Number Of Terminal End Buds Research Articles

Loss of tyrosine 211 phosphorylation of proliferating cell nuclear antigen (PCNA) enhances postnatal mammary gland development.

The intricately orchestrated progression of mammary tissue development involves the precise coordination of gland differentiation and cellular proliferation. Nevertheless, the understanding of the role and regulatory mechanisms governing the DNA replication machinery in mammary gland development remains limited. Given the essential role of DNA replication in the viability of living cells, any genetic disturbance to its replicative function, in any form, will impede organ development. This circumstance poses a technical challenge in elucidating the potential function of cell proliferation in mammary morphogenesis. PCNA is crucial in DNA replication, playing a pivotal role in the development of complete eukaryotic organisms. The phosphorylation of PCNA at tyrosine 211 (Y211) has been demonstrated to play a significant role in supporting replication forks and, consequently, cell proliferation. Therefore, the utilization of a knock-in mouse model, wherein the Y211 residue of PCNA is replaced with phenylalanine (211F), presents an opportunity to evaluate the impact of reduced cell proliferation potential on mammary gland development. Interestingly, the lack of Y211 phosphorylation did not significantly impact the rates of proliferation or cell death in the mammary gland. In contrast, the absence of Y211PCNA led to an increased, rather than reduced, growth of the mammary gland. This was evident in assessments of gland length and the number of terminal end buds (TEBs) in both postnatal and virgin mammary glands. Notably, this observation correlated with an elevation in tissue stemness within the 211F glands compared to the WT glands. Additionally, it was consistent with the greater body weight gains observed in 211F pups compared to WT pups during the weaning period. Our findings unveil an unexpected aspect that may carry significance for mammary development. This newfound is associated with the regulation of a central component within the DNA replication machinery, providing insights into the intricate interplay governing mammary tissue expansion.

Subacute tributyltin exposure alters the development and morphology of mammary glands in association with CYP19A1 expression in female rats

Tributyltin (TBT) is an endocrine-disrupting chemical (EDC) related to reproductive dysfunctions. However, few studies have investigated the effects of TBT exposure on mammary gland development. Thus, we assessed whether subacute TBT exposure causes irregularities in mammary gland development. We administered TBT (100 and 1,000 ng/kg/day for 30 days) to female rats from postnatal day (PND) 25 to PND 55, and mammary gland development, morphology, inflammation, collagen deposition, and protein expression were evaluated. Abnormal mammary gland development was observed in both TBT groups. Specifically, TBT exposure reduced the number of terminal end buds (TEBs), type 1 (AB1) alveolar buds, and type 2 (AB2) alveolar buds. An increase in the lobule and differentiation (DF) 2 score was found in the mammary glands of TBT rats. TBT exposure increased mammary gland blood vessels, mast cell numbers, and collagen deposition. Additionally, both TBT rats exhibited intraductal hyperplasia and TEB-like structures. An increase in estrogen receptor alpha (ERα), progesterone receptor (PR), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) - positive cells was observed in the mammary glands of TBT rats. A strong negative correlation was observed between CYP19A1- positive cells and TEB number. In addition, CYP19A1 - positive cells were positively correlated with mammary gland TEB-like structure, ductal hyperplasia, inflammation, and collagen deposition. Thus, these data suggest that TBT exposure impairs mammary gland development through the modulation of CYP19A1 signaling pathways in female rats.

Safety of lactational exposure to venlafaxine on the rat mammary gland development and carcinogenesis in F1 female offspring

