Activity Of Mammary Epithelial Cells Research Articles

Cyclical heat stress during lactation influences the microstructure of the bovine mammary gland.

This study aimed to evaluate the impact of heat stress on mammary epithelial cell (MEC) losses into milk, secretory mammary tissue structure, and mammary epithelial cell activity. Sixteen multiparous Holstein cows (632 ± 12 kg BW) approximately 100 d in milk housed in climate-controlled rooms were paired by body weight and randomly allocated to one of 2 treatments, heat stress (HS) or pair feeding thermoneutral (PFTN) using 2 cohorts. Each cohort was subjected to 2 periods of 4 d each. In period 1, both treatments had ad libitum access to a common total mixed ration and were exposed to a controlled daily temperature-humidity index (THI) of 64. In period 2, HS cows were exposed to controlled cyclical heat stress (THI: 74 to 80), while PFTN cows remained at 64 THI and daily dry matter intake was matched to HS. Cows were milked twice daily, and milk yield was recorded at each milking. Individual milk samples on the last day of each period were used to quantify MEC losses by flow cytometry using butyrophilin as a cell surface marker. On the final day of period 2, individual bovine mammary tissue samples were obtained for histomorphology analysis, assessment of protein abundance, and evaluation of gene expression of targets associated with cellular capacity for milk and milk component synthesis, heat response, cellular proliferation, and autophagy. Statistical analysis was performed using the GLIMMIX procedure of SAS. Milk yield was reduced by 4.3 kg by HS (n = 7) compared with PFTN (n = 8). Independent of treatment, MEC in milk averaged 174 cells/mL (2.9% of total cells). There was no difference between HS vs. PFTN cows for MEC shed or concentration in milk. Alveolar area was reduced 25% by HS, and HS had 4.1 more alveoli than PFTN. Total number of nucleated MEC per area were greater in HS (389 ± 1.05) compared with PFTN (321 ± 1.05); however, cell number per alveolus was similar between groups (25 ± 1.5 vs. 26 ± 1.4). There were no differences in relative fold expression for GLUT1, GLUT8, CSN2, CSN3, LALBA, FASN, HSPA5, and HSPA8 in HS compared with PFTN. Immunoblotting analyses showed a decrease abundance for phosphorylated STAT5 and S6K1, and an increase in LC3 II in HS compared with PFTN. These results suggest that even if milk yield differences and histological changes occur in the bovine mammary gland after 4 d of heat exposure, MEC loss into milk, nucleated MEC number per alveolus, and gene expression of nutrient transport, milk component synthesis, and heat stress related targets are unaffected. In contrast, the abundance of proteins related to protein synthesis and cell survival decreased significantly, while an upregulation of proteins associated with autophagy in HS compared with PFTN.

Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

Prolactin regulates LAT1 expression via STAT5 (signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows

The l-type amino acid transporter 1 (LAT1; also known as SLC7A5) is a transporter that allows the uptake of large neutral amino acids into mammalian cells. In dairy cows, LAT1 is highly expressed in lactating mammary tissues and involved in milk protein synthesis. Prolactin (PRL) has a lactogenic role and is capable of inducing milk production in ruminants. However, the relationship between PRL stimulation and LAT1 expression in dairy cow mammary gland has not been well understood. In this study, we showed that PRL stimulation increased expression of LAT1 and β-casein in mammary epithelial cells of dairy cows. The stimulatory effect of PRL on milk protein production was inhibited by LAT1-specific inhibitor or LAT1 knockdown, suggesting that PRL-induced milk protein production is involved in LAT1 expression. To determine whether the PRL signaling pathway participates in regulation of LAT1 expression, PRLR (PRL receptor) or STAT5 (signal transducer and activator of transcription 5) was knocked down by short interfering (si)RNA in mammary epithelial cells of dairy cows. Western blot results showed that knockdown of PRLR or STAT5 with siRNA markedly decreased PRL-stimulated LAT1 expression. In addition, we observed a marked increase in plasma membrane expression of LAT1 in PRL-stimulated cells compared with control cells. These observations indicated that PRL signaling can regulate LAT1 expression and activity in mammary epithelial cells of dairy cows, contributing to increased amino acid availability and milk protein synthesis in mammary gland of dairy cow.

