Mammary Epithelial Proliferation Research Articles

Abstract 76: Liver receptor homologue-1 increases incidence of DMBA-induced mammary tumors

Abstract Breast cancer is one of the most common malignancies globally. It accounts for approximately 15% of cancer-related deaths in Australian women. The orphan nuclear receptor liver receptor homolog-1 (LRH-1) promotes increased cell proliferation, motility and invasion in breast cancer cell lines. Additionally, high LRH-1 expression in human breast cancers is positively associated with estrogen receptor alpha status and aromatase activity. However, the role of LRH-1 in tumour growth is not well understood. Therefore, we aimed to generate a doxycycline (dox)-inducible mammary epithelial specific LRH-1 knock-in mouse in order to define the role of LRH-1 in mammary epithelial proliferation in vivo. In addition, the Dimethylbenz(a)anthracene (DMBA) induced mammary tumour model was utilized with the LRH-1 transgenic mice to determine the role LRH-1 plays in promoting mammary carcinogenesis. Given clear in vitro roles for LRH-1 in breast cancer cell proliferation, we hypothesise LRH-1 stimulates mouse mammary epithelial cell proliferation and promotes DMBA-mediated mammary tumorigenesis. We demonstrate increase in proliferation markers Ki-67, PCNA and PH3 immunoreactivity in luminal epithelial cells of dox-treated animals indicating that LRH-1 plays a role in mammary cell proliferation in vivo. In DMBA treated animals, we observed a five-fold increase of dense pre-neoplastic epithelial foci (p=0.0267), and a four-fold increase in microscopic lesions in dox-treated animals (p=0.0003). We also demonstrated that LRH-1 over-expression significantly reduced breast tumour-free survival in the transgenic DMBA model (no dox n=11; dox n=12, Mantel-Cox test p=0.0375). Tumour penetrance in DMBA animals not treated with dox was 9% (out of eleven animals) versus 25.0% (out of twelve animals) in the dox treated cohort. LRH-1 is known synergise with β-catenin to induce cyclin D1/E1 mediated cell proliferation in vitro. We analysed transcript levels of cyclin D1/E1 and show a significant increase in dox treated animals with or without DMBA. Finally, we demonstrate an increase in the oncogene β-catenin nuclear localisation in the dox treated animals with or without DMBA. Taken together, these data suggest that LRH-1 increases incidence of DMBA-induced mammary tumours. Further analyses on mechanism(s) mediated by LRH-1 are warranted to fully understand its role in mammary tumorigenesis. Note: This abstract was not presented at the meeting. Citation Format: Kyren A. Lazarus, Kristy Brown, Morag Young, Jason Cain, Samantha Jayasekara, Rhiannon Coulson, Neil Watkins, Colin Clyne, Ashwini Chand. Liver receptor homologue-1 increases incidence of DMBA-induced mammary tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 76. doi:10.1158/1538-7445.AM2014-76

Abstract 5094: Investigating the role of CDC25B in inhibition of cellular proliferation

Abstract CDC25B is a dual specificity phosphatase that activates cyclin-dependent kinases to trigger entry into mitosis and is required for re-entry into mitosis after DNA damage. As an oncogene, CDC25B is overexpressed in multiple tumor types. It has been specifically associated with prostate cancer, where overexpression is seen in prostate tumor tissues and higher levels correlate with increased tumor grade. In the prostate, in addition to its role as a cell cycle regulator, CDC25B acts as an androgen receptor coactivator increasing the response of reporter genes to the addition of androgen analogues. In a transgenic mouse model, overexpression of CDC25B in the mammary glands led to accelerated mammary epithelial proliferation resulting in hyperplasia, while in vitro, co-transfection of CDC25B with Ras allows for transformation of MEF's. Surprisingly, given the establishment of CDC25B as an oncogene, colony formation assays using ectopic expression of CDC25B in transformed cell lines reveal an anti-proliferative effect. Our data reveal a role for Ras inhibition as a cause of this anti-proliferative effect. Co-transfection of CDC25B and Ras results in a dose-dependent decrease in expression of the ectopic Ras protein in several cell lines tested. Preliminary experiments suggest that while mRNA levels remain stable, CDC25B inhibits Ras protein expression. As Ras is pivotal in multiple tumors, this effect of CDC25B may be broadly applicable. These data therefore reveal a new role for CDC25B, which may need to be overcome to allow transformation of tumor cells. Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5094. doi:10.1158/1538-7445.AM2014-5094

RANKL expression in normal and malignant breast tissue responds to progesterone and is up-regulated during the luteal phase.

