Beatrice A. Howard

Human Breast Development

This review is intended to give an overview of current knowledge on human breast development. It focuses on the limitations of our understanding on the origins of human breast cancer in the context of this mainly morphological and static assessment of what is known about human breast development. The world literature is very limited and caution is needed in drawing analogies with the mouse. There is an increasing emphasis on research to understand normal stem cells in the breast on the assumption that these are the targets for the initiation of breast cancer. It is thus a priority to understand normal human breast development, but there are major obstacles to such studies mainly due to ethical considerations and to tissue acquisition.

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.

Signalling Pathways Implicated in Early Mammary Gland Morphogenesis and Breast Cancer

Specification of mammary epithelial cell fate occurs during embryogenesis as cells aggregate to form the mammary anlage. Within the embryonic mammary bud, a population of epithelial cells exists that will subsequently proliferate to form a ductal tree filling the stromal compartment, and which can produce milk upon terminal differentiation after birth. Subsequently, these structures can be remodelled and returned to a basal state after weaning before regenerating in future pregnancies. The plasticity of the mammary epithelial cell, and its responsiveness to hormone receptors, facilitates this amazing biological feat, but aberrant signalling may also result in unintended consequences in the form of frequent malignancies. Reflecting this intimate connection, a considerable number of signalling pathways have been implicated in both mammary gland morphogenesis and carcinogenesis.

The basic pathology of human breast cancer

This article illustrates the most common benign and malignant lesions in the breast, and is intended for the biologist working in the area of breast cancer and breast biology, not for the practicing pathologist. The atlas covers benign proliferative lesions, atypical lesions, variants of in situ cancer, the main types of invasive cancers, spindle cell lesions, and examples of vascular and lymphatic spread. Some entities are included to illustrate a point of particular relevance to the biology and histogenesis of the lesions. Some controversial diagnostic areas are considered, along with the relative risk of developing breast cancer associated with some of the proliferative lesions. The content of this atlas should be read in conjunction with the companion article by Howard and Gusterson in this issue. Their article covers the cellular origin of epithelial and stromal tumors and presents a description of some of the common benign proliferative lesions that are considered to be components of the normal spectrum of changes seen at postmortem or in biopsies.

Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment

IntroductionThe mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised.MethodsHere, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made.ResultsSeveral of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species.ConclusionsThese results provide new insights into genetic regulatory mechanisms of mammary development, particularly identification of novel potential regulators of mammary fate and mesenchymal-epithelial cross-talk. Since cancers may represent diseases of mesenchymal-epithelial communications, we anticipate these results will provide foundations for further studies into the fundamental links between developmental, stem cell and breast cancer biology.

Neuregulin3 alters cell fate in the epidermis and mammary gland

BackgroundThe Neuregulin family of ligands and their receptors, the Erbb tyrosine kinases, have important roles in epidermal and mammary gland development as well as during carcinogenesis. Previously, we demonstrated that Neuregulin3 (Nrg3) is a specification signal for mammary placode formation in mice. Nrg3 is a growth factor, which binds and activates Erbb4, a receptor tyrosine kinase that regulates cell proliferation and differentiation. To understand the role of Neuregulin3 in epidermal morphogenesis, we have developed a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) of mouse epidermis and the outer root sheath of developing hair follicles.ResultsTransgenic females formed supernumerary nipples and mammary glands along and adjacent to the mammary line providing strong evidence that Nrg3 has a role in the initiation of mammary placodes along the body axis. In addition, alterations in morphogenesis and differentiation of other epidermal appendages were observed, including the hair follicles. The transgenic epidermis is hyperplastic with excessive sebaceous differentiation and shows striking similarities to mouse models in which c-Myc is activated in the basal layer including decreased expression levels of the adhesion receptors, α6-integrin and β1-integrin.ConclusionThese results indicate that the epidermis is sensitive to Nrg3 signaling, and that this growth factor can regulate cell fate of pluripotent epidermal cell populations including that of the mammary gland. Nrg3 appears to act, in part, by inducing c-Myc, altering the proliferation and adhesion properties of the basal epidermis, and may promote exit from the stem cell compartment. The results we describe provide significant insight into how growth factors, such as Nrg3, regulate epidermal homeostasis by influencing the balance between stem cell renewal, lineage selection and differentiation.

