Mammary Epithelial Proliferation Research Articles

PSIII-12 Influence of early gestational heat stress on biomarkers of mammary gland development in replacement gilts genomically selected for thermotolerance or thermosensitivity

Abstract Heat stress (HS) alters physiological and metabolic processes in lactating sows leading to decreased milk production, which is a major factor limiting growth and survivability of piglets. While genomic selection for thermotolerance may be a viable solution to alleviate the negative effects of HS on pig welfare, it may be linked to a reduction in milk production and subsequently, litter growth performance. Therefore, the study objective was to evaluate the effects of genomic selection for thermotolerance and its interaction with early gestational HS on biomarkers of mammary gland development in replacement gilts. We hypothesized that HS exposure as well as genomic selection for thermotolerance would negatively affect mammary epithelial proliferation rate. A total of 36 Landrace (33%) × Large White (67%) crossbred gilts divergently selected for thermotolerance (TOL; n = 18) or thermosensitivity (SEN; n = 18) were balanced by body weight, bred to a single Duroc sire, and then exposed to either thermoneutral (TN; constant 17 to 22°C) or cyclical HS (26 to 36°C) conditions until d 65 of gestation. From d 66 of gestation until farrowing, all pregnant gilts were exposed to TN conditions. Of the 36 total gilts bred, only 28 became pregnant yielding 15 HS gilts (n = 8 SEN and 7 TOL) and 13 TN gilts (n = 7 SEN and 6 TOL). On d 105 of gestation, a mammary biopsy was taken from all gilts, mammary tissue was placed into 10% buffered formalin, and KI67 immunohistochemical staining was performed to identify proliferating mammary epithelial cells (MEC). Proliferating and non-proliferating MEC populations were counted using ImageJ tool. Data were analyzed using PROC GLM in SAS 9.4 with individual gilt as the experimental unit. Overall, early gestational HS decreased (P < 0.01; 16.53 ± 2.12%) the percent of proliferating MEC relative to those kept under TN conditions (26.42 ± 2.39%). However, no effect of genomic line divergence was detected with any comparison (P > 0.05). In conclusion, early gestation HS, but not genomic selection for thermotolerance, had a negative impact on biomarkers of mammary gland development in replacement gilts. These data may suggest that early gestation HS could have a reductive effect on milk production capacity, which may negatively affect litter growth.

Abstract 5259: Bazedoxifene plus conjugated estrogen improves metabolic health and reduces mammary gland epithelial proliferation in an ovary intact rat model of obesity and breast cancer risk