The receptor activator of nuclear factor-κB ligand (RANKL) acts as a paracrine factor in progesterone-induced mammary epithelial proliferation and tumorigenesis. This evidence comes mainly from mouse models. Our aim was to examine whether RANKL expression in human normal and malignant breast is under the control of progesterone throughout the menstrual cycle. Breast epithelial samples were obtained by random fine needle aspiration (rFNA) of the contralateral unaffected breasts (CUB) of 18 breast cancer patients, with simultaneous serum hormone measurements. Genes correlated with serum progesterone levels were identified through Illumina microarray analysis. Validation was performed using qRT-PCR in rFNA samples from CUB of an additional 53 women and using immunohistochemistry in tissue microarrays of 61 breast cancer samples. Expression of RANKL, DIO2, and MYBPC1 was correlated with serum progesterone in CUB, and was significantly higher in luteal phase. RANKL and MYBPC1 mRNA expression were highly correlated between CUB and matched tumor samples. RANKL protein expression was also significantly increased in the luteal phase and highly correlated with serum progesterone levels in cancer samples, especially in hormone receptor positive tumors. The regulatory effects of progesterone on the expression of RANKL, DIO2, and MYBPC1 were confirmed in three-dimensional cultures of normal breast organoids. In normal breast and in breast cancer, RANKL mRNA and protein expression fluctuate with serum progesterone with highest levels in the luteal phase, suggesting that RANKL is a modulator of progesterone signaling in normal and malignant breast tissue and a potential biomarker of progesterone action and blockade.

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice.The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.

Abstract 574: Investigating the role of CDC25B in inhibition of cellular proliferation.

Abstract CDC25B is a dual specificity phosphatase that activates cyclin-dependent kinases to trigger entry into mitosis. Overexpression of CDC25B has been associated with breast and head and neck cancers as well as higher histological tumor grade in non-small cell lung cancers. In a transgenic mouse model, overexpression of Cdc25B in the mammary glands lead to accelerated mammary epithelial proliferation resulting in hyperplasia while in vitro, co-transfection of CDC25B with Ras allows for transformation of MEF's. However, in vitro experiments using ectopic expression of CDC25B in transformed cell lines have shown an anti-proliferative effect. This may be related to inappropriate cyclin-dependent kinase activation and premature mitotic entry, leading to both p53-dependent and independent checkpoints. In addition, S phase cells overexpressing CDC25B have higher rates of apoptosis due to premature entry into mitosis. CDC25B has also been shown to cause an early progression to S phase that is associated with increased amounts of DNA damage. Our goal is to expand on these results to further clarify the anti-proliferative effects of CDC25B as well as detail the mechanism by which tumor cells bypass these effects in vitro and in vivo. Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [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 574. doi:10.1158/1538-7445.AM2013-574

Abstract 185: Secoisolariciresinol diglucoside (SDG, flaxseed lignan) improves biomarkers of early mammary gland cancer progression in a rat model of breast and ovarian cancer.