The chronic use of selective serotonin reuptake inhibitors or serotonin-norepinephrine reuptake inhibitors (SNRIs) may result in human gynecomastia, mammoplasia, galactorrhea, and elevated breast cancer risk. As antidepressants are frequently used for postpartum depression (PPD) treatment, this study investigated the adverse effects of lactational exposure to venlafaxine (VENL, a selective SNRI) on mammary gland development and carcinogenesis in F1 female offspring. Thus, lactating Wistar rats (F0) received VENL by oral gavage at daily doses of 3.85, 7.7, or 15.4 mg/kg (N = 9, each group) from lactational day (LD 1) until the weaning of the offspring (LD 21). F1 female offspring were euthanized for mammary gland, and ovary histological analyses on the post-natal day (PND) 22 and 30 (1 pup/litter/period, N = 9, each group). At PND 22, other females (2 pups/litter, N = 18, each group) received a single dose of carcinogen N-methyl-N-nitrosourea (MNU, 50 mg/kg) intraperitoneally (i.p.) for tumor susceptibility assay until PND 250. Tumor incidence and latency were recorded and representative tumor samples were collected for histopathology. The results indicate that lactational exposure to VENL did not alter the development of the mammary gland (epithelial ductal tree or the mean number of terminal end buds), or the ovary (weight and primary, secondary, tertiary, and Graafian follicles) in prepubertal F1 female offspring. In addition, VENL exposure did not influence tumor incidence or tumor latency in adult female offspring that received MNU. Thus, the findings of this animal study indicated that lactational VENL exposure, a period similar to human PPD, did not exert an adverse effect on the mammary gland development at the prepubertal phase or on chemically induced mammary tumorigenesis in adult F1 female rats.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis.

Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.

Histological Findings of Mammary Gland Development and Risk of Breast Cancer in BRCA1 Mutant Mouse Models.

PurposeThe breast cancer susceptibility gene, BRCA1, is involved in normal development and carcinogenesis of mammary glands. Here, we aimed to evaluate the relationship between histological findings of mammary gland development and breast cancer risk in BRCA1 mutant mice.MethodsFive BRCA1 mutant mice and five non-mutant FVB/NJ mice were used for each group of 1-month-old (pubertal), 3-month-old (fertile), and 8-month-old (menopausal) mice. In another experiment, 15 BRCA1 mutant mice were followed up to 8 months after birth and classified into tumor-bearing (11 mice) and tumor-free (4 mice) groups. Excised mammary gland tissues were stained with Carmine Alum, and the number of terminal end buds (or alveolar buds), branching density, and duct elongation were measured using image analysis programs. Differences between the two groups were assessed using paired t-test.ResultsOne-month-old BRCA1 mutant mice showed a higher number of terminal end buds (23.8 ± 1.0 vs. 15.6 ± 0.8, p = 0.0002), branching density (11.7 ± 0.4 vs. 9.6 ± 0.5%, p = 0.0082), and duct elongation (9.7 ± 0.7 vs. 7.3 ± 0.4 mm, p = 0.0186) than controls. However, there was no difference between the 3- and 8-month-old groups. In BRCA1 mutant mice, the tumor-bearing group showed a significantly higher number of alveolar buds (142.7 ± 5.5 vs. 105.5 ± 5.4, p = 0.0008) and branching density (30.0 ± 1.0 vs. 24.1 ± 1.1%, p = 0.008) than the tumor-free group; however, duct elongation was not different (23.9 ± 0.6 vs. 23.6 ± 0.6 mm, p = 0.8099) between the groups.ConclusionBRCA1 mutant mice exhibited early pubertal mammary gland development and delayed age-related mammary gland involution was associated with breast cancer. Our results may have clinical implications for predicting breast cancer risk and developing prevention strategies for BRCA1 mutation carriers.

Effects of Maternal Grape Juice Intake on Unfolded Protein Response in the Mammary Glands of Offspring of High Fat Diet Fed Rat Dams.

Maternal high fat diet (HFD) and obesity during pregnancy increase female offspring′s mammary cancer risk in animal studies. We aimed to observe whether the consumption of grape juice during pregnancy can reverse this risk. During pregnancy and lactation, female Wistar rats were fed either a control or HFD and also received grape juice or tap water. At the age of 50 days, female offspring were euthanized, and mammary glands were collected to assess changes in biomarkers of increased mammary cancer risk. Maternal HFD increased the number of terminal end buds in offspring’s mammary glands and promoted cell proliferation (ki67). Maternal grape consumption blocked these effects. Apoptosis marker caspase 7, but not caspase 3, was reduced in the HFD offspring. HFD offspring also exhibited a reduction in the indicators of cell cycle regulation (p27, p21) and an ability to maintain DNA integrity (reduced p53). Maternal grape juice did not have any effect on these endpoints in the HFD offspring but reduced caspase 7 and p53 levels in the control offspring, perhaps reflecting reduced cellular stress. Maternal HFD increased oxidative stress marker GPx1 mRNA expression, and grape juice increased the levels of GPx2 in both the control and HFD offspring. HFD increased XBP1/Xbp1s, Atf4 and Atf6 mRNA expression and reduced ATF6 and CHOP protein levels. Maternal grape juice reversed the increase in XBP1/Xbp1s, Atf4 and Atf6 in the HFD offspring. PPARγ was downregulated in the HFD group, and grape juice reversed this effect. Grape juice also reduced the levels of HER2 and IRS, both in the control and HFD offspring. In conclusion, maternal grape juice supplementation reversed some of the biomarkers that are indicative of increased breast cancer risk in the HFD offspring.

