Normal Mammary Gland Development Research Articles

Loss of Nuclear Localized Parathyroid Hormone-Related Protein in Primary Breast Cancer Predicts Poor Clinical Outcome and Correlates with Suppressed Stat5 Signaling.

Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin-Stat5-PTHrP axis that is progressively lost in more aggressive tumors.

Krüpple-like factor 5 is essential for mammary gland development and tumorigenesis.

Krüpple-like factor 5 (KLF5) is required for the development of the embryo and multiple organs, such as the lung and intestine. KLF5 plays a pro-proliferative and oncogenic role in several carcinomas, including breast cancer. However, its role in normal mammary gland development and oncogenesis has not been elucidated in vivo. In this study, we used mammary gland-specific Klf5 conditional knockout mice derived by mating Klf5-LoxP and MMTV-Cre mice. The genetic ablation of Klf5 suppresses mammary gland ductal elongation and lobuloalveolar formation. Klf5 deficiency inhibits mammary epithelial cell proliferation, survival, and stem cell maintenance. Klf5 promotes mammary stemness, at least partially, by directly promoting the transcription of Slug. Finally, Klf5 depletion suppressed PyMT-induced mammary gland tumor cell stemness, tumor initiation, and growth in vivo. Slug also mediated these functions of Klf5 in vivo. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Joint linkage and linkage disequilibrium mapping reveals association of BRMS1L with total teat number in a large intercross between Landrace and Korean native pigs

Previously, we reported a quantitative trait locus (QTL) that affects total teat number (TTN) on pig chromosome 7 (SSC7) in a large F2 intercross population between Landrace and Korean native pigs. The aim of this study was to refine the QTL associated with TTN and to identify positional candidate gene(s) within the refined genomic region. TTN was recorded in 1105 F2 progeny. All experimental animals were genotyped using 980 informative single nucleotide polymorphism (SNP) markers located on SSC7. A haplotype-based linkage and association analysis using the PHASEBOOK programme was applied to refine the QTL region. Additionally, linear mixed-effect models were used to assess the effect of a positional candidate gene on TTN and other economically important traits [i.e., thoracic vertebrae number (THO), carcass body length (CBL) and weight (CW), back fat thickness (BFT) and intramuscular fat content (IMF) in loin muscles]. Joint linkage and association analysis refined the critical region to a 4.50 Mb region that included a novel positional candidate gene, BRMS1L that encodes the breast cancer metastasis-suppressor 1-like protein, which could possibly be implicated in normal mammary gland development. Significant association of an SNP marker (g. −1087 G > A) in the 5′-flanking region of BRMS1L with TTN (P = 1.10 × 10−8), THO (P = 5.80 × 10−4), and CBL (P = 0.038) was observed. Based on these data, we propose BRMS1L as a positional candidate gene for TTN in pigs. After validation of the association in other independent populations, these results could be useful in optimizing breeding programmes that improve TTN and other economically important traits in swine.

Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids.

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type–specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.

Abstract 1138: LBH, a novel WNT effector in promoting basal-like breast cancer

Abstract BACKGROUND: Clinically aggressive basal-like breast cancers (BLBC) disproportionally contribute to cancer deaths, but lack effective treatment options due to absence of expression of key therapeutic targets (ER, PR, HER2). New evidence suggests BLBC may originate from luminal breast epithelial cells through luminal-to-basal cell lineage conversion and dedifferentiation. However, the factors that reprogram cell fate and differentiation states during BLBC development remain poorly understood. Our laboratory has identified a novel WNT/β-Catenin target transcriptional regulator, Limb-Bud-and-Heart (LBH), which is majorly overexpressed in aggressive BLBC harboring WNT hyperactivation. We previously showed in a conditional LBH knockout mouse model in vivo that LBH is a key stem cell and basal lineage regulator in normal mammary gland development. However, it's role in breast cancer remains elusive. Here, we crossed conditional LBH knockout (KO) mice with a mouse model for WNT-driven breast cancer (MMTV-Wnt1) to test if LBH is a critical effector of WNT-driven basaloid breast cancer, and whether its inhibition may affect BLBC tumorigenesis. RESULTS: In our in vivo mouse model, epithelial-specific LBH inactivation using a Keratin 14/K14-Cre significantly attenuated Wnt1-induced mammary gland hyperplasia and delayed tumor onset. Yet, tumor volumes and burden were not changed, indicating that LBH is not essential for WNT-induced tumorigenesis. However, LBH-deficient MMTV-Wnt1-transgenic tumors exhibited pronounced histopathological differences compared to LBH WT MMTV-Wnt1-transgenic tumors. Whereas Wnt1+;K14CreLbh WT tumors were highly vascularized, disorganized, with mixed basal and luminal cell identity; tumors from Wnt1+;K14CreLbh KO mice were more organized, differentiated, and predominantly luminal Keratin 8 positive. We are currently investigating if the attenuated hyperplasia and tumor onset caused by LBH loss may be due to a deficiency in cancer stem cells in this breast cancer model. CONCLUSION: Our data indicate that LBH is an essential cell fate regulator downstream of WNT signaling that maintains basal lineage identity and the undifferentiated nature of BLBC. Thus, LBH inhibition may be a novel strategy to reduce the high mortality of BLBC patients through differentiation therapy. Citation Format: Kilan C. Ashad-Bishop, Karoline J. Briegel. LBH, a novel WNT effector in promoting basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1138.