Abstract Breast cancer prevention efforts increasingly are focused on potentially beneficial dietary modifications due to their ease of implementation, wide acceptance and excellent subject compliance. SDG is a bioactive component of flaxseed suggested to have beneficial SERM-like effects in females at high risk for the development of breast cancer. In parallel with a human phase II prevention trial, female Sprague Dawley rats (n=10-12/group) received 0, 1, 10 or 20 mg/kg/day SDG in the feed. Simultaneously, rats were induced to concurrent mammary and ovarian cancer progression in a previously validated model using sustained release 17β-estradiol and local ovarian DMBA treatment starting at 6 weeks of age. Serum was collected at 3 weeks to measure steady state SDG metabolite concentrations. Mammary gland and ovarian tissues were collected at 3 months post-treatment and examined for changes in epithelial cell proliferation (Ki-67, cell counts), histopathology and dysplasia scores as well as a panel of cancer risk and progression biomarker genes. Carcinogen treatment markedly elevated dysplasia scores while SDG decreased dysplasia in a dose-dependent fashion, with an approximately 50% improvement the highest dose. Mammary epithelial cell numbers were increased by carcinogen and this effect was mitigated similarly at all doses of SDG and reflected in reduced Ki-67 expression. Overall, SDG decreased mammary epithelial proliferation and dysplasia resulting from carcinogen treatment. This preclinical benefit occurred at serum concentrations similar to our human pilot study, in which dietary SDG reduced KI-67 expression in mammary epithelial cells, and decreased the proportion of women with RPFNA atypia. Supported in part by funds from Komen for the Cure grant KG101039. Citation Format: Devora Delman, Bruce F. Kimler, Carol J. Fabian, Brian K. Petroff. Secoisolariciresinol diglucoside (SDG, flaxseed lignan) improves biomarkers of early mammary gland cancer progression in a rat model of breast and ovarian cancer. [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 185. doi:10.1158/1538-7445.AM2013-185

Effect of androgens on different breast cancer cells co-cultured with or without breast adipose fibroblasts

About 70% of breast tumors express androgen receptors. In addition, there is clinical evidence suggesting that androgens can inhibit mammary epithelial proliferation. Vice versa, there is also significant evidence indicating that androgens can increase the risk of breast cancer via multiple mechanisms, e.g. direct conversion to estrogens that can bind to the estrogen receptor and thereby stimulate cell proliferation. We examined the effect of testosterone (T) and dihydroxytestosterone (DHT) on cell proliferation, pS2 and Ki-67 expression in three different breast cancer cell lines alone or in co-culture with primary human breast adipose fibroblasts (BAFs) obtained from breast cancer patients. In the co-cultures, T induced cell proliferation, pS2 and Ki-67 expression in the estrogen receptor positive (ER+) MCF-7 and T47D cells. This was not observed in the (ER−) MDA-MB-231 cells. The differences might be explained by the high expression of aromatase, which converts androgens to estrogens in BAFs followed by ER-mediated cell proliferation. In line with this absence of increased cell proliferation, pS2 and Ki-67 expression was observed in the presence of DHT, which is not a substrate for aromatase. In contrast, DHT caused a significant suppression of cell proliferation (68% and 38%), pS2 and Ki-67 expression in the (ER+) MCF-7 and T47D cells. More importantly, DHT decreased cell proliferation in (ER−) MDA-MB-231 cells by 38%. The results suggest that androgens that cannot be aromatized, like DHT, may provide a perspective for treatment of breast cancer patients, especially those with triple negative breast cancer.

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2-/- mammary epithelial cells. Consequently, mammary glands of Agr2-/- females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.

Specific β-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration.

Genistein‐mediated inhibition of mammary stromal adipocyte differentiation limits expansion of mammary stem/progenitor cells by paracrine signaling

