 Receptor (VDR) signaling along with paracrine secretion of 1,25(OH)2D3 to surrounding mammary epithelial cells to regulate growth of normal and transformed breast epithelial cells. We utilized mammary glands from VDR knockout (KO) and wild type (WT) mice for various procedures including ex vivo co-cultures with mammary epithelium and adipocytes, gene and protein expression assays, immunohistochemistry, and whole mount analysis. We discovered that breast adipocytes express essential signaling components of the vitamin D3 endocrine system and have the ability to bioactivate 25(OH)D3 to 1,25(OH)2D3 inducing mammary specific vitamin D3-responsive gene expression. Breast adipocytes likely participate to maintain localized vitamin D3 homeostasis and contribute to 1,25(OH)2D3-induced growth regulation of mammary epithelial cells in response to various hormonal stimulations, modulating mammary development and differentiation. Thus, our work suggests that breast adipose tissue serves as a vital endocrine mediator of Vitamin D3 signaling and offers a possible mechanism by which elevated 25(OH)D3 serum concentrations reduce the susceptibility to breast cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A57.

Morphogenesis of the developing mammary gland: Stage-dependent impact of adipocytes

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (apoptosis through triggered activation of caspase-8), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points and fewer terminal end buds (TEBs) and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.

Establishment of a mammary stromal fibroblastic cell line for in vitro studies in mice of mammary adipocyte differentiation.

Mammary stromal adipose tissue remodeling is important for appropriate mammary gland development during pregnancy, lactation, and involution. However, the precise mechanisms underlying mammary stromal adipose tissue remodeling remain unclear. We have established a mammary stromal, fibroblastlike cell line (MSF) from primary mouse mammary culture by introducing a temperature-sensitive simian virus-40 large tumor antigen. Among several hormones related to mammary gland development, hydrocortisone was found to commit MSF cells to a preadipocyte lineage, whereas insulin was found to induce extracellular matrix-dependent adipogenic differentiation of the cells, as assessed by lipid accumulation and marker gene expression. Interestingly, such hormone-induced adipogenic differentiation of MSF cells, but not 3T3-L1 cells, was suppressed by prolactin through its receptor and downstream STAT5. Furthermore, coculture of MSF cells with mammary epithelial HC11 cells and culture in HC11-conditioned medium also suppressed adipogenic differentiation of MSF cells. We have demonstrated that adipogenic differentiation of at least some populations of mammary stromal cells is modulated by lactogenic hormones and humoral factors from epithelial cells, suggesting that the response of these mammary cells may differ from adipocytes at other sites. We believe that the MSF cell line will prove a useful model to elucidate mammary stromal adipose development in vitro as well as represent an important first step toward developing stable adipocyte cell lines that faithfully represent their site of origin.

Sheep and goats differences in CLA and fatty acids milk fat content in relation with mRNA stearoyl-CoA desaturase and lipogenic genes expression in their mammary gland

An experiment was conducted with 12 lactating dairy ewes and 12 goats with the objective to determine whether, under the same dietary treatments, the differences in their fatty acid (FA) profile with emphasis on cis-9 trans-11 CLA milk fat content, are reflected in the transcript levels of genes involved in FA and cis-9, trans-11 CLA biosynthesis. The animals were fed with two diets (A, B) in different days of milk (DIM) due to the different milk yield, body weight etc, in order to have the same food intake and to avoid dietary effects. Diet A was fed to the animals on a group basis as it is traditionally used in practice, while diet B was chosen to avoid individual feed intake variation which is usually observed in group feeding. The results showed that there are significantly lower mRNA levels of acetyl-CoA carboxylase (ACC) in sheep mammary gland compared with those of goats, independently from the diet fed. The same trend was observed with the mRNA level of FA synthase (FAS), but the results were significant only for diet A. The mRNA level of lipoprotein lipase (LPL) in the mammary gland did not differ between sheep and goats fed with diet A. In addition, the concentration of cis-9 trans-11 CLA content was significantly higher in sheep milk fat compared with those of goats. This is in accordance with the significant higher levels on mRNA of stearoyl-CoA desaturase (SCD) which were observed in their mammary adipocytes of sheep compared with those of goats, independently of the fed diet (A or B). In conclusion, these findings demonstrate that the differences between sheep and goats, concerning cis-9, trans-11 CLA and FA milk fat content, under the same dietary treatments could be explained in part by the differences in mRNA of SCD and lipogenic genes in their mammary gland.

Early Soy Exposure via Maternal Diet Regulates Rat Mammary Epithelial Differentiation by Paracrine Signaling from Stromal Adipocytes1–3

Diet-mediated changes in transcriptional programs that promote the early differentiation of the mammary gland may lead to reduced breast cancer risk. The disparity in adult breast cancer incidence between Asian women and Western counterparts is attributed partly to high soy food intake. Here, we conducted genome-wide profiling of mammary tissues of weanling rats exposed to soy protein isolate (SPI) or control casein (CAS) via maternal diet to evaluate the contribution of early exposure on mammary gene expression. Of the identified 18 up- and 39 downregulated genes with SPI relative to CAS, a subset was associated with lipid metabolic pathways, consistent with reduced mammary adipocyte size and suggesting stromal adipocyte-specific genomic changes. Female offspring of rats fed SPI tended to have fewer terminal end buds (P = 0.06) and had significantly lower body weight and abdominal fat mass. To demonstrate the functional consequence of SPI-mediated adipocyte metabolic changes on neighboring mammary epithelium, the expression of in vivo regulated genes in 3T3-L1 adipocytes treated with soy isoflavone genistein and effects of the resultant conditioned medium (CM) on the differentiation of HC11 mammary epithelial cells were evaluated by quantitative RT-PCR and/or Western immunoblots. In differentiated 3T3-L1, genistein decreased fatty acid synthase and stearoyl-CoA desaturase and increased hydroxysteroid 11-β dehydrogenase 1 expression. CM from genistein-treated adipocytes had higher adiponectin levels and augmented prolactin-induced, glucocorticoid-regulated β-casein levels. These findings suggest that soy-associated components, by targeting mammary adipocytes, alter paracrine signaling to enhance mammary epithelial differentiation, with important implications for the prevention of breast cancer associated with obesity and obesity-related diseases.