PO-168 Loss of the MCC gene expression promotes invasiveness of colon cancer cells

IntroductionBreast cancer is the most common cancer in women in developed countries. Many reports have suggested that oestrogens play a key role in normal mammary gland development and in breast cancer progression and metastasis. About 70% of breast carcinoma expresses the oestrogen receptor alpha. The selective oestrogen receptor modulator tamoxifen, functions as an antagonist in most ER-positive breast tumours. However, 20%–30% of tumours are resistant to tamoxifen therapy. Foetal oestrogen estetrol seems to have a protective role during pregnancy and anti-estrogenic properties on breast tissue. Estetrol is an interesting compound for therapeutic applications due to its high oral bioavailability and long terminal elimination half-life, with no active oestrogen metabolites. Traditional two dimensional (2D) cell cultures lack proper environmental context leading to changes on cell function. On the contrary, three-dimensional (3D) cell culture has gained increasing interest in drug discovery due to its realistic modelling of complex environment that drives to cellular heterogeneity, nutrient and oxygen gradients of tumours. The aim of the current study was to evaluate whether estetrol have an anti-tumour activity in T47D breast cancer cells on 2D and 3D culture systems.Material and methodsT47D cells were growth in DMEM high glucose, phenol red-free media with 10% FBS. After starvation, cells were treated with increasing concentrations (1, 10, 100, 1000 nM) of Estetrol (E4), 17β-oestradiol (E2) and Tamoxifen (Tmx). All experiments were realised in DMEN with 5% of charcoal stripped FBS. Cell proliferation (trypan blue), clonogenic and wound healing micro-inserts assays were performed on 2D cell culture. T47D spheroids were performed on 96-well low attachment plate. 3D cell proliferation was measured as Feret diameter and spheroid-based migration assay was measured as radial migration index.Results and discussionsWe observed that 100 and 1000 nM of E4 significantly reduced cell proliferation, survival fraction and migration compared to the control on 2D cell model. As expected, Tmx was more effective on the reduction of cell proliferation, survival fraction and migration. On the other side, E4 and Tmx significantly reduced 3D cell proliferation and migration compared to the control. We can conclude that E4 do not stimulate cell proliferation and migration, on the contrary reduced it.ConclusionWe can conclude that E4 could have an anti-oestrogenic activity on T47D breast cancer cell line.

PTEN PDZ-binding domain suppresses mammary carcinogenesis in the MMTV-PyMT breast cancer model

Phosphatase and tension homolog (PTEN) is a potent tumor suppressor that possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus, whose functions during tumorigenesis remains unclear. Here, we crossed a mouse strain with germline deletion of PTEN PDZ-BD with MMTV-PyMT breast cancer model, and found that knockout (KO) mice display normal development of mammary glands, but have both increased breast tumorigenicity and lung metastasis. Orthotopic allograft experiments suggest the loss of PTEN PDZ-BD in breast cancer cells rather than in tumor microenvironment plays a prominent role in increasing tumor burden. Through RNA-sequencing, we observed a significant downregulation of myoepithelial marker genes in both KO primary breast cancer and orthotopic allografts. Moreover, these myoepithelial marker genes are significantly downregulated in human breast cancer tissues, and are associated with poorer clinical prognosis. In addition, several homeobox genes were also identified to be downreguated in KO breast cancer, whose expressions showed significant positive correlation with myoepithelial marker genes. Overall, our findings suggest a novel tumor suppressive role of PTEN PDZ-BD in a murine model of breast cancer, and the mechanism involves the dysregulation of homeobox genes which may result in defective myoepithelial differentiation in breast cancer cells.