Mammary adiposity may contribute to breast cancer development and progression by releasing cytokines and other inflammatory mediators that promote mammary epithelial proliferation. We evaluated the effects of soy isoflavone genistein (GEN) on the adipogenic differentiation of a SV40‐immortalized mouse mammary stromal fibroblast like cells (MSF) treated with differentiating agents insulin, hydrocortisone and troglitazone (DM). MSF cultured in DM with and without extracellular matrix for 14 days showed reduced differentiation (2–3 fold) into mature adipocytes with 40nM but not 2uM GEN, as evaluated by Oil Red O staining. Addition of sera from GEN‐fed adult mice to MSF similarly reduced adipogenic differentiation (4–5 fold), relative to sera from casein‐fed mice. Lipid accumulation was decreased by GEN (40nM>2uM). Expression levels of lipogenic enzyme genes fatty acid synthase and PPAR‐γ were down‐regulated by 40nM GEN. Estrogen receptor (ER)‐β, an inhibitor of PPAR‐γ transcriptional activity, but not ER‐α transcript levels were increased by 40 nM GEN. Conditioned media from GEN‐treated MSF reduced anchorage‐independent mammosphere formation of human breast cancer cells MCF‐7. Thus, dietary factor control of mammary adiposity, possibly mediated by ER‐β, may limit mammary stem/progenitor cell expansion and protect against breast cancer.Grant Funding Source: USDA‐CRIS 6251‐5100002‐06S; CUMG/ACHRI

Folate exacerbates the effects of ethanol on peripubertal mouse mammary gland development

Alcohol consumption is linked with increased breast cancer risk in women, even at low levels of ingestion. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels resulting in diminished DNA synthesis and repair, and/or acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty, and that folate deficiency alone might mimic and/or exacerbate the effects of ethanol. To test this hypothesis, weight-matched 28–35 day old CD2F1 female mice were pair-fed liquid diets ±3.2% ethanol, ±0.1% folate for 4 weeks. Folate status was confirmed by assay of liver and kidney tissues. In folate deficient mice, no significant ethanol-induced changes to the mammary gland were observed. Folate replete mice fed ethanol had an increased number of ducts per section, due to an increased number of terminal short branches. Serum estrogen levels were increased by ethanol, but only in folate replete mice. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree.

P2-01-02: Pharmacological Inhibition of RANKL Attenuates Mammary Tumor Development and Lung Metastases in the MMTV-neu Transgenic Spontaneous Tumor Model.

Abstract Background: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary glands during pregnancy. In a mouse model of mammary carcinogenesis induced by the hormone medroxyprogesterone (MPA) and a carcinogen (DMBA), treatment with a RANKL inhibitor RANK-Fc reduces hormone-induced mammary epithelial proliferation, incidence of preneoplasias, and mammary adenocarcinomas. This study assessed the expression of mouse RANK and RANKL in FVB wild-type (WT) and MMTV-neu mice and whether RANKL inhibition could inhibit mammary tumor formation and lung metastases in this model. Materials and Methods: MMTV-neu mice were treated with the RANKL inhibitor RANK-Fc (10 mg/kg, 3x/week) or vehicle (muFc) beginning at 20 weeks. Mammary tumor formation was determined by palpation and confirmed by histologic examination. The presence of preneoplastic lesions and histotype of tumors was determined on H&E-stained tissues. Entire lungs from tumor-bearing MMTV-neu mice were step-sectioned at 75 μm and assessed histologically for the presence of metastases. Epithelial proliferation was measured by BrdU labeling of the mammary epithelium. The expression of RANK, RANKL, cyclin D1, progesterone receptor (PR), F4/80, FOXP3, and CD3 was determined by immunohistochemistry (IHC). Results: RANK-Fc treatment of MMTV-neu mice resulted in a significant reduction in total number of mammary tumors and lung metastases quantified at necropsy but did not impact median time to spontaneous mammary tumor onset. RANKL inhibition resulted in reduced pre-neoplasias and corresponding reductions in proliferation and cyclin D1 within normal mammary epithelia but not in adenocarcinomas. Treatment of MMTV-neu mice with RANK-Fc also reduced the incidence and number of lung metastases per mouse. RANK protein was detected in normal mammary epithelium, preneoplastic mammary intraepithelial neoplasia (MIN), the epithelial component of adenocarcinomas and lung metastases, and monocytic element adjacent to primary MMTV-neu tumors. RANKL protein was detected in normal mammary epithelium but not in the epithelia, stromal component or infiltrating cells of adenocarcinomas or lung metastases. Progesterone receptor expression was observed in normal mammary epithelium of MMTV-neu and FVB WT mice but decreased over time in MMTV-neu mice and was not detected in preneoplasias, adenocarcinomas, or lung metastases. CD3 was detected in mammary tumors and lung metastases but neither RANKL nor FOXP3 were present. Staining of lymph nodes was used as a positive control for RANKL and FOXP3. Expression of FOXP3 and RANKL were not measurably colocalized in dual staining of lymph nodes. Discussion: RANKL is expressed in normal mammary epithelia of MMTV-neu mice but is not detected in any stage of atypia, including MIN, mammary adenocarcinoma, or lung metastases. Selective pharmacological inhibition of RANKL by RANK-Fc treatment attenuated mammary tumor development and lung metastases in the MMTV-neu transgenic spontaneous tumor model. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-02.

