Normal Mammary Gland Development Research Articles

Abstract PO4-28-06: A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression

Abstract Characterizing the mechanisms underlying the regulation of cellular differentiation and mitochondrial dynamics are critical to understanding normal development and breast cancer in the mammary gland. Our previous studies have highlighted the significance of Singleminded-2s (Sim2s), a member of the bHLH/PAS family, in regulating mitochondrial dynamics during mammary gland development and the progression of estrogen receptor-positive (ER+) breast cancer. Sim2s is temporally regulated, with maximal expression occurring during mid-lactation. Cross-fostered pups nursed by miceover-expressing Sim2s under the mouse mammary tumor virus (MMTV-Sim2s) display significantly higher weights by mid-lactation compared to pups nursed by control dams. Overexpression of Sim2s leads to alterations in mitochondrial morphology and dynamics, characterized by increased OPA1 expression and decreased DRP1 levels, resulting in enhanced mitochondrial fusion and elongation. Furthermore, we have extended our investigations to an ER+ breast cancer cell line (MCF7). We have identified SIM2s as a tumor suppressor expressed in mammary epithelial cells, known to inhibit epithelial-mesenchymal transition (EMT) and metastasis. In our previous studies, we discovered that loss of SIM2s expression in the MCF7 cell line promotes mitochondrial fragmentation, as evidenced by a decrease in OPA1 expression and an increase in DRP1 levels. In conjunction with these findings, literature suggests that sirtuins also play a role in the regulation of mitochondrial dynamics similar to Sim2s. Specifically, there is an upregulation of Sirtuin 1 in cancer when Sim2 is lost, while Sirtuin 3 increases during differentiation and normal development progression. Therefore, our aim is to elucidate the potential interaction between Singleminded-2s and sirtuins, a family of NAD+-dependent deacetylases, in the regulation of mitochondrial dynamics. We hypothesize that Sim2s may modulate mitochondrial function through its association with sirtuins, thereby influencing normal mammary gland development and breast cancer progression. Citation Format: Hannah Carter. A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-06.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues

ObjectiveUncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. MethodsWe employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. ResultsUcp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. ConclusionsOur findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Defining and targeting macrophage heterogeneity in the mammary gland and breast cancer.

Macrophages are innate immune cells that are associated with extensive phenotypic and functional plasticity and contribute to normal development, tissue homeostasis, and diseases such as cancer. In this review, we discuss the heterogeneity of tissue resident macrophages in the normal mammary gland and tumor-associated macrophages in breast cancer. Tissue resident macrophages are required for mammary gland development, where they have been implicated in promoting extracellular matrix remodeling, apoptotic clearance, and cellular crosstalk. In the context of cancer, tumor-associated macrophages are key drivers of growth and metastasis via their ability to promote matrix remodeling, angiogenesis, lymphangiogenesis, and immunosuppression. We identified and summarized studies in Pubmed that describe the phenotypic and functional heterogeneity of macrophages and the implications of targeting individual subsets, specifically in the context of mammary gland development and breast cancer. We also identified and summarized recent studies using single-cell RNA sequencing to identify and describe macrophage subsets in human breast cancer samples. Advances in single-cell RNA sequencing technologies have yielded nuances in macrophage heterogeneity, with numerous macrophage subsets identified in both the normal mammary gland and breast cancer tissue. Macrophage subsets contribute to mammary gland development and breast cancer progression in differing ways, and emerging studies highlight a role for spatial localization in modulating their phenotype and function. Understanding macrophage heterogeneity and the unique functions of each subset in both normal mammary gland development and breast cancer progression may lead to more promising targets for the treatment of breast cancer.

Loss of the extracellular matrix glycoprotein EMILIN1 accelerates Δ16HER2-driven breast cancer initiation in mice