Regulation of OPN expression in MDA‐MB‐435: role of genistein

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.Support or Funding InformationDBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reprogramming of the estrogen responsive transcriptome contributes to tamoxifen-dependent protection against tumorigenesis in the p53 null mammary epithelial cells.

The tumor suppressor gene p53 is frequently mutated in human breast cancer and is a marker for poor prognosis and resistance to chemotherapy. Transplantation of p53 null mouse mammary epithelium into syngeneic wild-type mice leads to normal mammary gland development followed by spontaneous mammary tumors that recapitulate many of the phenotypic, molecular and genetic features of human breast cancer. Transient exposure of p53 null mice to the anti-estrogen, tamoxifen leads to sustained and robust protection against tumor development. However the mechanism underlying this anti-tumor activity remains poorly understood. Here we demonstrate that transient exposure to tamoxifen leads to a reduction in mammary ductal side-branching and epithelial cell proliferation after tamoxifen withdrawal. Global gene expression analysis showed that transient tamoxifen exposure leads to persistent changes in the expression of a subset of estrogen regulated gene signatures in mammary epithelial cells (MECs). Among these was the protein tyrosine phosphatase, non-receptor type 5 (Ptpn5). We show that Ptpn5 is a novel tamoxifen regulated target gene which is upregulated in MECs after transient tamoxifen exposure and displays tumor suppressor activity in human breast cancer cells. Further, PTPN5 expression is strongly associated with good clinical outcome in tamoxifen treated human breast cancer patients suggesting that PTPN5 may represent a novel biomarker of tamoxifen response in human breast cancer.

HSD17B1 expression induces inflammation-aided rupture of mammary gland myoepithelium.

Hydroxysteroid (17-beta) dehydrogenase type 1 (HSD17B1) converts low-active estrogen estrone to highly active estradiol. Estradiol is necessary for normal postpubertal mammary gland development; however, elevated estradiol levels increase mammary tumorigenesis. To investigate the significance of the human HSD17B1 enzyme in the mammary gland, transgenic mice universally overexpressing human HSD17B1 were used (HSD17B1TG mice). Mammary glands obtained from HSD17B1TG females at different ages were investigated for morphology and histology, and HSD17B1 activity and estrogen receptor activation in mammary gland tissue were assessed. To study the significance of HSD17B1 enzyme expression locally in mammary gland tissue, HSD17B1-expressing mammary epithelium was transplanted into cleared mammary fat pads of wild-type females, and the effects on mammary gland estradiol production, epithelial cells and the myoepithelium were investigated. HSD17B1TG females showed increased estrone to estradiol conversion and estrogen-response element-driven estrogen receptor signaling in mammary gland tissue, and they showed extensive lobuloalveolar development that was further enhanced by age along with an increase in serum prolactin concentrations. At old age, HSD17B1TG females developed mammary cancers. Mammary-restricted HSD17B1 expression induced lesions at the sites of ducts and alveoli, accompanied by peri- and intraductal inflammation and disruption of the myoepithelial cell layer. The lesions were shown to be estrogen dependent, as treatment with an antiestrogen, ICI 182,780, starting when lesions were already established reversed the phenotype. These data elucidate the ability of human HSD17B1 to enhance estrogen action in the mammary gland in vivo and indicate that HSD17B1 is a factor inducing phenotypic alterations associated with mammary tumorigenesis.

Prep1 (pKnox1) transcription factor contributes to pubertal mammary gland branching morphogenesis.