Infant Formula Feeding Changes the Proliferative Status in Piglet Neonatal Mammary Glands Independently of Estrogen Signaling

Background:Soy infant formula contains isoflavones, which are able to bind to and activate estrogen receptor (ER) pathways. The mammary gland is sensitive to estrogens, raising concern that the use of soy formulas may promote premature development. Objective:We aimed to determine if soy formula feeding increases mammary gland proliferation and differentiation in comparison to other infant postnatal diets. Methods:White-Dutch Landrace piglets aged 2 d received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/(kgd); M + E2), or milk formula supplemented with genistein (84 mg/L of diet; M + G) until day 21. Mammary gland proliferation and differentiation was assessed by histology, and real-time RT-PCR confirmation of differentially expressed genes identified by microarray analysis. Results:Mammary terminal end bud numbers were 19–31% greater in the Milk, Soy, and M+G groups relative to the Sow and M+E2, P <0.05. Microarray analysis identified differentially expressed genes between each formula-fed group relative to the Sow (±1.7-fold, P <0.05). Real-time RT-PCR confirmed 2- to 4-fold increases in mRNA transcripts of genes involved in cell proliferation, insulin-like growth factor 1 (IGF1), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 18 (FGF18), in all groups relative to the Sow, P <0.05. In contrast, genes involved in cell differentiation and ductal morphogenesis, angiotensin II receptor type 2 (AGTR2), microtubule associated protein 1b (MAP1B), and kinesin family member 26b (KIF26B), were significantly upregulated by 2-, 4-, and 13-fold, respectively, in the M+E2 group. Additionally, mRNA expression of ER-specific gene targets, progesterone receptor (PGR), was increased by 12-fold, and amphiregulin (AREG) and Ras-like estrogen regulated growth inhibitor (RERG) expression by 1.5-fold in the M+E2 group, P <0.05. In the soy and M+G groups, mRNA expressions of fatty acid synthesis genes were increased 2- to 4-fold. Conclusions:Our data indicate soy formula feeding does not promote ER-signaling in the piglet mammary gland. Infant formula feeding (milk- or soy-based) may initiate proliferative pathways independently of estrogenic signaling.

Differential effects of the 1,25D3-MARRS receptor (ERp57/PDIA3) on murine mammary gland development depend on the vitamin D3 dose

1,25 dihydroxyvitamin D3 (1,25D3) is the most potent biologically active form of vitamin D3. Its actions on the mammary gland include cell growth inhibition and anti-cancer effects. This study’s purpose was to explore the role of the 1,25D3-membrane associated rapid response steroid (MARRS) receptor in the mammary gland using a tissue-specific knockout mouse model and a vitamin D3 dietary intervention. Three genotype groups were created using the Cre/loxp system to knock-down (+/−) and knockout (−/−) the MARRS receptor in epithelial cells of mammary glands (MG). Abdominal MGs were collected from 6-week old female mice (n = 94) on diets of 10,000 IU/kg (excess), 1,000 IU/kg (sufficient) or 0 IU/kg (deficient) of D3. There was a significant interaction between genotype and diet regarding number of terminal end buds (TEBs) (p = 0.001) and ductal coverage of the fat pad (p = 0.03). MARRS -/- mice on the sufficient diet had significantly fewer TEBs (p = 0.001) compared to MARRS +/+ on the same diet, but the opposite effect was seen in mice on the excess diet. There were no effects of genotype on TEBs when animals were vitamin D3 deficient. These results suggest that there is an effect of MARRS on mammary gland development that is dependent on 25(OH)D status, specifically, altering the number of highly proliferative TEBs. Increased numbers of TEBs have been correlated with increased breast cancer risk later in life. Therefore the results of this study warrant further examination of 25(OH)D status and recommendations in adolescent humans to reduce dietary effects on future breast cancer risk.