Abstract Background: The majority of women risk eligible for primary breast cancer prevention with tamoxifen or aromatase inhibitors refuse uptake due to concerns about side effects, especially vasomotor symptoms. Tamoxifen may also have detrimental metabolic effects in overweight/obese women. Duavee™, a complex of bazedoxifene (BZA) and conjugated estrogen (CE), is FDA approved for relief of hot-flashes and prevention of osteoporosis. Preclinical studies suggest favorable metabolic effects in ovariectomized animals and potential for breast cancer risk reduction. A single arm clinical trial found reduction in Ki-67 and mammographic density such that BZA+CE is being further evaluated in a Phase IIB trial of peri and early postmenopausal women with vasomotor symptoms who are at high risk for breast cancer. Here, in a companion study, we assessed the effect of BZA+ CE on metabolic health and cancer risk in a rat model of carcinogen induced ER+ tumors and high fat diet induced obesity. Methods: Rats received 50 mg/kg N-methylnitrosourea at 7 weeks to increase mammary tumor risk and were fed a high fat diet (46% kcal fat) to promote obesity. At 16 weeks lean and obese rat were selected based on % body fat and were then randomized to 8 weeks of daily oral vehicle control or 3mg BZA + 0.07mg CE/kg body weight. Systemic metabolic variables were measured at 16 and 24 weeks, and RNA from mammary glands analyzed by gene expression microarray (Affymetrix). Results: BZA+CE resulted in systemic as well as mammary changes indicative of improved metabolic health, including: reduction in total and visceral fat (p<0.01), glucose (p<0.05), cholesterol (p<0.001), and triglycerides(p=0.08), as well as an increase in the adiponectin:leptin ratio (p<0.01). BZA+CE vs control increased the ratio of small (insulin sensitive) to large adipocytes in the mammary gland. Gene set enrichment analysis (GSEA) showed BZA+CE downregulated proliferation-associated pathways which had been upregulated in obese rats, consistent with anti-cancer effects. Obese rats treated with BZA+CE also had upregulation of metabolic pathways, including adipocyte differentiation, fatty acid metabolism, and inflammatory response pathways. Conclusions: Unlike tamoxifen, BZA+CE improved whole body and mammary gland metabolic health, particularly in obese rats. Transcriptional profiling and GSEA demonstrated a blunting of cell cycle and proliferation and enrichment of metabolic and immune-related pathways. Together this supports BZA+CE (Duavee) as an agent to be explored for breast cancer risk reduction in obese individuals. Funding: This work was supported in part by P30 CA168524, R00 CA169430, and R25 CA203650. Citation Format: Ramsey M. Jenschke, Karen A. Corleto, Carol J. Fabian, Stephen D. Hursting, Bruce F. Kimler, Danilo Landrock, Tara N. Mahmood, Katherine L. Cook, Erin D. Giles. Bazedoxifene plus conjugated estrogen improves metabolic health and reduces mammary gland epithelial proliferation in an ovary intact rat model of obesity and breast cancer risk. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5259.

C/EBP\u03b2 isoform-specific regulation of migration and invasion in triple-negative breast cancer cells

The transcription factor C/EBPβ is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPβ-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPβ-isoforms in breast cancer development may reveal new avenues of treatment.

Organization of mammary blood vessels as affected by mammary parenchymal region and estradiol administration in Holstein heifer calves

Mammary blood flow is central to mammary growth, development, and productivity, but the development of the vasculature network is poorly understood. The objective of this study was to determine how the vascular system adapts to mammary growth by inducing different levels of mammary growth and examining 2 regions of mammary parenchymal tissue. Holstein heifer calves (n = 12) received daily injections on the days immediately preceding euthanasia at 82 d of age. Treatments were control (CON), short-term estradiol (STE), and long-term estradiol (LTE). The CON calves received corn oil injections, the STE calves received 9 injections of corn oil followed by 3 injections of estradiol, and the LTE calves received 12 estradiol injections. Mammary tissues were collected from the center and edge parenchymal regions of all right rear mammary glands to quantify the tissue area of various tissue structures, the percentage of proliferating epithelial cells, and the number and form of blood vessels. Results showed that LTE calves had a greater tissue area occupied by epithelium than CON and STE calves, and the epithelial area in CON and STE calves was similar. Edge parenchyma had a greater percentage of proliferating epithelial cells than center parenchyma across all treatment groups. In the edge region, LTE calves had the greatest percentage of proliferating epithelial cells, coinciding with greater epithelial area. The number of blood vessels per unit of tissue area was greater in center than in edge parenchyma; the corresponding vessel surface area per unit of tissue area followed the same pattern. Mammary blood vessel measures were not markedly influenced by estradiol treatment. These results highlight the marked difference in the number and organization of blood vessels in different mammary parenchyma regions but indicate that the effects of estradiol on stimulating mammary epithelial proliferation does not directly translate to increasing numbers of blood vessels.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Inhibition of the RUNX1-CBFβ transcription factor complex compromises mammary epithelial cell identity: a phenotype potentially stabilized by mitotic gene bookmarking.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report that interaction between RUNX1 and its heterodimeric partner CBFβ is essential for sustaining mammary epithelial cell identity. Disruption of RUNX1-CBFβ interaction, DNA binding, and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. During interphase, RUNX1 is organized as punctate, predominantly nuclear, foci that are dynamically redistributed during mitosis, with a subset localized to mitotic chromosomes. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 associates with RNA Pol II-transcribed protein coding and long non-coding RNA genes and RNA Pol I-transcribed ribosomal genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. A subset of these genes remains occupied by the protein during the mitosis to G1 transition. Together, these findings establish that the RUNX1-CBFβ complex is required for maintenance of the normal mammary epithelial phenotype and its disruption leads to EMT. Importantly, our results suggest, for the first time, that RUNX1 mitotic bookmarking of a subset of epithelial-related genes may be an important epigenetic mechanism that contributes to stabilization of the mammary epithelial cell identity.