Prep1 (pKnox1) is a homeodomain transcription factor essential for in utero and post-natal development and an oncosuppressor gene in human and adult mice. We have analyzed its role in the development of the mouse mammary gland. We used Prep1i/i hypomorphic and Prep1F/F-Ker5CRE crosses to analyze the role of Prep1 in vivo in adult mouse mammary gland development. We also cultured mammary gland stem/progenitor cells in mammospheres to perform biochemical studies. Prep1 was expressed in mammary gland progenitors and fully differentiated mammary gland cells. Using different Prep1-deficient mouse models we show that in vivo Prep1 contributes to mammary gland branching since the branching efficiency of the mammary gland in Prep1-deleted or Prep1 hypomorphic mice was largely reduced. In-vitro, Prep1 sustained functions of the mammary stem/progenitor compartment. Prep1-deficient mammary stem/progenitor cells showed reduced ability to form mammospheres; they were not able to branch in a 3D assay, and exhibited reduced expression of Snail1, Snail2 and vimentin. The branching phenotype associated with increased Tp53-dependent apoptosis and inability to properly activate signals involved in branching morphogenesis. Finally, Prep1 formed complexes with Snail2, a transcription factor essential in branching morphogenesis, and its absence destabilizes and promotes Snail2 proteasome-mediated degradation. We conclude that Prep1 is required for normal adult mammary gland development, in particular at its branching morphogenesis step. By binding Snail2, Prep1 protects it from the proteasomal degradation.

The ANK repeats of Notch-4/Int3 activate NF-\u03baB canonical pathway in the absence of Rbpj and causes mammary tumorigenesis

Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.

Pubertally Initiated High-Fat Diet Promotes Mammary Tumorigenesis in Obesity-Prone FVB Mice Similarly to Obesity-Resistant BALB/c Mice

Premenopausal breast cancer is associated with increased animal fat consumption among normal-weight but not overweight women. Our previous findings in obesity-resistant BALB/c mice showed that a diet high in saturated animal fat (HFD) promotes mammary tumorigenesis in both DMBA carcinogenesis and Trp53-null transplant models. Having made these observations in BALB/c mice, which have very modest HFD weight gain, we determined the effects of HFD in FVB mice, which gain significant weight on HFD. Three-week-old FVB mice fed a low-fat diet or HFD were subjected to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis. Like BALB/c mice, HFD promoted mammary tumorigenesis. Development of tumors largely occurred prior to mice becoming obese, indicating the role of animal-derived HFD rather than resulting obesity in tumor promotion. Also similar to BALB/c mice, early-occurring adenosquamous mammary tumors were abundant among HFD-fed FVB mice. Tumors from HFD mice also had increased intra-tumor M2 macrophages. Prior to tumor development, HFD accelerated normal mammary gland development and increased mammary M2 macrophages, similarly to BALB/c mice. The promotional effects of puberty-initiated HFD on carcinogen-induced mammary cancer are thus largely weight gain-independent. Like BALB/c mice, HFD promoted adenosquamous tumors, suggesting a role for early age HFD in promoting this subtype of triple negative mammary cancer. M2 macrophage recruitment was common to both mouse strains. We speculate that a similar effect of HFD on immune function may contribute to epidemiological findings of increased breast cancer risk in young, premenopausal, normal-weight women who consume a diet high in saturated animal fat.

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.

Sectioning Mammary Gland Whole Mounts for Lesion Identification.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&E section. The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

A new method to study the change of miRNA-mRNA interactions due to environmental exposures.

MotivationIntegrative approaches characterizing the interactions among different types of biological molecules have been demonstrated to be useful for revealing informative biological mechanisms. One such example is the interaction between microRNA (miRNA) and messenger RNA (mRNA), whose deregulation may be sensitive to environmental insult leading to altered phenotypes. The goal of this work is to develop an effective data integration method to characterize deregulation between miRNA and mRNA due to environmental toxicant exposures. We will use data from an animal experiment designed to investigate the effect of low-dose environmental chemical exposure on normal mammary gland development in rats to motivate and evaluate the proposed method.ResultsWe propose a new network approach—integrative Joint Random Forest (iJRF), which characterizes the regulatory system between miRNAs and mRNAs using a network model. iJRF is designed to work under the high-dimension low-sample-size regime, and can borrow information across different treatment conditions to achieve more accurate network inference. It also effectively takes into account prior information of miRNA–mRNA regulatory relationships from existing databases. When iJRF is applied to the data from the environmental chemical exposure study, we detected a few important miRNAs that regulated a large number of mRNAs in the control group but not in the exposed groups, suggesting the disruption of miRNA activity due to chemical exposure. Effects of chemical exposure on two affected miRNAs were further validated using breast cancer human cell lines.Availability and implementationR package iJRF is available at CRAN.Supplementary information Supplementary data are available at Bioinformatics online.