Dietary zinc deficiency or supplementation during gestation increases breast cancer susceptibility in adult female mice offspring following a J-shaped pattern and through distinct mechanisms

Zinc is required for fetal development and is involved in key processes associated with breast carcinogenesis. We evaluated whether maternal zinc deficiency or supplementation during gestation influences female offspring susceptibility to breast cancer in adulthood. C57BL/6 mice consumed during gestation control (30 p.p.m. zinc), zinc-deficient (8 p.p.m) or zinc-supplemented (45 p.p.m.) diets. Maternal zinc supplementation increased in female mice offspring the incidence of chemically-induced mammary adenocarcinomas that were heavier, compared to control group. This was accompanied by a decreased number of terminal end buds, increased cell proliferation and apoptosis, and increased tumor suppressors p21, p53 and Rassf1, Zfp382 and Stat3 expression in mammary glands, as well as increased zinc status. Although maternal zinc deficiency did not alter the incidence of these lesions, it also induced heavier mammary adenocarcinomas, compared to control group. These effects were accompanied by a decreased number of terminal end buds, increased proto-oncogenes c-Myc and Lmo4 expression and H3K9Me3 and H4K20Me3 epigenetic marks in mammary glands of offspring, and decreased zinc status and increased levels of oxidative marker malondialdehyde. The data suggest that both maternal zinc deficiency and supplementation during gestation programmed increased breast cancer susceptibility in adult mice offspring following a J-shaped pattern through distinct mechanisms.

Abstract 646: Pre conception paternal DDT exposure and programming of metabolic dysfunction and breast cancer risk in offspring

Abstract Background: Breast cancer is the most common cancer in American women, being the second leading cause of cancer-related death in this group. Exposure to environmental chemicals, such as the pesticide DDT (dichlorodiphenyltrichloroethane), has been long suspected to be a contributing factor in the development of metabolic disease and breast cancer. For instance, early life (in utero and early childhood) exposure to DDT has been associated with increased breast cancer risk in women, and these findings are supported by animal studies. While maternal DDT exposure in pregnancy has been linked to metabolic syndrome and breast cancer risk in offspring, the effects of paternal DDT exposure in male germ-line reprogramming and phenotypes in their progeny has not been investigated. Here, we evaluated the effects of pre-conception paternal exposure to DDT on offspring’s metabolism and susceptibility of breast cancer, using a mouse model. Methods: Male mice were exposed to DDT (1.7mg/kg body weight) by oral gavage for two weeks. At the end of this period, DDT and control-vehicle (CO) were housed with female mice, with free access to a standard chow diet, for three days. Pregnancy onset was assessed by the presence of a vaginal plug. The weight and number of pups per litter were determined two days after birth. Pups were weaned from mothers at 21 days of age, fed a standard chow diet for the extent of the study and weighed weekly. Mammary tumors were induced by subcutaneous administration of 15mg of medroxyprogesterone to six-week-old female offspring, followed by oral administration of 1mg 7,12-dimethylbenz[a]anthracene once-a-week for 3 weeks. Results: Paternal DDT exposure reprogramed the sperm small non-coding RNA content. In line with that, DDT offspring had decreased birthweight (p=0.04) and weaning weight (p=0.01) compared to CO. Further, ancestral DDT exposure caused metabolic dysfunction in offspring (p&amp;lt;0.05) at 7 and 22 weeks of age. Further, female offspring of DDT exposed fathers showed alterations in mammary gland development with a non-significant increase in numbers of terminal end buds and lower rates of apoptosis. In line with that, our preliminary data suggest that ancestral paternal exposures to DDT increases breast cancer risk in offspring: Compared to CO, the DDT offspring presented a non-significant increase in mammary tumor incidence and shorter latency to first-tumor onset. In addition, tumor growth (volume) was significantly increased in DDT offspring compared to CO (p&amp;lt;0.01). In conclusion, our findings support a role for DDT exposure from the paternal lineage in metabolic dysfunction and increased breast cancer risk in their offspring. Citation Format: Raquel Santana Da Cruz, Hong Cao, Camille Castilho Fontelles, Apsra Nasir, M Idalia Cruz, Sonia de Assis. Pre conception paternal DDT exposure and programming of metabolic dysfunction and breast cancer risk in offspring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 646.