The extracellular matrix (ECM) is an important component of the tumor microenvironment and undergoes extensive remodeling during both initiation and progression of breast cancer (BC). EMILIN1 is an ECM glycoprotein, whose function has been linked to cancer and metastasis. However, EMILIN1 role during mammary gland and BC development has never been investigated. In silico and molecular analyses of human samples from normal mammary gland and BC showed that EMILIN1 expression was lower in tumors than in healthy mammary tissue and it predicted poor prognosis, particularly in HER2-positive BC. HER2+ BC accounts for 15-20% of all invasive BC and is characterized by high aggressiveness and poor prognosis. The Δ16HER2 isoform, a splice variant with very high oncogenic potential, is frequently expressed in HER2+ BC and correlates with metastatic disease. To elucidate the role of EMILIN1 in BC, we analyzed the phenotype of MMTV-Δ16HER2 transgenic mice, developing spontaneous multifocal mammary adenocarcinomas, crossed with EMILIN1 knock-out (KO) animals. We observed that Δ16HER2/EMILIN1 KO female mice exhibited an accelerated normal mammary gland development and a significantly anticipated appearance of palpable tumors (13.32 vs 15.28 weeks). This accelerated tumor initiation was corroborated by an increased number of tumor foci observed in mammary glands from Δ16HER2/EMILIN1 KO mice compared to the wild-type counterpart. Altogether our results underscore the centrality of ECM in the process of BC initiation and point to a role for EMILIN1 during normal mammary gland development and in protecting from HER2-driven breast tumorigenesis.

Cellular Plasticity in Mammary Gland Development and Breast Cancer

Cellular plasticity is a phenomenon where cells adopt different identities during development and tissue homeostasis as a response to physiological and pathological conditions. This review provides a general introduction to processes by which cells change their identity as well as the current definition of cellular plasticity in the field of mammary gland biology. Following a synopsis of the evolving model of the hierarchical development of mammary epithelial cell lineages, we discuss changes in cell identity during normal mammary gland development with particular emphasis on the effect of the gestation cycle on the emergence of new cellular states. Next, we summarize known mechanisms that promote the plasticity of epithelial lineages in the normal mammary gland and highlight the importance of the microenvironment and extracellular matrix. A discourse of cellular reprogramming during the early stages of mammary tumorigenesis that follows focuses on the origin of basal-like breast cancers from luminal progenitors and oncogenic signaling networks that orchestrate diverse developmental trajectories of transforming epithelial cells. In addition to the epithelial-to-mesenchymal transition, we highlight events of cellular reprogramming during breast cancer progression in the context of intrinsic molecular subtype switching and the genesis of the claudin-low breast cancer subtype, which represents the far end of the spectrum of epithelial cell plasticity. In the final section, we will discuss recent advances in the design of genetically engineered models to gain insight into the dynamic processes that promote cellular plasticity during mammary gland development and tumorigenesis in vivo.

The Influence of the Normal Mammary Microenvironment on Breast Cancer Cells.

The tumor microenvironment is recognized as performing a critical role in tumor initiation, progression, and metastasis of many cancers, including breast cancer. The breast cancer microenvironment is a complex mixture of cells consisting of tumor cells, immune cells, fibroblasts, and vascular cells, as well as noncellular components, such as extracellular matrix and soluble products. The interactions between the tumor cells and the tumor microenvironment modulate tumor behavior and affect the responses of cancer patients to therapies. The interactions between tumor cells and the surrounding environment can include direct cell-to-cell contact or through intercellular signals over short and long distances. The intricate functions of the tumor microenvironment in breast cancer have led to increased research into the tumor microenvironment as a possible therapeutic target of breast cancer. Though expanded research has shown the clear importance of the tumor microenvironment, there is little focus on how normal mammary epithelial cells can affect breast cancer cells. Previous studies have shown the normal breast microenvironment can manipulate non-mammary stem cells and tumor-derived cancer stem cells to participate in normal mammary gland development. The tumorigenic cells lose their tumor-forming capacity and are "redirected" to divide into "normal", non-tumorigenic cells. This cellular behavior is "cancer cell redirection". This review will summarize the current literature on cancer cell redirection and the normal mammary microenvironment's influence on breast cancer cells.

RAC1B function is essential for breast cancer stem cell maintenance and chemoresistance of breast tumor cells

Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.

Fascin is essential for mammary gland lactogenesis

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin−/− dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin−/− mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin−/− epithelial fraction was significantly reduced. Further in-depth analyses of fascin−/− mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and β-casein in fascin−/− mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

Induction of alternative NF-κB within TAg-induced basal mammary tumors in activation-resistant inhibitor of κ-B kinase (IKKα) mutant mice.