Prenatal Morphogenesis of Mammary Glands in Mouse and Rabbit

Our understanding of prenatal morphogenesis of mammary glands has recently greatly advanced. This review focuses on morphogenesis proper, as well as cellular processes and tissue interactions involved in the progression of the embryonic mammary gland through sequential morphogenic stages in both the mouse and rabbit embryo. We provide a synthesis of both historical and more recent studies of embryonic mammary gland development, as well as arguments to revise old concepts about mechanisms of mammary line and rudiment formation. Finally, we highlight outstanding issues that remain to be addressed.

Embryonic mammary signature subsets are activated in Brca1 -/- and basal-like breast cancers

IntroductionCancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents.MethodsWe defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers.ResultsSmall subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell proliferation and cell survival.ConclusionsSpecific subsets of embryonic mammary genes, rather than the entire embryonic development transcriptomic program, are activated in tumorigenesis. Genes involved in embryonic mammary development are consistently upregulated in some breast cancers and warrant further investigation, potentially in drug-discovery research endeavors.

The ERα coactivator, HER4/4ICD, regulates progesterone receptor expression in normal and malignant breast epithelium

The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERα) coactivator with activities in breast cancer and the developing mammary gland that appear to overlap with progesterone receptor (PgR). In fact, 4ICD has recently emerged as an important regulator and predictor of tamoxifen response, a role previously thought to be fulfilled by PgR. Here we investigated the possibility that the 4ICD coactivator regulates PgR expression thereby providing a mechanistic explanation for their partially overlapping activities in breast cancer. We show that 4ICD is both sufficient and necessary to potentiate estrogen stimulation of gene expression. Suppression of HER4/4ICD expression in the MCF-7 breast tumor cell line completely eliminated estrogen stimulated expression of PgR. In addition, the HER4/4ICD negative MCF-7 variant, TamR, failed to express PgR in response to estrogen. Reintroduction of wild-type HER4 but not the γ-secretase processing mutant HER4V673I into the TamR cell line restored PgR expression indicating that 4ICD is an essential PgR coactivator in breast tumor cells. These results were substantiated in vivo using two different physiologically relevant experimental systems. In the mouse mammary gland estrogen regulates expression of PgR-A whereas expression of PgR-B is estrogen independent. Consistent with a role for 4ICD in estrogen regulated PgR expression in vivo, PgR-A, but not PgR-B, expression was abolished in HER4-null mouse mammary glands during pregnancy. Coexpression of PgR and 4ICD is also commonly observed in ERα positive breast carcinomas. Using quantitative AQUA IHC technology we found that 4ICD potentiated PgR expression in primary breast tumors and the highest levels of PgR expression required coexpression of ERα and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERα coactivator and 4ICD cooperates with ERα to potentiate PgR expression in the normal and malignant breast. We propose that direct coupling of these signaling pathways may have important implications for mammary development, breast carcinogenesis, and patient response to endocrine therapy.

The characterization of a mouse mutant that displays abnormal mammary gland development.