Abstract 644: Ancestral paternal obesity: Systemic versus local effects on offspring mammary tissue development and tumorigenesis

Abstract Background: Obesity and overweight are risk factors for breast cancer, particularly in the post-menopausal years. We recently reported that having a history of ancestral overweight from the paternal lineage is enough to increase breast cancer susceptibility in daughters. Using a mouse model, we demonstrated that paternal consumption of an obesity inducing diet (OID) altered mammary gland development, increased mammary carcinogenesis and disrupted metabolic parameters in the female offspring, compared to the female offspring of fathers who consumed only control (CO) diet. Given both the systemic and local the mammary tissue alterations observed, we aimed to investigate in more details why daughters of overweight fathers are at increased risk for breast cancer. More specifically, we used mammary gland transplantation experiments to study whether this ancestrally-induced breast cancer predisposition is linked to systemic factors and/or mammary tissue confined changes in daughters. Material and methods: Male mice were exposed either to a control (CO) or to high-fat (OID) diet. CO and OID male mice were then mated with female mice fed CO diet exclusively. Female offspring of both CO and OID male mice underwent a mammary gland transplantation surgery. Briefly, female offspring had their mammary fat pad area between the nipple and the proximal lymph node excised. Afterwards, mammary tissue fragments (1 mm3) of a donor mouse, either CO or OID female offspring, were implanted into a pocket made in the cleared fat pad. The transplantations were performed from CO female offspring donors to both CO [CO(CO)] and OID [OID(CO)] female offspring hosts, as well as from OID female offspring donors to both CO [CO(OID)] and OID [OID(OID)] female offspring hosts. Approximately 10 weeks post-surgery, the mammary glands were collected, photographed and analyzed using ImageJ software to determine branching density, epithelial elongation and number of Terminal End Buds. Cell proliferation was assessed by ki-67 and cell apoptosis was measured on ImageJ software. Results: Our preliminary data shows that CO(OID) had more (p≤0.05) articulated mammary branching morphogenesis than CO(CO). OID(CO) displayed marginal increase (p≤0.09) in the mammary gland area, as well as in the mammary ductal elongation, compared to CO(OID). Additionally, the number of apoptotic cells within mammary ducts and lobules is higher in CO(OID) compared to OID(CO). In contrast, the number of proliferating cells is higher in OID(CO) mammary gland compared to CO(OID). Conclusion: Altogether, our findings suggest that daughters of overweight fathers have both systemic and mammary gland confined factors alterations that ultimately lead to higher breast cancer risk in adulthood. However, further data is needed to corroborate this hypothesis. Our ongoing mammary tumor transplantation studies should provide further insight. Citation Format: Camile C. Fontelles, Anni Wärri, Raquel S. Da Cruz, Maria I. Cruz, Ersilia Barin, Sonia De Assis. Ancestral paternal obesity: Systemic versus local effects on offspring mammary tissue development and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 644.

Gestational and Lactational Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Downregulates Junctional Proteins, Thyroid Hormone Receptor α1 Expression, and the Proliferation-Apoptosis Balance in Mammary Glands Post Puberty.

Mammary gland development requires hormonal regulation during puberty, pregnancy, and lactation. Brominated flame retardants (BFRs) are endocrine disruptors; they are added to consumer products to satisfy flammability standards. Previously, we showed that gestational and lactational exposure to an environmentally relevant mixture of BFRs disrupts proteins of the adherens junctions in rat dam mammary glands at weaning. Here, we hypothesize that perinatal exposure to the same BFR mixture also disrupts junctional proteins and signaling pathways controlling mammary gland development in pups. Dams were exposed through diet to a BFR mixture based on the substances in house dust; doses of the mixture used were 0, 0.06, 20, or 60 mg/kg/day. Dams were exposed continuously beginning prior to mating until pups' weaning; female offspring were euthanized on postnatal day (PND) 21, 46, and 208. The lowest dose of BFRs significantly downregulated adherens junction proteins, E-cadherin, and β-catenin, and the gap junction protein p-Cx43, as well as thyroid hormone receptor alpha 1 protein at PND 46. No effects were observed on estrogen or progesterone receptors. The low dose also resulted in a decrease in cleaved caspase-3, a downward trend in PARP levels, proteins involved in apoptosis, and an upward trend in proliferating cell nuclear antigen, a marker of proliferation. No effects were observed on ductal elongation or on the numbers of terminal end buds. Together, our results indicate that gestational and lactational exposure to an environmentally relevant mixture of BFRs disrupts cell-cell interactions, thyroid hormone homeostasis and the proliferation-apoptosis balance at PND 46, a critical stage for mammary gland development.