Activation of peroxisome proliferator-activated receptor gamma in mammary epithelial cells upregulates the expression of tumor suppressor Cyld to mediate growth inhibition and anti-inflammatory effects

Several studies have implicated the downregulation of the tumor suppressor Cyld expression in breast cancer development. However, the mechanisms that regulate Cyld expression in mammary epithelial cells are largely unknown. In order to investigate them, a bioinformatic analysis of the promoter region of Cyld was performed and identified putative nuclear hormone receptor response elements that included peroxisome proliferator-activated receptor gamma (PPAR-γ)-responsive elements. In the present study, we showed that upon activation of the nuclear hormone receptor PPAR-γ by the agonist troglitazone (TZD), there was a significant increase in Cyld mRNA in human mammary epithelial cell lines. The effect of TZD could be attributed to the transactivation of the Cyld promoter as indicated by the upregulation of a luciferase reporter that was driven by the −1995 to +95 region of the human Cyld gene. Furthermore, the upregulation of Cyld expression by TZD was dependent on PPAR-γ since downregulation of PPAR-γ expression by RNAi compromised the induction of Cyld expression by TZD. CYLD induction mediated, at least in part, the TZD-mediated downregulation of tumor necrosis factor α (TNFα)-induced interleukin 8 (IL-8). In addition, downregulation of CYLD compromised the cytotoxic effects of TZD in immortalized mammary epithelial cells. Our results demonstrated that PPAR-γ is a novel regulator of Cyld transcription and identified CYLD as a mediator of the PPAR-γ-dependent anti-inflammatory and anti-proliferative activity in mammary epithelial cells, which underscores its potential to be used as a target for the development of breast cancer therapeutic approaches.

The Proliferative Activity of Mammary Epithelial Cells in Normal Tissue Predicts Breast Cancer Risk in Premenopausal Women

The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67(+) cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12-48.0]. Conversely, the frequency of ER(+) or p27(+) cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER(+): OR = 0.70, 95% CI, 0.33-1.50; p27(+): OR = 0.89, 95% CI, 0.45-1.75). Notably, high Ki67(+)/low p27(+) and high Ki67(+)/low ER(+) cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67(+)/low p27(+) and low Ki67(+)/low ER(+) cell frequencies, respectively, among premenopausal women (Ki67(hi)/p27(lo): OR = 5.08, 95% CI, 1.43-18.1; Ki67(hi)/ER(lo): OR = 4.68, 95% CI, 1.63-13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926-34. ©2016 AACR.

Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.

Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis.

Prolyl‐isomerase Pin1 controls normal and cancer stem cells of the breast

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers.

Increased Proliferative Background in Healthy Women with BRCA1/2 Haploinsufficiency Is Associated with High Risk for Breast Cancer

Previous studies indicated that BRCA haploinsufficiency was associated with activation of the EGF receptor (EGFR) signaling pathway and increased proliferative activity in mammary epithelial cells of healthy women. We hypothesized that these processes might be reflected in the expression of serologic soluble EGFR (sEGFR) and thymidine kinase 1 (TK1) activity, which signal the initial and final steps of the proliferative pathway, respectively. We found that healthy carriers of BRCA1/2 mutations (n = 80) showed a significantly higher TK1 activity than age-matched controls (P = 0.0003), and TK1 activity was similar in women with BRCA1 and BRCA2 mutations (P = 0.74). The sEGFR concentration was significantly higher in women with BRCA1 than in controls and BRCA2 mutation (P = 0.013 and 0.002, respectively). During follow-up, four of 80 BRCA1/2 mutation carriers developed breast cancer. These women showed a significantly higher TK1 activity and somewhat higher sEGFR concentrations than the other 76 BRCA1/2 carriers (P = 0.04 and 0.09, respectively). All tumors were negative for ovarian hormone receptors, but showed a high EGFR expression. This study was limited by the short-term follow-up (mean, 27 months; range, 5-45), which resulted in a small sample size. Women with BRCA1 and BRCA2 mutations that had undergone risk-reducing bilateral salpingo-oophorectomy (BSO) showed significantly lower sEGFR compared with those without surgery (P = 0.007 and 0.038, respectively). Larger, prospective studies are warranted to investigate whether TK1 and sEGFR measurements may be useful for identifying healthy BRCA1/2 carriers with high risk of developing breast cancer; moreover, sEGFR measurements may serve as effective tools for assessing risk before and after BSO.