The alternative NF-κB pathway is activated by the NF-κB-inducing kinase (NIK) mediated phosphorylation of the inhibitor of κ-B kinase α (IKKα). IKKα then phosphorylates p100/NFKB2 to result in its processing to the active p52 subunit. Evidence suggests that basal breast cancers originate within a subpopulation of luminal progenitor cells which is expanded by signaling to IKKα. To determine the role of IKKα in the development of basal tumors. Kinase dead IkkαAA/AA mice were crossed with the C3(1)-TAg mouse model of basal mammary cancer. Tumor growth and tumor numbers in WT and IkkαAA/AA mice were assessed and immunopathology, p52 expression and stem/progenitor 3D colony forming assays were performed. Nik-/- mammary glands were isolated and mammary colonies were characterized. While tumor growth was slower than in WT mice, IkkαAA/AA tumor numbers and pathology were indistinguishable from WT tumors. Both WT and IkkαAA/AA tumors expressed p52 except those IkkαAA/AA tumors where NIK, IKKαAA/AA and ErbB2 were undetectable. Colonies formed by WT and IkkαAA/AA mammary cells were nearly all luminal/acinar however, colony numbers and sizes derived from IkkαAA/AA cells were reduced. In contrast to IkkαAA/AA mice, virgin Nik-/- mammary glands were poorly developed and colonies were primarily derived from undifferentiated bipotent progenitor cells. C3(1)-TAg induced mammary tumors express p100/p52 even without functional IKKα. Therefore the development of basal-like mammary cancer does not strictly rely on IKKα activation. Signal-induced stabilization of NIK may be sufficient to mediate processing of p100NFKB2 which can then support basal-like mammary tumor formation. Lastly, in contrast to the pregnancy specific role of IKKα in lobuloalveogenesis, NIK is obligatory for normal mammary gland development.

Abstract 2536: Investigating the mechanisms of HER2+ breast cancer cell redirection

Abstract Introduction: Breast cancer research has advanced understanding of breast cancer development and progression greatly over the past few decades. Despite progress in research, breast cancer is the second leading cause of cancer death in North America and is the most frequent type of cancer for women. Better treatments and diagnostic tools have increased breast cancer survival rates, but there is still a fundamental lack of understanding in tumor progression. Previous studies show the normal mammary microenvironment can influence non-mammary cells and tumor-derived cancer cells to participate in normal mammary gland development. The tumorigenic cells lose their tumor-forming capabilities and are “redirected” into phenotypically normal, non-tumorigenic cells. The purpose of this study is to obtain knowledge of the mechanisms that play a role in cancer cell redirection and therefore be able manipulate those mechanisms for therapeutic treatment in a clinical setting. Our hypothesis is that microenvironmental elements control whether a tumorigenic cell will enter a redirected state. We have developed and validated an in vitro model to mimic the mammary microenvironment to study cancer cell redirection. We found that when cancer cells that overexpress HER2+ are redirected in our cancer redirection model, phospho-HER2+ is silenced. We use HER2+ phosphorylation as a marker for cancer cell redirection though not a mechanism for cancer cell redirection. Materials and Methods: HER2+ breast cancer cells and normal breast epithelial cells (BECs) were co-cultured in ratios of 1:1 or 1:50. Monocultures of breast cancer cells and BECs were used as controls. Western analysis and immunostaining was used to assess attenuation of HER2+ and phospho-HER2+ receptors and RNAseq was used for pathway analyses. Images were taken using a Leica confocal microscope. For comparison of 2 or more groups, a one-way analysis of variance was performed. A p-value of less than 0.05 was considered significant. Results and Discussion: We found that the redirected cells underwent a phenotype shift in which the redirected cells adopted a normal mammary epithelial phenotype based on gene expression profiles. Furthermore, when HER2+ breast cancer cells were redirected in vitro they lost their tumor-forming potential in vivo. Signal pathway analyses revealed that redirected cancer cells are adopting a normal phenotype compared to breast cancer cells. Conclusions: These results indicate that epithelial cells provide signals that influence HER2+ breast cancer cells to undergo a shift in phenotype. The phenotypic shift in cancer cell redirection includes multiple intracellular signaling pathways that may be the key towards effective cancer treatment. Acknowledgements: This research was supported by South Carolina Idea Networks of Biomedical Research Excellence (SC INBRE). Citation Format: Caroline J. Campbell, Brian W. Booth. Investigating the mechanisms of HER2+ breast cancer cell redirection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2536.

Vitamin D and Breast Cancer: Mechanistic Update.