Cancer cell-intrinsic function of CD177 in attenuating β-catenin signaling.

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in breast cancer. Additionally, CD177 expression is correlated with good prognosis in several other solid cancers including prostate, cervical, and lung. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Loss of CD177 leads to hyperproliferative mammary epithelium and contributes to breast cancer pathogenesis. Mechanistically, we found that CD177-deficiency is associated with an increase in β-Catenin signaling. Here we identified CD177 as a novel regulator of mammary epithelial proliferation and breast cancer pathogenesis likely via the modulation of Wnt/β-Catenin signaling pathway, a key signaling pathway involved in multiple cancer types.

Abstract B57: Microtubule dynamics regulates mammary gland morphogenesis and tumorigenesis via stathmin

Abstract Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. The dynamics of MT is governed by the activity of MT-stabilizing and destabilizing proteins, generally known as MT-acting proteins. Little is known about the role of the MT-dynamics and of MT-acting proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and postpartum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells of the developing alveolus, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore decided to examine the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of preneoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, preneoplastic lesions expressed high level of stathmin compared with normal tissue, supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, by syngeneic injection of tumor cells in recipient mice of the two different genotypes, we could assess that stathmin plays a cell-autonomous function in tumor initiation. Our study has shed new light on the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also considering that several microtubule-acting drugs are currently employed in the therapy of BC patients. Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Giorgia Mungo, Sara D’Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Microtubule dynamics regulates mammary gland morphogenesis and tumorigenesis via stathmin [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B57.

Abstract 1460: Stathmin regulates mammary gland morphogenesis and tumorigenesis

Abstract Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. Little is known about the role of the MT-dynamics and of MT-regulating proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and post-partum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore examined the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of pre-neoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, pre-neoplastic lesions expressed high level of stathmin compared with normal tissue supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, we assessed that stathmin plays a cell autonomous function in tumor initiation. Our study has shed new light in the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules, causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also in consideration that several microtubules acting drugs are currently employed in the therapy of BC patients Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Francesca Citron, Sara D'Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Stathmin regulates mammary gland morphogenesis and tumorigenesis [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 1460.

Sterilization of Silastic Capsules Containing 17β-Estradiol for Effective Hormone Delivery in Mus musculus.

Silastic capsules are frequently used to study the physiologic effects of estrogen exposure in animal models. The Office of Laboratory Animal Welfare requires the sterilization of nonpharmaceutical-grade compounds before use. We compared 2 commonly used terminal sterilization methods-ionizing radiation (IR) and ethylene oxide (EO)-for their utility in sterilizing silastic capsules containing 0.05 or 0.1 mg 17β-estradiol (E2). E2-specific ELISA demonstrated that serum estrogen levels did not differ between mice implanted with 0.05-mg E2 capsules that were sterilized with IR or EO and those implanted with nonsterilized capsules. Likewise, mammary gland morphology and progesterone receptor expression and proliferation in mammary epithelium were similar among mice treated with E2 capsules, regardless of sterilization method, and pregnant day 15 mice. In addition, IR-sterilized 0.1-mg E2 pellets provided high serum E2. We conclude that neither ionizing radiation nor ethylene oxide degraded E2 or the cellulose matrix, suggesting that these methods of sterilization are appropriate to provide effective sterile hormone capsules for animal research.