Deficiencies and supernumerary mammary glands in inbred strains of mice have been reported and were postulated to have a genetic basis (Gardner and Strong 1935; Little and McDonald 1965). Here we describe a naturally occurring mouse mutation named scaramanga that displays abnormal embryonic and postnatal mammary gland development found in the A/J strain of mice. We have performed a genetic analysis of this mutation and have determined that a single autosomal recessive locus appears to be responsible for this trait. The question of how body patterns are formed is a central theme of developmental biology. Little is known about the molecular events that specify the site of organ development within a larger, morphologically indistinguishable region. In mice, five pairs of mammary glands are arranged in an ordered array along the ventral surface between the forelimbs and hindlimbs. How this order and spacing are achieved is unknown. Like many other organs, the mammary gland develops as a result of reciprocal epithelial-mesenchymal interactions (Cunha and Hom 1996); the cellular and molecular mechanisms of these cell-cell interactions in the mammary glands at the molecular level are poorly understood. Epithelial-mesenchymal interactions also control postnatal growth and ductal branching morphogenesis of the mammary gland (Cunha and Hom 1996). It is thought that perturbations of epithelial-mesenchymal interactions in adulthood may play a role in carcinogenesis (Cunha 1994). Since breast cancer is a major cause of mortality of women in developed countries, a better basic understanding of mammary gland development would be useful. Abnormalities in human breast development exist that are characterized by additional nipples (supernumerary), the presence of additional breasts (polymastia), the absence of breasts (amastia), the abnormal positioning of breasts, and the presence of more than one nipple per breast (polythelia; Klinkerfuss 1924; Gates 1946; Fraser 1956; Goldenring and Crelin 1961). Supernumerary nipples and polymastia have been reported in many mammals, including mice, sheep, guinea pigs, as well as humans (Bell 1898; Goertzen and Ibsen 1951). Polymastia and supernumerary nipples can be sporadic, but when occurring as an hereditary trait, behave in an autosomal dominant fashion in humans and in guinea pigs (Klinkerfuss 1924; Gates 1946; Goertzen and Ibsen 1951). Mouse mutants and human syndromes have been described that display multiple developmental defects that include abnormal mammary gland development. Ulnar-mammary syndrome and scalp-earnipple syndromes affect a variety of other organs as well as the mammary gland (Finlay and Marks 1978; Edwards et al. 1994; Bamshad et al. 1995). There are reports of an increased incidence of renal and urological abnormalities and renal carcinomas with the presence of supernumerary nipples in humans (Meggyessy and Mehes 1987; Urbani and Betti 1995). A few mouse mutants display aberrant mammary gland development during the early stages of mammary gland formation. Mammary gland development is incomplete or abrogated before morphogenesis in bothLef1and p63-deficient mice (van Genderen et al. 1994; Mills et al. 1999; Yang et al. 1999). Both parathyroid hormone-related protein (PTHrP) knockout mice and parathyroid hormone (PTH)/PTHrP receptor knockout mice fail to undergo the initial round of branching growth that is responsible for transforming the mammary bud into the rudimentary duct system, and no mammary epithelial ducts or nipple structures form (Wysolmerski et al. 1998). Studies of other mouse mutants that display abnormal mammary gland formation in early patterning should lead to further insights into how pattern is initially generated in these specialized structures. Most wild and inbred strains of mice, including the C57BL/6J (or B6) strain, have 10 nipples (Fig. 1A). Three pairs of thoracic (#1, #2, and #3) and two pairs of inguinal mammary glands (#4 and #5) are present in a linear order on the ventral surface of the female mouse. However, we have observed that the A/J inbred strain of mice displays abnormal mammary development. A/J mice display supernumerary mammary glands and nipples, misplaced nipples, and mammary gland deficiencies (Fig. 1B, Table 1). Ninety-five percent of A/J mice display a mammary gland pattern phenotype. We have not observed any other developmental defects in these mice, and it is possible that a novel gene is involved. We have used classical genetic analysis to study this gene, which we have namedska (for scaramanga). Correspondence to: B.A. Howard, e-mail: beatrice@icr.ac.uk Fig. 1. Mammary gland development in the B6 and A/J strains of mice. Mammary glands are numbered from 1 to 5. (A) In adult female B6 mice, five pairs of mammary glands are evenly spaced along the ventral surface. (B) In adult female A/J mice, abnormal mammary development occurs; in this case, an absent left #3 mammary gland is displayed, which is indicated by an arrow. Mammalian Genome 11, 234–237 (2000).