OR26-2 Phosphorylation of Estrogen Receptor Alpha is Required for Mammary Gland Development and MCF-7 Breast Tumor Response to Estradiol and Tamoxifen

Serine-167 (S167) is a major phosphorylation site in estrogen receptor alpha (ERα) and a predictor for endocrine therapy response in breast cancer. We previously showed using stable transfection that mutation of S167 to alanine (S167A) impacted MCF-7 breast cancer cell growth and morphology. Using CRISPR, we evaluated S167A mutation in the endogenous ERα gene in MCF-7 breast cancer cells and xenograft tumors. In vitro, MCF-7 cells with wildtype ERα (WT) exhibited >60% increase in growth after 4 days incubation with 10-9 M 17-β estradiol (E2), and >300% decrease in growth after 4 days incubation with 10-8 M 4-hydroxy-tamoxifen (TAM) as assessed by MTS assay. In marked contrast, S167A cells showed resistance to both E2 growth stimulation and TAM growth arrest. WT cells exhibited >300% increase in migration after 24 hr E2 as assessed by Boyden Chamber assay. S167A cells exhibited >3 fold increase in basal migration compared to WT cells but were insensitive to E2 stimulation of migration. RNAseq was performed on WT and S167A cells incubated with E2 or vehicle (6 hr). Sequencing data was assessed using FastQC and sequenced libraries mapped to the human genome (UCSC hg38) using STAR RNAseq aligner. RNASeq data was normalized using TMM. Differential expression analysis by edgeR revealed significant differences in gene expression between WT and S167A cells. Vehicle-treated S167A cells exhibited 623 differentially regulated genes compared to WT cells. Notably, S167A cells exhibited marked E2 resistance with only 9 genes regulated by E2 in S167A cells compare to 93 genes in WT cells. In tumor xenograft experiments using ovariectomized nude mice, similar to WT tumors the S167A tumors did not grow in the absence of implanted 17 beta-estradiol (0.72 mg/pellet; 60 day release) demonstrating dependency of S167A tumors on E2 for tumor establishment and growth. However, unlike WT tumors that exhibited tumor regression with E2 + TAM (5 mg/pellet; 60 day release) treatment (week 4, P<0.05), S167A tumors were resistant to TAM and exhibited continued growth similar to E2 alone treatment. Serine-212 (S216 in mouse) is a phosphorylation site located between the two zinc fingers in the DNA binding domain and is highly conserved in the nuclear receptor superfamily. To understand the importance of ERα phosphorylation for mammary gland development, ERα knock-in (KI) mice (Esr1S216A) that express the ERα S216A mutation were evaluated. Mammary glands were whole-mounted with carmine-alum from 5 week-old pubertal female ERα wildtype (WT) and KI mice (n=3). KI mice exhibited a significant decrease in ductal elongation (P<0.05), number of terminal end buds (P<0.01) and branching morphogenesis (P<0.01) compared to WT mice. Collectively, these results demonstrate that S216 phosphorylation is critical for mouse mammary gland development and S167 phosphorylation is required for MCF-7 breast tumor response to estradiol and tamoxifen.

Role of the ERp57 protein (1,25D3-MARRS receptor) in murine mammary gland growth and development

The protein disulfide isomerase ERp57 (GRp58/PDIA3/1,25D3-MARRS) has been implicated in a multitude of signaling pathways throughout the entire body. Most thoroughly studied for its protein-folding role, ERp57 has also been found to have multiple binding partners, and have significant effects on cellular growth. ERp57 has been studied n the context of several neurodegenerative disorders, metabolic conditions, and can be used as a prognosis marker in certain cancers. One role, as an alternate vitamin D binding receptor, has prompted research in tissues with known vitamin D activity, such as the intestine and bone. Vitamin D has been studied in relation to mammary gland growth and development, but it is not yet known if ERp57 plays an independent role in this tissue. In this study, ERp57 was knocked out in murine mammary gland epithelial cells of 30 4-week old mice. Several markers of mammary gland growth were measured, including number of terminal end buds (TEB), ductal coverage of the fat pad, and ductal extension. It was found the knockout animals had decreased numbers of TEBs (p = 0.019), and decreased ductal extension (p = 0.018) compared to wildtype animals, with no differences in gross body weight. Immunohistochemistry analysis of mammary glands showed ERp57 localized to the apical side of alveolar branches, and on leading edges of TEBs. These results provide further evidence for ERp57 functioning separately to the VDR, and further insights into the roles of ERp57.