Assessing estrogen signaling aberrations in breast cancer risk using genetically engineered mouse models

Aberrations in estrogen signaling increase breast cancer risk. Molecular mechanisms that impact breast cancer initiation, promotion, and progression can be investigated using genetically engineered mouse models. Increasing estrogen receptor alpha (ERα) expression levels twofold is sufficient to initiate and promote breast cancer progression. Initiation and promotion can be increased by p53 haploinsufficiency and by coexpressing the nuclear coactivators amplified in breast cancer 1 (AIB1) or the splice variant AIB1Δ3. Progression to invasive cancer is found with coexpression of these nuclear coactivators as well as following a single dose of 7,12-dimethylbenz(a)anthracene. Loss of signal transducer and activator of transcription 5a reduces the prevalence of initiation and promotion but does not protect from invasive cancer development. Cyclin D1 loss completely interrupts mammary epithelial proliferation and survival when ERα is overexpressed. Loss of breast cancer gene 1 increases estrogen signaling and cooperates with ERα overexpression in initiation, promotion, and progression of mammary cancer.

A mouse transgenic approach to induce β-catenin signaling in a temporally controlled manner

Although constitutive murine transgenic models have provided important insights into β-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated β-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of β-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89β-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of β-catenin (ΔN89β-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89β-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89β-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89β-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89β-catenin transgene results in palpable mammary tumors (within 7-9months) in the doxycycline-treated virgin MTB/TETO-ΔN89β-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89β-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89β-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate "proof-of-principle" for using the TETO-ΔN89β-catenin transgenic responder to activate (and then de-activate) β-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.

Elevated Circulating IGF-I Promotes Mammary Gland Development and Proliferation

Animal studies have shown that IGF-I is essential for mammary gland development. Previous studies have suggested that local IGF-I rather than circulating IGF-I is the major mediator of mammary gland development. In the present study we used the hepatic IGF-I transgenic (HIT) and IGF-I knockout/HIT (KO-HIT) mouse models to examine the effects of enhanced circulating IGF-I on mammary development in the presence and absence of local IGF-I. HIT mice express the rat IGF-I transgene under the transthyretin promoter in the liver and have elevated circulating IGF-I and normal tissue IGF-I levels. The KO-HIT mice have no tissue IGF-I and increased circulating IGF-I. Analysis of mammary gland development reveals a greater degree of complexity in HIT mice as compared to control and KO-HIT mice, which demonstrate similar degrees of mammary gland complexity. Immunohistochemical evaluation of glands of HIT mice also suggests an enhanced degree of proliferation of the mammary gland, whereas KO-HIT mice exhibit mammary gland proliferation similar to control mice. In addition, HIT mice have a higher percentage of proliferating myoepithelial and luminal cells than control mice, whereas KO-HIT mice have an equivalent percentage of proliferating myoepithelial and luminal cells as control mice. Thus, our findings show that elevated circulating IGF-I levels are sufficient to promote normal pubertal mammary epithelial development. However, HIT mice demonstrate more pronounced mammary gland development when compared to control and KO-HIT mice. This suggests that both local and endocrine IGF-I play roles in mammary gland development and that elevated circulating IGF-I accelerates mammary epithelial proliferation.

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.