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac-treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected inestrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

CUZD1 is a critical mediator of the JAK/STAT5 signaling pathway that controls mammary gland development during pregnancy.

In the mammary gland, genetic circuits controlled by estrogen, progesterone, and prolactin, act in concert with pathways regulated by members of the epidermal growth factor family to orchestrate growth and morphogenesis during puberty, pregnancy and lactation. However, the precise mechanisms underlying the crosstalk between the hormonal and growth factor pathways remain poorly understood. We have identified the CUB and zona pellucida-like domain-containing protein 1 (CUZD1), expressed in mammary ductal and alveolar epithelium, as a novel mediator of mammary gland proliferation and differentiation during pregnancy and lactation. Cuzd1-null mice exhibited a striking impairment in mammary ductal branching and alveolar development during pregnancy, resulting in a subsequent defect in lactation. Gene expression profiling of mammary epithelium revealed that CUZD1 regulates the expression of a subset of the EGF family growth factors, epiregulin, neuregulin-1, and epigen, which act in an autocrine fashion to activate ErbB1 and ErbB4 receptors. Proteomic studies further revealed that CUZD1 interacts with a complex containing JAK1/JAK2 and STAT5, downstream transducers of prolactin signaling in the mammary gland. In the absence of CUZD1, STAT5 phosphorylation in the mammary epithelium during alveologenesis was abolished. Conversely, elevated expression of Cuzd1 in mammary epithelial cells stimulated prolactin-induced phosphorylation and nuclear translocation of STAT5. Chromatin immunoprecipitation confirmed co-occupancy of phosphorylated STAT5 and CUZD1 in the regulatory regions of epiregulin, a potential regulator of epithelial proliferation, and whey acidic protein, a marker of epithelial differentiation. Collectively, these findings suggest that CUZD1 plays a critical role in prolactin-induced JAK/STAT5 signaling that controls the expression of key STAT5 target genes involved in mammary epithelial proliferation and differentiation during alveolar development.

Mosaic loss of non-muscle myosin IIA and IIB is sufficient to induce mammary epithelial proliferation.

The mammary epithelium elaborates through hormonally regulated changes in proliferation, migration and differentiation. Non-muscle myosin II (NMII) functions at the interface between contractility, adhesion and signal transduction. It is therefore a plausible regulator of mammary morphogenesis. We tested the genetic requirement for NMIIA and NMIIB in mammary morphogenesis through deletion of the three NMII heavy chain-encoding genes (NMHCIIA, NMHCIIB and NMHCIIC; also known as MYH9, MYH10 and MYH14, respectively) that confer specificity to the complex. Surprisingly, mosaic loss, but not ubiquitous loss, of NMHCIIA and NMHCIIB induced high levels of proliferation in 3D culture. This phenotype was observed even when cells were cultured in basal medium, which does not support tissue level growth of wild-type epithelium. Mosaic loss of NMIIA and NMIIB combined with FGF signaling to induce hyperplasia. Mosaic analysis revealed that the cells that were null for both NMIIA and NMIIB, as well as wild-type cells, proliferated, indicating that the regulation of proliferation is both cell autonomous and non-autonomous within epithelial tissues. This phenotype appears to be mediated by cell-cell contact, as co-culture did not induce proliferation. Mosaic loss of NMIIA and NMIIB also induced excess proliferation in vivo Our data therefore reveal a role for NMIIA and NMIIB as negative regulators of proliferation in the mammary epithelium.