Conditional knockout of N-Myc and STAT interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors

The process of organ development requires a delicate balance between cellular plasticity and differentiation. This balance is disrupted in cancer initiation and progression. N-Myc and STAT interactor (NMI: human or Nmi: murine) has emerged as a relevant player in the etiology of breast cancer. However, a fundamental understanding of its relevance to normal mammary biology is lacking. To gain insight into its normal function in mammary gland, we generated a mammary-specific Nmi knockout mouse model. We observed that Nmi protein expression is induced in mammary epithelium at the onset of pregnancy, in luminal cells and persists throughout lactation. Nmi knockout results in a precocious alveolar phenotype. These alveoli exhibit an extensive presence of nuclear β-catenin and enhanced Wnt/β-catenin signaling. The Nmi knockout pubertal ductal tree shows enhanced invasion of the mammary fatpad and increased terminal end bud numbers. Tumors from Nmi null mammary epithelium show a significant enrichment of poorly differentiated cells with elevated stem/progenitor markers, active Wnt/β-catenin signaling, highly invasive morphology as well as, increased number of distant metastases. Our study demonstrates that Nmi has a distinct role in the differentiation process of mammary luminal epithelial cell compartment and developmental aberrations resulting from Nmi absence contribute to metastasis and demonstrates that aberration in normal developmental program can lead to metastatic disease, highlighting the contribution and importance of luminal progenitor cells in driving metastatic disease.

Relationship between morphological development and sex hormone receptor expression of mammary glands with age in male rats.

The aim of this study is to investigate the changes with age on morphology and sex hormone receptor expression in the mammary glands of male Sprague-Dawley rats, focusing on male-specific cells, “oxyphilic cells”, observed after sexual maturity. The mammary glands of male rats at 14, 21, 35, 50, 75 and 100 days old were examined by gross observation, microscopic observation using whole mount specimens, histological and immunohistochemical sections. Grossly, mammary glands showed brown color at 50–100 days old. In whole mount specimens, terminal end buds (TEBs) were observed at 14–50 days old and the number of TEBs was highest at 35 days old. Histologically, the male mammary glands contained small epithelial cells with scanty cytoplasm at 14–35 days old while ductal and lobular epithelial cells were changed into oxyphilic cells with abundant cytoplasm at 50–100 days old. Immunohistochemicaly, androgen receptor (AR), estrogen receptor (ER) and progesterone receptor (PgR) expressions were found in both mammary glands found at a young age and oxyphilic cells. In oxyphilic cells, AR expression was dominant compared to ER and PgR expressions and increased with age. From these results, the development at 50–100 days old might be strongly related to AR. Ultrastructural observation of oxyphilic cells confirmed a number of lipid droplets, deformed and/or enlarged mitochondria, lysosomes and peroxisomes in their cytoplasm.

Early postnatal exposure to endosulfan interferes with the normal development of the male rat mammary gland

Our aim was to evaluate whether postnatal exposure to endosulfan (ENDO) modifies mammary gland (MG) development in pre- and post-pubertal male rats. From postnatal day 1 (PND1) to PND7, male rats were injected subcutaneously every 48h with either corn oil (vehicle) or 600μg ENDO/kg.bw. On PND21 and PND60, MG and blood samples were collected. Estradiol (E2) and testosterone (T) serum levels, MG histology, collagen fiber organization, proliferation index, and estrogen (ESR1) and androgen receptor (AR) expressions were evaluated. On PND21, E2 and T levels were similar between groups, whereas MG area, perimeter, number of terminal end buds and ESR1 expression were increased in ENDO-exposed rats. These changes were associated with alveolar development and increased organized collagen in the stroma. On PND60, a higher proliferation index in ENDO-exposed rats was correlated with a more developed lobuloalveolar structure. Hyperplastic alveoli and, hyperplastic ducts surrounded by a dense stroma were also observed in this group. T levels and ESR1 expression were similar between groups, whereas E2 levels and AR expression were decreased in ENDO-exposed rats. The exposure to ENDO in the first week of life interferes with the normal development of the MG and induces pre-malignant lesions in post-pubertal male rats.