Abstract 1000: Estrogen receptor coactivator MED1 in breast tumorigenesis and therapeutic resistance

Abstract Recent studies have established Mediator Subunit 1 (MED1) as a key ER transcriptional coactivator for in both normal mammary gland development and breast cancer. Significantly, the MED1 gene is located at the chromosome 17q12 region, also known as the HER2 amplicon, and co-amplifies with HER2 in almost all instances. Importantly, we found that MED1 serves as a key crosstalk point for the HER2 and ER pathways in anti-estrogen resistance of breast cancer. Significantly, MED1 expression highly correlates with poor disease-free survival of breast cancer patients; and most recent studies have discovered increased frequency of MED1 mutations in circulating tumor cells of human patients following anti-estrogen and anti-HER2 treatments. To determine the role of MED1 in HER2-driven tumorigenesis, we have crossed the MMTV-HER2 mammary tumor model with our established MED1 mutant knockin and newly generated MED1 mammary specific overexpression mouse models. Collectively, our studies revealed critical roles for MED1 in tumor progression, metastasis, cancer stem cell formation and therapeutic resistance in HER2-mediated mammary tumorigenesis. These studies not only for the first time reported key roles for a HER2 amplicon co-amplified gene in HER2-driven tumorigenesis but also support MED1 as a potential therapeutic target. To test that, we have assembled highly innovative RNA nanotechnology-based pRNA-HER2apt-siMED1 nanoparticles. We found these RNA nanoparticles have a very high Tm value, and is ultra-stable under RNase A, 8 M urea, serum and PBS conditions. Importantly, pRNA-HER2apt-siMED1 nanoparticles could specifically target HER2+ human breast cancer, efficiently deplete the expression of MED1 and decrease ER-mediated gene transcription both in vitro and in vivo. Most significantly, pRNA-HER2apt-siMED1 nanoparticles not only greatly reduce the growth, metastasis and cancer stem cell formation of HER2+ breast cancer, but are also able to overcome their therapeutic resistance. (This work is supported by Susan G. Komen for the Cure Foundation Career Catalyst Grant, American Cancer Society Research Scholar Grant and NIH/NCI Grant Number R01CA197865). Citation Format: Xiaoting Zhang. Estrogen receptor coactivator MED1 in breast tumorigenesis and therapeutic resistance [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 1000. doi:10.1158/1538-7445.AM2017-1000

Abstract P4-05-01: Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development

Abstract The mammary gland is one of the few organs that continues to develop postnatally through stages including puberty, pregnancy, lactation, and involution. The gland is composed of epithelial and stromal cells that include fibroblasts, adipocytes, endothelial cells, nerve cells, and macrophages. Terminal end bud (TEB) structures are found exclusively in the pubertal developmental stage. The formation of TEBs and side branching drives mammary gland epithelial cell invasion into the mammary fat pad, continuing until the entire fat pad is filled. Pubertal mammary gland morphogenesis integrates a balance of epithelial cell proliferation, differentiation, and apoptosis. Several studies have shown that the interaction between mammary epithelial and stromal cells is crucial for the proper postnatal development of the mammary ductal tree. Interestingly, studies have shown that processes important in mammary gland development are often deregulated during breast cancer tumorigenesis. Thus, understanding the complex signaling network as well as the interactions between the different cell types during mammary gland development will be vital for elucidating the mechanisms underlying breast cancer progression and metastasis. Glycation is the non-enzymatic glycosylation of sugar moieties to biological macromolecules such as protein and DNA which produces reactive metabolites known as advanced glycation end products (AGE's). AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar laden and cheap processed/manufactured foods which are high in reactive AGE metabolites. AGE containing food can lead to the accumulation of AGEs in the body overtime leading to pro-inflammatory and pro-oxidant effects when signaling through receptor for advanced glycation end products (RAGE). Leading too many complications associated with diseases including diabetes, Alzheimer's, heart disease and cancer. Preliminary data in our lab has shown that AGEs also have an effect on phosphorylation and signaling of estrogen receptor α (ERα), a key receptor and signaling pathway in the regulation of mammary gland development during puberty. This observation, together with the links between diet, mammary gland development and immune cell recruitment lead us to examine the biological effects of a diet high in AGEs on pubertal mammary gland development in mice. We observed a significant disruption of normal pubertal mammary gland development in mice fed a high AGE diet when compared to mice fed a control diet. Mice fed the high AGE diet showed increases in TEB number as well as width, length and area. We also observed an increase in ductal branching and a decrease in ductal extension. Future studies will assess the role of macrophage recruitment to the developing gland, specifically around the TEBs based on its reported role in normal TEB function. We also plan to assess ERa signaling in mice fed the high AGE diet based on the reported role of estrogen signaling in ductal elongation. Citation Format: Krisanits BA, Nogueira LM, Findlay VJ, Turner DP. Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-01.

Abstract ES1-2: Paracrine signaling and intratumoral heterogeneity

Abstract Breast cancer is no longer considered a single disease, but instead comprises multiple subtypes with genetically and most likely epigenetically heterogeneous tumors composed of numerous clones. Both the hierarchical cancer stem cell (CSC) and clonal evolution models have been invoked to help explain this intratumoral heterogeneity. Several recent studies have helped define the functional interactions among the different cellular subpopulations necessary for the evolution of this complex ecosystem. These interactions involve paracrine interactions that include locally acting Wnt family members, reminiscent of the signaling pathways important for normal mammary gland development and stem cell self-renewal. In addition, they involve interactions between breast cancer cells and the immune microenvironment. In this presentation, we will discuss the interactions among various cell populations in both normal and tumor tissues. A better understanding of these interactions, especially in the metastatic setting, will be important for the development of improved combinatorial therapies designed to prevent relapse and to ultimately decrease mortality. Citation Format: Rosen JM. Paracrine signaling and intratumoral heterogeneity [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES1-2.

Abstract P4-05-02: Conditional inactivation of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) in the mouse mammary epithelium alters mammary gland development

Abstract The biologically active form of vitamin D (1,25(OH)2D) regulates proliferation, differentiation, and apoptosis in diverse cell types. We have previously identified anti-proliferative activities of 1,25(OH)2D in human breast tissue, as well as 1,25(OH)2D target gene expression consistent with reports that elevated vitamin D levels may protect against cancer. In mouse studies, vitamin D signaling modulates normal mammary gland development, including ductal outgrowth and branching, and protects against tumorigenesis. Degradation of 1,25(OH)2D is initiated by the enzyme Cyp24a1 in target tissues, providing critical local control of 1,25(OH)2D bioactivity. In vitro, blockade of Cyp24a1 activity potentiates the anti-proliferative effects of 1,25(OH)2D. However, the extent to which endogenous Cyp24a1 activity within the mammary epithelium regulates local 1,25(OH)2D levels to modulate normal mammary gland development, with possible implications for cancer, has not been investigated. We generated a novel mouse model with conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium (MMTV-Cre x Cyp24a1lox/lox). Ablation of Cyp24a1 activity in the mammary epithelium does not alter either gland or body weight at 4, 6 or 10 weeks of age. Preliminary analyses of mammary gland whole mounts indicate that virgin knockout mice form fewer terminal end buds compared to glands from wild-type littermates at 4 and 6 weeks of age (P<0.05). Moreover, the width of the ducts proximal to the central lymph node of knockout mice was less than that of wild-type mice at 4 and 10 weeks of age (P<0.05 and P<0.01, respectively). In addition, the number of secondary and tertiary branching points is reduced in mammary glands from knockout mice at 6 weeks of age (P<0.05 and P<0.01, respectively). In summary, our findings suggest that Cyp24a1 activity within epithelial cells plays a crucial role to modulate postnatal mammary gland development, presumably by limiting the local accumulation of 1,25(OH)2D. Citation Format: Sheng L, Turner AG, Tarulli GA, Barratt K, Kremer R, Morris HA, Callen DF, Anderson PH. Conditional inactivation of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) in the mouse mammary epithelium alters mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-02.