Aberrant regulation of RANKL/OPG in women at high risk of developing breast cancer.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their lifetime in North America and Europe. Receptor Activator of NF-kB Ligand (RANKL), its receptor RANK and the natural antagonist osteoprotegerin (OPG) are essential regulators of bone resorption. We have initially shown that RANKL/RANK are essential for hormone-driven mammary epithelial proliferation in pregnancy and RANKL/RANK have been implicated in mammary stem cell biology. Using genetic mouse-models, we and others identified the RANKL/RANK system as a key regulator of sex hormone, BRCA1-mutation, and oncogene-driven breast cancer and we proposed that RANKL/RANK might be involved in the initiation of breast tumors. We now report that in postmenopausal women without known genetic predisposition, high RANKL and progesterone serum levels stratify a subpopulation of women at high risk of developing breast cancer 12-24 months before diagnosis (5.33-fold risk, 95%CI 1.5-25.4; P=0.02). In women with established breast cancer, we demonstrate that RANKL/OPG ratios change dependent on the presence of circulating tumor cells (CTCs). Finally, we show in a prospective human breast cancer cohort that alterations in RANKL/OPG ratios are significantly associated with breast cancer manifestation. These data indicate that the RANKL/RANK/OPG system is deregulated in post-menopausal women at high risk for breast cancer and in women with circulating tumor cells. Thus, serum levels of RANKL/OPG are potentially indicative of predisposition and progression of breast cancer in humans. Advancement of our findings towards clinical application awaits prior validation in independent patient cohorts.

Abstract P3-11-05: RANK ligand is a target for breast cancer prevention in BRCA1 mutation carriers

Abstract Background: BRCA1 mutation carriers often undergo prophylactic mastectomy to minimize their risk of breast cancer. The value of targeting ovarian hormones to prevent breast tumorigenesis remains contentious and the identification of an effective and acceptable chemoprevention strategy remains a 'holy grail' for the field. Recently, luminal progenitor cells have been identified as the likely cell-of-origin for BRCA1-associated breast tumors1. In addition, deregulated progesterone signaling has been implicated as a potential mechanism underlying tumor development in Brca1-deficient mammary glands2, although its role in luminal progenitor activation in BRCA1 mutation carriers is unknown. RANKL (Receptor Activator of Nuclear Factor-kappa B Ligand) has been identified as a key paracrine effector of progesterone-induced mammary epithelial proliferation in both mouse and human tissue3-5. Notably, RANKL and its receptor RANK play a critical role in the development of breast cancer, with inhibition of RANKL resulting in attenuation of tumorigenesis in mouse models of hormone-driven mammary carcinogenesis6,7. Methods: We explored a role for the RANK/RANKL pathway during the preneoplastic phase in freshly isolated, histologically normal specimens from BRCA1 mutation carriers using a combination of strategies. RANK and RANKL expression in breast cancer was also evaluated in formalin fixed paraffin embedded (FFPE) archival sections by IHC. All samples were obtained with relevant IRB approval. A role for RANKL inhibition in attenuating tumor onset was studied using models that recapitulate human basal-like cancer. Results: A RANK+ subset of luminal progenitor cells was identified in histologically normal breast tissue from BRCA1-mutation carriers. The RANK+ luminal progenitors exhibited higher proliferative activity compared to RANK- progenitors. RNA profiling revealed a distinctive molecular signature, consistent with the RANK+ subset being a possible target for neoplastic transformation. In established BRCA1-associated breast tumors, a four-fold higher incidence of RANK expression was observed, compared to tumors from non-carriers. In ongoing work, histologically normal pre-neoplastic BRCA1mut/+ tissue is being studied using ex vivo breast organoid assays to determine whether RANKL inhibition can attenuate breast epithelial proliferation. Conclusions: Our data raise the possibility that RANK signaling is implicated in the initiation of tumorigenesis in BRCA1 mutation carriers (and possibly other high risk women) and that RANKL is a promising chemoprevention target. The findings are of sufficient interest to have led to a clinical trial, BRCA-D (Registered as ACTRN12614000694617). A finalized abstract will be submitted in early September, during the Late-Breaking Abstract submission period.

Osteoprotegerin (OPG), The Endogenous Inhibitor of Receptor Activator of NF-κB Ligand (RANKL), is Dysregulated in BRCA Mutation Carriers

Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling.We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n=88) and serum RANKL and OPG in postmenopausal women (n=150).BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p=0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p=0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho=−0.216; p=0.002). Serum OPG levels were inversely correlated (rho=−0.545, p<0.001) with mammary epithelial proliferation measured by Ki67 expression and increased (p=0.01) in postmenopause.The P–RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention.