ABSTRACTThe presence of the vitamin D receptor (VDR) in mammary gland and breast cancer has long been recognized, and multiple preclinical studies have demonstrated that its ligand, 1,25‐dihydroxyvitamin D (1,25D), modulates normal mammary gland development and inhibits growth of breast tumors in animal models. Vitamin D deficiency is common in breast cancer patients, and some evidence suggests that low vitamin D status enhances the risk for disease development or progression. Although many 1,25D‐responsive targets in normal mammary cells and in breast cancers have been identified, validation of specific targets that regulate cell cycle, apoptosis, autophagy, and differentiation, particularly in vivo, has been challenging. Model systems of carcinogenesis have provided evidence that both VDR expression and 1,25D actions change with transformation, but clinical data regarding vitamin D responsiveness of established tumors is limited and inconclusive. Because breast cancer is heterogeneous, the relevant VDR targets and potential sensitivity to vitamin D repletion or supplementation will likely differ between patient populations. Detailed analysis of VDR actions in specific molecular subtypes of the disease will be necessary to clarify the conflicting data. Genomic, proteomic, and metabolomic analyses of in vitro and in vivo model systems are also warranted to comprehensively understand the network of vitamin D–regulated pathways in the context of breast cancer heterogeneity. This review provides an update on recent studies spanning the spectrum of mechanistic (cell/molecular), preclinical (animal models), and translational work on the role of vitamin D in breast cancer. © 2021 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Current Status of Breast Organoid Models.

Breast cancer (BC) is the most frequently diagnosed malignancy among women globally. Although mouse models have been critical in advancing the knowledge of BC tumorigenesis and progression, human breast models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of BC risk factors. As such, it is essential to identify an ex vivo human breast tissue mimetic model that can accurately pinpoint the effects of these factors in BC development. While two-dimensional models have been invaluable, they are not suitable for studying patient-specific tumor biology and drug response. Recent developments in three-dimensional (3D) models have led to the prominence of organized structures grown in a 3D environment called “organoids.” Breast organoids can accurately recapitulate the in vivo breast microenvironment and have been used to examine factors that affect signaling transduction, gene expression, and tissue remodeling. In this review, the applications, components, and protocols for development of breast organoids are discussed. We summarize studies that describe the utility of breast organoids, including in the study of normal mammary gland development and tumorigenesis. Finally, we provide an overview of protocols for development of breast organoids, and the advantages and disadvantages of different techniques in studies are described. The included studies have shown that breast organoids will continue to serve as a crucial platform for understanding of progression of BC tumors and the testing of novel therapeutics.

Notch Signalling in Breast Development and Cancer.

The Notch signalling pathway is a highly conserved developmental signalling pathway, with vital roles in determining cell fate during embryonic development and tissue homeostasis. Aberrant Notch signalling has been implicated in many disease pathologies, including cancer. In this review, we will outline the mechanism and regulation of the Notch signalling pathway. We will also outline the role Notch signalling plays in normal mammary gland development and how Notch signalling is implicated in breast cancer tumorigenesis and progression. We will cover how Notch signalling controls several different hallmarks of cancer within epithelial cells with sections focussed on its roles in proliferation, apoptosis, invasion, and metastasis. We will provide evidence for Notch signalling in the breast cancer stem cell phenotype, which also has implications for therapy resistance and disease relapse in breast cancer patients. Finally, we will summarise the developments in therapeutic targeting of Notch signalling, and the pros and cons of this approach for the treatment of breast cancer.

CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. SIGNIFICANCE: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G2-M checkpoint and proliferative signaling.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope.

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Dorsal raphe serotonin neurotransmission is required for the expression of nursing behavior and for pup survival

Proper maternal care is an essential factor of reproductive success in mammals, involving a repertoire of behaviors oriented toward the feeding and care of the offspring. Among the neurotransmitters involved in the initiation of these behaviors, serotonin (5-HT) seems to play an important role. Here we compared pup-oriented maternal behaviors in mice with constitutive 5-HT depletion, the tryptophan hydroxylase 2-knock-out (Tph2-KO) and the Pet1-KO mice. We report that the only common pup-oriented defect in these 2 hyposerotoninergic models is a defective nursing in parturient mice and altered nursing-like (crouching) behavior in virgin mice, while pup retrieval defects are only present in Tph2-KO. Despite a normal mammary gland development and milk production, the defect in appropriate nursing is responsible for severe growth retardation and early lethality of pups born to hyposerotonergic dams. This nursing defect is due to acute rather constitutive 5-HT depletion, as it is reproduced by adult knockdown of Tph2 in the dorsal raphe nucleus in mothers with a prior normal maternal experience. We conclude that 5-HT innervation from the dorsal raphe is required for both the initiation and maintenance of a normal nursing behavior. Our findings may be related to observations of reduced maternal/infant interactions in human depression.