A dioxin-like compound induces hyperplasia and branching morphogenesis in mouse mammary gland, through alterations in TGF-β1 and aryl hydrocarbon receptor signaling

Hexachlorobenzene (HCB) is a widespread environmental pollutant and a dioxin-like compound that binds weakly to the aryl hydrocarbon receptor (AhR). Because AhR and transforming growth factor β1 (TGF-β1) converge to regulate common signaling pathways, alterations in this crosstalk might contribute to developing preneoplastic lesions. The aim of this study was to evaluate HCB action on TGF-β1 and AhR signaling in mouse mammary gland, through AhR+/+ and AhR−/− models. Results showed a differential effect in mouse mammary epithelial cells (NMuMG), depending on the dose: 0.05μM HCB induced cell migration and TGF-β1 signaling, whereas 5μM HCB reduced cell migration, promoted cell cycle arrest and stimulated the dioxin response element (DRE) -dependent pathway. HCB (5μM) enhanced α-smooth muscle actin expression and decreased TGF-β receptor II mRNA levels in immortalized mouse mammary fibroblasts AhR+/+, resembling the phenotype of transformed cells. Accordingly, their conditioned medium was able to enhance NMuMG cell migration. Assays in C57/Bl6 mice showed HCB (3mg/kg body weight) to enhance ductal hyperplasia, cell proliferation, estrogen receptor α nuclear localization, branch density, and the number of terminal end buds in mammary gland from AhR+/+ mice. Primary culture of mammary epithelial cells from AhR+/+ mice showed reduced AhR mRNA levels after HCB exposure (0.05 and 5μM). Interestingly, AhR−/− mice exhibited an increase in ductal hyperplasia and mammary growth in the absence of HCB treatment, thus revealing the importance of AhR in mammary development. Our findings show that environmental HCB concentrations modulate AhR and TGF-β1 signaling, which could contribute to altered mammary branching morphogenesis, likely leading to preneoplastic lesions and retaining terminal end buds.

Abstract 4826: Maternal high butter fat intake heightens mammary cancer risk in offsprings gestationally exposed to bisphenol A at environmentally relevant dose

Abstract In utero exposure to bisphenol A (BPA) at 250ug/kg BW was shown to augment mammary cancer risk in rodent models. High fat diet is widely believed to be a major risk of breast cancer (BCa). It is therefore important to investigate whether maternal high fat intake could potentially heighten mammary cancer risk associated to gestational BPA exposure. In this study, we exposed Sprague Dawley rats with different doses of BPA (2.5-2500ug/kg BW) mixed with high butter-based diet (HBF) in the period from preconception to birth. DMBA-PND50 protocol was followed. In the presence of HBF, we observed non-monotonic effects of BPA in cancer incidence as well as in the number of terminal end buds of PND21 mammary glands. Both results were peaked at a dosage of 25ug/kg BW BPA and the effects were significant when compared with HBF alone group. We further analyzed transcriptomic data of microdissected epithelia of PND21 mammary glands from BPA (25ug/kg BW) group as well as HBF group and revealed that two specific cancer networks involving ERK and androgen receptor signaling were dysregulated. DNA methylation is one of the key mechanisms to dysregulate and impair the transcription of two selected top genes, Car7 and Kcnv2, as supported by our bisulfite-sequencing data. These data suggest that in utero BPA exposure could epigenetically modify gene expression and predispose cancer risk during early development. More importantly, some BPA genes are of high human relevance because seven selected BPA genes could dichotomize patients into two groups with significant difference in overall survival in a TCGA cancer cohort. The prognostic power of the genes was further enhanced in the survival analysis of Caucasian with ER positive patients. In conclusion, our data strongly suggest that BPA predisposes higher cancer risk by dysregulating gene expression during early mammary gland development in the presence of HBF, which could facilitate cancer development later in life. Citation Format: Yuet-Kin Leung, Vinothini Govindarajah, Ana Cheong, Dan Song, Xuegong Zhu, Jun Ying, Ady Kendler, Mario Medvedovic, Scott Belcher, Shuk-Mei Ho. Maternal high butter fat intake heightens mammary cancer risk in offsprings gestationally exposed to bisphenol A at environmentally relevant dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4826. doi:10.1158/1538-7445.AM2017-4826