Pubertal high fat diet: effects on mammary cancer development

IntroductionEpidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD.MethodsPubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis.ResultsHFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages.ConclusionsTaken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.

Distinct FAK Activities Determine Progenitor and Mammary Stem Cell Characteristics

Mammary stem cells (MaSC) and progenitor cells are important for mammary gland development and maintenance and may give rise to mammary cancer stem cells (MaCSC). Yet, there remains limited understanding of how these cells contribute to tumorigenesis. Here, we show that conditional deletion of focal adhesion kinase (FAK) in embryonic mammary epithelial cells (MaEC) decreases luminal progenitors and basal MaSCs, reducing their colony-forming and regenerative potentials in a cell-autonomous manner. Loss of FAK kinase activity in MaECs specifically impaired luminal progenitor proliferation and alveologenesis, whereas a kinase-independent activity of FAK supported ductal invasion and basal MaSC activity. Deficiency in luminal progenitors suppressed tumorigenesis and MaCSC formation in a mouse model of breast cancer. In contrast with the general inhibitory effect of FAK attenuation, inhibitors of FAK kinase preferentially inhibited proliferation and tumorsphere formation of luminal progenitor-like, but not MaSC-like, human breast cancer cells. Our findings establish distinct kinase-dependent and -independent activities of FAK that differentially regulate luminal progenitors and basal MaSCs. We suggest that targeting these distinct functions may tailor therapeutic strategies to address breast cancer heterogeneity more effectively.

Ultra-structure study of mammary gland in Zaraibi goats during different stages of lactation.

ABSTRACTThis work was carried out on 32 Egyptian Nubian (Zaraibi) goats to investigate the effect of stage of lactation on the ultra-structure of secretory mammary cells and its relationship with milk production. Biopsies were taken surgically from the mammary gland from three goats for histological and fine structure studies. The histological structures of mammary gland showed clear differences between lactation stages, being more developed in the early and mid stages, compared to late stage of lactation. The number of alveolar secretory cells increased from early to mid stage of lactation by 17.6% then reduced at the late stage by 25% from that at mid stage. The milk yield increased by 32.2% from early to mid stage, and then reduced to 61.3% from mid to late stage. Ultra-structure features in mammary epithelial cells showed different features at successive stages of lactation. The nuclei of the active mammary epithelial cells were ovoid or nearly spheroid in shape and indentation in cells membranes. The stage of lactation had an effect on nucleus diameter and sectional area. The nucleus diameter was smallest during early and mid stages, while being greatest at late stage of lactation. The sectional area of the nucleus showed increased values in late stage of lactation than in other stages. The majority of cell organelles were distributed in the cytoplasm. These organelles showed different phenomenon during different stages of lactation.The mitochondria showed a clear change in shape (ovoid or elongated), number and sectional area during different stages of lactation. The number of mitochondria per plate was maximal during mid-stage, followed by early stage (44.2 and 37.9, respectively). The total mitochondria sectional area at late stage was 27.9 um2. This area increased at early and mid stage of lactation by 3.8 and 37.9%, respectively. A well- developed laminated endoplasmic reticulum (ER) was frequently found in the cytoplasm of the mammary epithelial cells. Stage of lactation influenced the diameter of ER. At early and mid stages of lactation, the ER was well developed to occupy large part of the cell, with diameters of 0.55 and 51 μm, respectively. This value was minimal during the late stage of lactation (0.36 um2). Protein granules had an ovoidal shape and released in different sizes. The sectional area of the protein was the smallest during late lactation (0.05 μm2) and increased during early and mid stages of lactation (0.1 μm2). The lipid droplets were appeared in different sizes, the sectional area of the lipid droplets were the smallest during late lactation (1.46 μm2), and increased during early and mid lactation (4.52 and 2.58 μm2, respectively). In conclusion, the results of the present study indicated that stage of lactation influence the activity of mammary epithelial cells. The activity increases at early and mid lactation which is reflected on ultrastructure of epithelial cells of mammary gland of Egyptian Nubian (Zaraibi) goats.