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.

The many facets of Notch signaling in breast cancer: toward overcoming therapeutic resistance.

Breast cancer is the second leading cause of cancer-related death in women and is a complex disease with high intratumoral and intertumoral heterogeneity. Such heterogeneity is a major driving force behind failure of current therapies and development of resistance. Due to the limitations of conventional therapies and inevitable emergence of acquired drug resistance (chemo and endocrine) as well as radio resistance, it is essential to design novel therapeutic strategies to improve the prognosis for breast cancer patients. Deregulated Notch signaling within the breast tumor and its tumor microenvironment (TME) is linked to poor clinical outcomes in treatment of resistant breast cancer. Notch receptors and ligands are also important for normal mammary development, suggesting the potential for conserved signaling pathways between normal mammary gland development and breast cancer. In this review, we focus on mechanisms by which Notch receptors and ligands contribute to normal mammary gland development and breast tumor progression. We also discuss how complex interactions between cancer cells and the TME may reduce treatment efficacy and ultimately lead to acquired drug or radio resistance. Potential combinatorial approaches aimed at disrupting Notch- and TME-mediated resistance that may aid in achieving in an improved patient prognosis are also highlighted.

Classical and Non-Classical Progesterone Signaling in Breast Cancers.

Much emphasis is placed on estrogen (E2) and estrogen receptor (ER) signaling as most research is focused on understanding E2 and ER’s ability to enhance proliferative signals in breast cancers. Progesterone (P4) is important for normal mammary gland development, function and menstrual control. However, P4 and its receptors (PRs) in breast cancer etiology continue to be understudied and its role in breast cancer remains controversial. The Women’s Health Initiative (WHI) clinical trial clearly demonstrated the importance of progestogens in breast cancer development. P4 has historically been associated with classical-signaling through nuclear receptors, however non-classical P4 signaling via membrane receptors has been described. Progestogens have the ability to bind to nuclear and membrane receptors and studies have demonstrated that both can promote breast cancer cell proliferation and breast tumor growth. In this review, we attempt to understand the classical and non-classical signaling role of P4 in breast cancers because both nuclear and membrane receptors could become viable therapeutic options for breast cancer patients.

Premenopausal Plasma Osteoprotegerin and Breast Cancer Risk: A Case-Control Analysis Nested within the Nurses' Health Study II.

Emerging evidence supports a role of the receptor activator of NF-κB (RANK) pathway in normal mammary gland development and breast carcinogenesis. Osteoprotegerin (OPG) is the endogenous decoy receptor for RANK-ligand (RANKL), which inhibits RANK-signaling. Whether OPG may be a biomarker of breast cancer risk remains unclear. We evaluated the association between plasma OPG and breast cancer risk in a case (n = 297)-control (n = 297) study nested within the Nurses' Health Study II. Cases were women who were cancer-free and premenopausal at blood collection who developed invasive breast cancer. OPG was quantified using an ELISA. Conditional logistic regression was used to estimate multivariable odds ratios (OR) and 95% confidence intervals (CI) for the association between OPG levels and breast cancer risk, adjusting for potential confounders. Unconditional logistic regression, additionally adjusting for matching factors, was used for stratified analyses. Overall, there was no substantial evidence for an association between plasma OPG levels and breast cancer risk, although the point estimate for the highest (vs. lowest) quartile was below 1 (OR = 0.78; 95% CI, 0.46-1.33; P trend = 0.30). There was no evidence of heterogeneity by various reproductive, hormonal, or tumor characteristics, including hormone receptor status and grade (all P heterogeneity ≥ 0.17). Findings from this prospective study do not provide substantial evidence for an association between circulating OPG and breast cancer risk among premenopausal women; however, we were underpowered in stratified analyses. Results do not provide strong evidence for OPG as a potential biomarker of breast cancer risk among premenopausal women.