Antitumor Agents. 282. 2′-(R)-O-Acetylglaucarubinone, a Quassinoid from Odyendyea gabonensis As a Potential Anti-Breast and Anti-Ovarian Cancer Agent

A new quassinoid, designated 2'-(R)-O-acetylglaucarubinone (1), and seven known quassinoids (2-8) were isolated, using bioactivity-guided separation, from the bark of Odyendyea gabonensis (Pierre) Engler [syn. Quassia gabonensis Pierre]. The structure of 1 was determined by spectroscopic analysis and by semisynthesis from glaucarubolone. Complete (1)H and (13)C NMR assignments of compounds 1-8 were also established from detailed analysis of two-dimensional NMR spectra, and the reported configurations in odyendene (7) and odyendane (8) were corrected. Compound 1 showed potent cytotoxicity against multiple cancer cell lines. Further investigation using various types of breast and ovarian cancer cell lines suggested that 1 does not target the estrogen receptor or progesterone receptor. When tested against mammary epithelial proliferation in vivo using a Brca1/p53-deficient mice model, 1 also caused significant reduction in mammary duct branching.

Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Abstract 2872: Effects of ethanol and folate on peripubertal mouse mammary gland

Abstract Alcohol consumption has been clearly linked with increased breast cancer risk in women, with risks incurred even at low levels of consumption. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels and diminished DNA repair capacity, coupled with acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty. We also hypothesized that folate deficiency alone might mimic the effects of ethanol, and that folate deficiency would exacerbate the effects of ethanol. CD2/F1 female mice (N=8 per group) were pair-fed control and ethanol-containing liquid diets +/− 0.2g/L folate supplementation from 5 weeks until 9 weeks of age. Mice were sacrificed after this 4 week feeding regimen, and mammary glands were harvested for preparation of whole mounts and paraffin sections. Folate levels of liver and kidney were assessed to verify folate status. Analysis of mammary gland whole mounts revealed that mammary gland structure was significantly altered in folate-replete mice fed ethanol, compared to control-fed mice (no ethanol/ folate-replete). These ethanol/folate replete mice showed an increased total number of ductal branches, due to an increased number of terminal short branches; no effect was seen on the number of terminal end buds or primitive alveolar buds. Systematic histological analysis of paraffin sections using NIH Image J software revealed that diets containing ethanol in the presence of folate significantly increased the total number of ducts, ductal epithelial area, and number of ducts with epithelial multilayering. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree. Future studies are examining the effects of peripubertal ethanol and folate exposure on gene expression in the mouse mammary gland. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2872.

Cold‐inducible RNA binding protein modulates proliferation in the mouse mammary epithelium

Cold‐inducible RNA binding protein (CIRP) is upregulated in response to cellular stress, either facilitating or repressing translation of its mRNA targets. We showed that CIRP is upregulated in all breast cancer cell lines examined, while others have shown CIRP overexpression in primary breast tumors. In the MCF‐7 breast cancer cell line, CIRP upregulates the expression of another stress‐induced RNA binding protein, HuR, as well as the cell cycle regulator cyclin E1. Both HuR and cyclin E1 are also overexpressed in breast cancer, with higher expression correlating with a poor prognosis. In order to determine the in vivo function of CIRP in the breast epithelium, we produced a transgenic mouse expressing CIRP in the mammary epithelium under control of the mouse mammary tumor virus (MMTV) regulatory sequences. Real‐time RT‐PCR showed maximal expression of the transgene in the pregnant mouse at gestation day 16, and a second peak of expression after birth, at lactation day 14. Immunohistochemistry for the proliferation marker Ki‐67 showed decreased proliferation in the mammary epithelium of CIRP transgenic mice compared to wild type littermates at gestation day 16 (45% CIRP vs. 56% wild type, p=0.05) and lactation day 1 (7% CIRP vs. 25% wild type, p=0.0068). These results suggest that CIRP negatively regulates proliferation in the intact mammary gland. We are currently examining effects of CIRP expression on apoptosis in the involuting mammary gland. This work was supported by a grant from the National Cancer Institute‐National Institutes of Health to RSH (R01CA095898).