EFFECTS OF MILKING FREQUENCY ON MILK PRODUCTION AND HISTOLOGICAL STRUCTURE OF UDDER IN ZARAIBI DAIRY GOATS

ABSTRACTThis work was carried out on 32 Egyptian Nubian (Zaraibi) goats to investigate both the effect of the milking frequency on milk yield and milk composition and the effect of stage of lactation on histological structure and histochemistry of the secretory mammary cells and its relationship with milk production in Zaraibi goats. Biopsies were taken surgically from the mammary gland from 3 does milked once daily (1x) and 3 does milked twice daily (2x) at the three stages of lactation, early, mid and late, for histological and histochemical studies. The histological structure showed clear differences due to milking frequency and lactation stage, being more developed at early and mid stages and twice daily milking compared to late stage of lactation and once daily milking. The number of the alveolus secretory cells per alveolus increased from the early to the mid stage of lactation by17.6% and then reduced at the late stage by 25% compared to mid stage, while no difference noticed between twice and once daily milking. Once daily milking (1x) reduced milk yield by 6%, and increased fat percentage to 4.0% compared to 3.67% in twice daily milking group (2x). Milk of once milking (1x) group contained higher percentage of total solids 11.38% than twice milking (10.93%), but milk protein and lactose did not differ between 1x and 2x milking. Lactation curve showed 32.2% increase in yield during mid stage than early stage, while late stage attained 61.3% reduction in milk yield compared to mid stage. Protein and lactose percentages did not change throughout different stages of lactation, while fat and total solids percentages showed the highest values at early stage of lactation (4.0 and 11.5%, respectively), and the lowest at mid and late stage of lactation. The total sectional areas (u/plate) of the alveoli were the smallest during late lactation (495399 u /plate) compared to that during early and mid stages of lactation (705206 and 759901u/plate, respectively). Numerous loci of alcaline phosphatase (AP) were apparent on the outer surface of the alveolar secretory cells at the early and mid stages of lactation—reflecting high activity of this enzyme at these two stages. This was accompanied by a high level of milk secretion reaching1778.2±38.9 and 2351.4±68.4 g/head/day, respectively.In contrast, at the late stage of lactation, the size of alveoli was reduced and few alveoli showed weak AP activity. This coincided with the reduction in milk yield (910g/head/day). It could be concluded that stages of lactation influence the cell number and activity of mammary epithelial cells.

Changes in mammary secretory tissue during lactation in ovariectomized dairy cows

In dairy animals, the milk yield (MY) changes during a lactation and is influenced by several physiological, livestock management and environmental factors. The MY produced by a mammary gland depends on synthetic activity of mammary epithelial cells (MECs) as well as MEC number and mammary secretory tissue organization. It has been suggested that ovarian steroids (estradiol and progesterone) have a negative effect on MY in lactating cows. In a previous study, we showed that the suppression of ovarian secretions by an ovariectomy improved lactation persistency in dairy cows. Here we were interested in the effects of ovariectomy on plasma estradiol and progesterone concentrations and on changes that occur in mammary secretory tissue during lactation. We demonstrated that the ovariectomy of lactating cows at the time of the lactation peak induced a rapid and dramatic drop in plasma progesterone and a smaller reduction in plasma estradiol. Interestingly, the study of the changes in mammary secretory tissue over time revealed that the improvement of MY measured in the ovariectomized cows was associated with a limited increase in estradiol receptivity in MECs, a reduced mammary tissue remodeling and reduced blood protein concentration in milk, in late lactation. These results suggest that ovarian secretions, particularly estradiol and progesterone, act to enhance processes for mammary gland involution in late-lactating dairy cows.

P4-02-08: Obesity-Induced Aromatase Expression in the Breast Microenvironment Promotes Estrogen Receptor Activity Independent of Circulating Estradiol Levels.

Abstract Epidemiological studies indicate that obesity increases the risk of postmenopausal breast cancer by approximately 50%. In the past, researchers have hypothesized that elevated estrogen synthesis by the peripheral adipose tissue may be the principal mediator of breast tumorigenesis in this population, which primarily develops estrogen receptor alpha (ERα) positive breast cancer. However, obesity is also accompanied by an elevation in growth factor and cytokine signaling, and these pathways have been linked to tumorigenesis. In addition, certain growth factor and cytokine family members can promote aromatase expression in both the epithelial and stromal tumor compartments. Consequently, we hypothesized that obesity increases the risk of postmenopausal breast cancer via elevated aromatase expression and/or activity in the local mammary tissue. To test our hypothesis, we investigated how ERa activity in mammary epithelial cells was influenced by adipose stromal cells (ASC) cultured under obesity-associated conditions, including high cell density and exposure to elevated levels of circulating growth factors and cytokines. For the latter condition, sera was obtained from postmenopausal women, pooled by BMI category (lean: 18.5−24.9; obese: ≥30), and applied to the ASC, which were originally derived from women undergoing reduction mammoplasty. High ASC density was achieved via the protocol previously published by Dr. Li. Preliminary data indicated that both elevated cell density and sera from obese postmenopausal women induces greater aromatase expression in ASC, indicating that multiple factors may be contributing to the increased local aromatase expression seen with obesity. We are currently exploring the signaling pathways responsible for obesity's upregulation of ASC aromatase expression and will present these results at the meeting. Intriguingly, exposure to conditioned media from both the high density and obese sera-exposed ASC enhanced ERa activity in MCF-7 mammary epithelial cells, independent from exogenous estradiol but dependent on the presence of androgens, suggesting an important role for the aromatase enzyme in this observation. To expand on this finding, we plan to investigate the effect of ASC conditioned media on different markers of cancer aggression, including proliferation and survival, and assess the degree to which these effects depend on estradiol. Through further examination of obesity's impact on signaling pathways in both the epithelial and stromal tumor compartments, we ultimately hope to identify more effective chemopreventive and therapeutic regimens for the high-risk obese postmenopausal population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-08.

The effect of sulindac on growth differentiation factor 15 and 13,14-dihydro-15-keto prostaglandin A2 in nipple aspirate fluid

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated in several large epidemiologic studies with a lower risk of epithelial cancers including breast though no randomized, placebo controlled phase III studies have been conducted to date. NSAID use was associated with a significantly lower risk of breast cancer among participants in a multiethnic cohort, though the nearly 50% reduction in risk was limited to women with estrogen receptor positive tumors; an observation previously reported by Terry et al., and supported by Gierach et al., and findings of Harris et al for cyclooxygenase-2 (COX-2) inhibitors. These findings in epidemiologic studies are supported by work in human mammary tissues and cell culture where overexpression of COX-2, one of two cyclooxygenase targets of NSAIDs, acts as an early event in the transition of normal breast cells to malignancy. Further, COX-2-associated prostaglandin E2 (PGE2) production has been shown to increase aromatase activity in mammary epithelial cells possibly explaining the observed benefit of NSAID use in hormone receptor positive disease and the recent observation of lower circulating estradiol levels among regular users of NSAIDs. Thus, there exists sufficient rationale for the use of NSAIDs for the prevention of breast cancer. While the best studied target of NSAIDs is the COX isoenzymes, the anti-tumor activities of each of the NSAID agents via 'non COX' pathways remains unknown. Sulindac, a non selective NSAID with well recognized COX-independent induction of apoptosis has remained a strong candidate for chemoprevention largely because of its broader anti-tumor activity. Growth differentiation factor 15 (GDF-15), a potent proapoptotic molecule in the TGF-β superfamily and potential tumor suppressor is upregulated by several NSAIDS independent of its activity as a COX inhibitor. Sulindac sulfone, a metabolite of sulindac and a COX-independent mediator of apoptosis, has been shown to be a potent inducer of GDF-15 at pharmacologically relevant doses whereas Celecoxib, a COX-2 specific inhibitor, has low GDF-15 induction potency even at toxic doses. Other NSAIDs such as aspirin and indomethacin as well as a number of candidate dietary chemopreventives including resveratrol, indole-3-carbinol, 3,3' Diindolylmethane (DIM), and lycopene as well as the PPAR agonists troglitazone, and 15-deoxy-Δ12,14-prostaglandin J2 all show potent induction of GDF-15 leading to the suggestion that GDF-15 might serve as a complementary molecular target to COX-2 for cancer chemoprevention and serve as a surrogate biomarker of apoptotic response. To better understand the effect of NSAIDs in the breast, we conducted a phase 1b dose study using sulindac as a representative drug of the non selective NSAIDs with known activity to induce GDF-15 and inhibit COX-2. To assess sulindac and its metabolites and their effects on a stable COX-2 derived prostaglandins 13,14-dihydro-15-keto prostaglandin A2 [PGEM], and the NSAID inducible growth differentiation factor (GDF-15) in nipple aspirate fluid (NAF), we randomized 30 women at increased risk for breast cancer to 150 mg once daily or twice daily sulindac for 6 weeks. Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. Sulindac was associated in a dose independent manner with a nonsignificant decrease in NAF PGEM levels (p = 0.1). Serum levels of sulindac, but not NAF sulindac, were correlated with a decrease in NAF PGEM levels (p = 0.03). GDF-15, showed a borderline significant trend towards higher levels in NAF with 300 mg daily sulindac (p = 0.07). This study suggests that COX inhibition in the breast may be achieved at 150 mg sulindac once daily while a higher daily dose is needed to exhibit activities via COX independent pathways. The study also suggests that NAF levels of PGEM and GDF-15 could be considered as drug effect biomarkers for future early phase chemoprevention trials.

Up-regulation of SEPT9_v1 stabilizes c-Jun-N-Terminal kinase and contributes to its pro-proliferative activity in mammary epithelial cells

SEPT9_v1, the largest transcript of the septin gene family member, SEPT9, encodes a septin isoform implicated in the tumorigenic transformation of mammary epithelial cells. High levels of SEPT9_v1 expression also have been observed in both breast cancer cell lines, primary breast cancers as well as other solid tumor malignancies. We found a novel interaction between SEPT9_v1 and the c-Jun-N-terminal kinase (JNK), a mitogen-activated protein kinase important in cellular stress responses, cell proliferation, and cell survival. We found that up-regulation of SEPT9_v1 stabilizes JNK by delaying its degradation, thereby activating the JNK transcriptome. C-jun kinase assays in mammary epithelial cells expressing SEPT9_v1, compared to controls, exhibited increased JNK/c-Jun transcriptional activity. This increase was associated with increased levels of cyclin D1, a critical component of the proliferative response required for progression through G 1 of the cell cycle in many cell types. These findings demonstrate the first link between a septin protein and the JNK signaling pathway. Importantly, it suggests a novel functional role of SEPT9_v1 in driving cellular proliferation of mammary epithelial cells, a hallmark feature of oncogenesis that is directly relevant to breast cancer.

Protein kinase CK2 and cell polarity

There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellular processes, in the maintenance of mammalian cell morphology and cell polarity. Here, we show that in epithelial cells, a fraction of CK2 is associated to the plasma membrane and that this localization is controlled by cell-matrix interactions. In addition, inhibition of CK2 activity in mammary epithelial cells (MCF10A), using either the specific CK2 inhibitor TBB or siRNA-mediated CK2beta knockdown, induced differential phenotypes revealing an important role of this enzyme in epithelial cell morphology.

Progesterone increases metabolic rate and progesterone withdrawal enhances epithelial integrity of mammary epithelium

Progesterone receptors (PR) were documented in a bovine mammary epithelial cell line (BME-UV), which allowed us to test the hypothesis that progesterone (PRG) has direct effects on mammary epithelial function. PR were identified in BME-UV cells at the mRNA and protein (Western blot) levels. Thus, BME-UV cells were grown as confluent monolayers in either no added hormones, PRG, prolactin (PRL), and PRG + PRL over a 14 day culture period. Parturition was simulated in vitro by using PRG-free medium for the final day in culture. PRG exposure caused an immediate and substantial increase in glucose utilization that was maintained for at least 10 days of continued PRG exposure. Small effects on transepithelial electrical resistance (Rte) were identified in the presence of these hormones. However, treatment including PRG and PRL for the first 13 days of culture, but lacking PRG for the final 24 hours prior to assay substantially increased Rte. The results demonstrate that PRG exposure is directly associated with an increase in mammary cell metabolism. PRG also ‘primed’ the epithelial cells for a rapid transition to greater Rte when PRG was withdrawn. This suggests that PRG increases metabolic activity of mammary epithelial cells during pregnancy and that the sudden reduction in circulating PRG levels at parturition signals the rapid closure of mammary epithelial tight junctions. [USDA NRI, RRO17686 NCRR NIH Support]

Effect of Stage of Lactation and Parity on Mammary Gland Cell Renewal

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8±1.3 and 25.2±0.8 kg/d) and 50 DIM (38.0±1.2 and 29.8±1.1 kg/d), but it was the same for both groups at 250 DIM (23.9±1.5 and 23.8±1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, β-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.