Abstract 2809: Chemopreventive studies of multiple agents in the methylnitrosourea-induced ER+ mammary cancer model in animals on standard and Western diets

Abstract Multiple positive and negative preventive agents [tamoxifen, vorozole (aromatase inhibitor), targretin (RXR agonist), gefitinib (EGFR inhibitor), metformin, and Lipitor] were examined for efficacy in rats given either standard Teklad (4% fat) diet or a modified Western diet [high fat (42%)]. Female Sprague Dawley rats were placed on the high fat or standard diet at 43 days of age, and administered methylnitrosourea (MNU) at 50 days of age. Rats on high fat diet vs standard diet gained approximately 40% more weight, exhibited a 50% increase in cancer multiplicity, and a decrease in cancer latency. The strong preventive agents (tamoxifen, vorozole, targretin, and gefitinib) were highly effective in animals on standard or high fat diets (reducing cancer multiplicity &amp;gt;75% in rats on either diet). It was unclear whether the altered physiology of animals on high fat diet or altered PK of agents in animals with higher fat content might alter activity of these agents. We had previously found that both metformin and Lipitor were ineffective in rats on a standard fat diet, but thought that they might be effective in rats on a high fat diet. Interestingly, neither compound decreased tumorigenesis; but, at least marginally, increased tumor incidence and final tumor weights in rats on both diets. The ability of the various agents to alter proliferation of normal mammary epithelium in MNU-treated rats exposed for 2 weeks to the various agents was determined. It was found that inhibition of proliferation in mammary epithelium corresponded well to the efficacy of these agents in long-term chemopreventive assays. These results demonstrated that: (1) a high fat diet can be employed in this rat model to increase tumor multiplicity; (2) tamoxifen, vorozole, targretin and gefitinib were highly effective in rats on standard or high fat diet; (3) metformin and Lipitor were ineffective with either diet; and (4) there is a correlation between inhibition of proliferation in normal mammary epithelium and long-term preventive efficacy of agents; but this may reflect the homogeneity of the rat mammary cancers. Supported by NCI contract HHSN261201200021I. Citation Format: Clinton J. Grubbs, Mark S. Miller, Vernon E. Steele, Fariba Moeinpour, Harold Seifried, Brandy M. Heckman-Stoddard, Ronald A. Lubet. Chemopreventive studies of multiple agents in the methylnitrosourea-induced ER+ mammary cancer model in animals on standard and Western diets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2809. doi:10.1158/1538-7445.AM2015-2809

Abstract 3774: 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. Four isoforms of CDC25B are known to be expressed and some differences in their role in dividing cells versus continuation of the cell cycle after DNA damage have been shown. As an oncogene, CDC25B is upregulated by c-Myc and activated by Aurora-A. In humans, CDC25B is overexpressed in multiple tumor types and increased levels correlate with worsening tumor grade. In a transgenic mouse model, overexpression of CDC25B in the mammary glands led to accelerated mammary epithelial proliferation resulting in hyperplasia and the formation of tumors when exposed to the carcinogen DMBA. 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. Although CDC25B has been shown to cause DNA damage and activates p53, our data show that colony formation is decreased independent of p53 status. Preliminary data reveal a role for Ras inhibition as a cause of this anti-proliferative effect. Co-transfection of CDC25B and activated Ras results in decreased expression of the ectopic Ras protein in several cell lines tested. Furthermore overexpression of CDC25B abrogates the G2 arrest and decreases cell viability after pulse treatment with doxorubicin. Taken together, these data suggest a novel mechanism by which CDC25B may inhibit proliferation by decreasing Ras expression. Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2015-3774

The Notch pathway inhibits TGFβ signaling in breast cancer through HEYL-mediated crosstalk.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer.