Domenici, G., Aurrekoetxea-Rodríguez, I., Simões, B.M., Rábano, M., Lee, S.Y., Millán, J.S., Comaills, V., Oliemuller, E., López-Ruiz, J.A., Zabalza, I., et al.
(2019). A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells. Oncogene,
Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer..
Krasny, L., Bland, P., Kogata, N., Wai, P., Howard, B.A., Natrajan, R.C. & Huang, P.H.
(2018). SWATH mass spectrometry as a tool for quantitative profiling of the matrisome. J proteomics,
Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15-20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SIGNIFICANCE: The matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment..
Kogata, N., Bland, P., Tsang, M., Oliemuller, E., Lowe, A. & Howard, B.A.
(2018). Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells. Commun biol,
Embryonic mammary cells are a unique population comprised of undifferentiated, highly plastic progenitor cells that create normal mammary tissues. The mammary gland continues to develop after birth from descendants of embryonic mammary cells. Here, we establish cell lines from mouse mammary organs, immediately after they formed during prenatal development, to facilitate studies of primitive mammary cells, which are difficult to isolate in sufficient quantities for use in functional experiments. We show that some lines can be induced to secrete milk, a distinguishing feature of mammary epithelial cells. Targeted deletion of Sox9, from one clone, decreases the ability to respond to lactogenic stimuli, consistent with a previously identified role for Sox9 in regulating luminal progenitor function. Sox9 ablation also leads to alterations in 3D morphology and downregulation of Zeb1, a key epithelial-mesenchymal transition regulator. Prenatal mammary cell lines are an invaluable resource to study regulation of mammary progenitor cell biology and development..
Oliemuller, E., Kogata, N., Bland, P., Kriplani, D., Daley, F., Haider, S., Shah, V., Sawyer, E.J. & Howard, B.A.
(2017). SOX11 promotes invasive growth and ductal carcinoma in situ progression. J pathol,
Here, we show that SOX11, an embryonic mammary marker that is normally silent in postnatal breast cells, is expressed in many oestrogen receptor-negative preinvasive ductal carcinoma in situ (DCIS) lesions. Mature mammary epithelial cells engineered to express SOX11 showed alterations in progenitor cell populations, including an expanded basal-like population with increased aldehyde dehydrogenase (ALDH) activity, and increased mammosphere-forming capacity. DCIS.com cells engineered to express SOX11 showed increased ALDH activity, which is a feature of cancer stem cells. The CD44+/CD24-/ALDH+ cell population was increased in DCIS.com cells that expressed SOX11. Upregulating SOX11 expression in DCIS.com cells led to increased invasive growth both in vitro and when they were injected intraductally in a mouse model of DCIS that recapitulates human disease. Invasive lesions formed sooner and tumour growth was augmented in vivo, suggesting that SOX11 contributes to the progression of DCIS to invasive breast cancer. We identified potential downstream effectors of SOX11 during both microinvasive and invasive tumour growth stages, including several with established links to regulation of progenitor cell function and prenatal developmental growth. Our findings suggest that SOX11 is a potential biomarker for DCIS lesions containing cells harbouring distinct biological features that are likely to progress to invasive breast cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
Krasny, L., Paul, A., Wai, P., Howard, B.A., Natrajan, R.C. & Huang, P.H.
(2016). Comparative proteomic assessment of matrisome enrichment methodologies. Biochem j,
The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome..
Rakić, S., Kanatani, S., Hunt, D., Faux, C., Cariboni, A., Chiara, F., Khan, S., Wansbury, O., Howard, B., Nakajima, K., et al.
(2015). Cdk5 phosphorylation of ErbB4 is required for tangential migration of cortical interneurons. Cereb cortex,
Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism. Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation. Here, using a mouse model and a host of histological and molecular biological techniques, we report that the signaling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the tangential migration of interneurons toward and within the cerebral cortex by modulating the critical neurodevelopmental signaling pathway, ErbB4/phosphatidylinositol 3-kinase, that has been repeatedly linked to schizophrenia. This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals. .
Kogata, N. & Howard, B.A.
(2015). A protocol for studying embryonic mammary progenitor cells during mouse mammary primordial development in explant culture. Methods mol biol,
Embryonic explant culture is a powerful technique to observe tissue morphogenesis ex vivo, and is particularly useful for monitoring embryonic mammary gland development. It has been established that mammary cell lineage specification occurs during embryogenesis, although much remains to be elucidated with respect to how this occurs. During mammary specification, mammary progenitor cells are formed. Embryonic mammary development can proceed and be monitored in embryonic explant culture. Studies using explant culture will greatly enhance our understanding of the cellular mechanisms that regulate embryonic mammary primordial development and mammary progenitor cell specification. We present a protocol for culturing explants from mid-gestation mouse embryos so that morphogenetic processes and mammary epithelial progenitor cells can be studied during embryonic mammary development ex vivo. .
Howard, B.A. & Lu, P.
(2014). Stromal regulation of embryonic and postnatal mammary epithelial development and differentiation. Semin cell dev biol,
The stroma, which is composed of supporting cells and connective tissue, comprises a large component of the local microenvironment of many epithelial cell types, and influences several fundamental aspects of cell behaviour through both tissue interactions and niche regulation. The significance of the stroma in development and disease has been increasingly recognised. Whereas normal stroma is essential for various developmental processes during vertebrate organogenesis, it can be deregulated and become abnormal, which in turn can initiate or promote a disease process, including cancer. The mouse mammary gland has emerged in recent years as an excellent model system for understanding stromal function in both developmental and cancer biology. Here, we take a systematic approach and focus on the dynamic interactions that the stroma engages with the epithelium during mammary specification, cell differentiation, and branching morphogenesis of both the embryonic and postnatal development of the mammary gland. Similar stromal-epithelial interactions underlie the aetiology of breast cancer, making targeting the cancer stroma an increasingly important and promising therapeutic strategy to pursue for breast cancer treatment. .
Kogata, N., Oliemuller, E., Wansbury, O. & Howard, B.A.
(2014). Neuregulin-3 Regulates Epithelial Progenitor Cell Positioning and Specifies Mammary Phenotype. Stem cells and development,
Propper, A.Y., Howard, B.A. & Veltmaat, J.M.
(2013). Prenatal Morphogenesis of Mammary Glands in Mouse and Rabbit. Journal of mammary gland biology and neoplasia,
Howard, B.A. & Veltmaat, J.M.
(2013). Embryonic mammary gland development; a domain of fundamental research with high relevance for breast cancer research Preface. J mammary gland biol neoplasia,
Kogata, N. & Howard, B.A.
(2013). A whole-mount immunofluorescence protocol for three-dimensional imaging of the embryonic mammary primordium. J mammary gland biol neoplasia,
Whole-mount immunofluorescent staining facilitates the profiling of protein expression patterns within diverse and complex tissues. Thanks to the application of antibodies on whole mounted instead of sectioned specimens, this technique has many advantages with respect to the preservation of biological and pathological features of specimens when compared to conventional immunohistological methods. Here, we describe a protocol and optimal conditions of whole-mount immunofluorescence for studying the formation of mammary primordia. We also show an example three-dimensional reconstruction of a mammary primordium based on z-stacked images of a whole-mount stained specimen using confocal microscopy and image analysis software. .
Propper, A, , Howard, B.A. & Veltmaat, J.
(2013). Prenatal morphogenesis of mammary glands. Journal of mammary gland biology and neoplasia,
Zvelebil, M., Oliemuller, E., Gao, Q., Wansbury, O., Mackay, A., Kendrick, H., Smalley, M.J., Reis-Filho, J.S. & Howard, B.A.
(2013). Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers. Breast cancer res,
INTRODUCTION: Cancer 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. METHODS: We 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. RESULTS: Small 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. CONCLUSIONS: Specific 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..
Kogata, N., Zvelebil, M. & Howard, B.A.
(2013). Neuregulin3 and Erbb signalling networks in embryonic mammary gland development. Journal of mammary gland biology and neoplasia,
(2012). In the beginning: the establishment of the mammary lineage during embryogenesis. Semin cell dev biol,
The mammary primordium is comprised of an aggregate of immature, undifferentiated mammary epithelial cells and its associated mammary mesenchyme, a specialised tissue which harbours mammary-inductive capacity. The mammary primordium forms during embryogenesis as a result of inductive interactions between its two component tissues, the mammary mesenchyme and epithelium. These two tissues constitute a signalling centre that directs the formation of the mammary gland through a series of reciprocal mesenchymal-epithelial interactions. A rudimentary mammary ductal tree and stroma is formed prior to birth as a result of these interactions. The subsequent mammary outgrowths that arise upon hormonal stimulation during puberty originate from this rudimentary tissue. The initial appearance of the embryonic mammary primordium during embryogenesis represents the earliest morphological evidence of commitment to the mammary lineage. Classic tissue recombination studies of mouse mammary primordial cells have demonstrated that the epithelial cells are already functionally determined as mammary at the embryonic mammary bud stage. Recent studies have determined the molecular identity of the embryonic mammary cells by transcriptomic profiling and these have provided new insights into signalling components that mediate early embryonic mammary inductive signalling and lineage commitment. This review highlights what is currently known about the morphogenesis, function, and behaviour of embryonic mammary cells and examine current knowledge of the genetics underlying mammary cell fate and establishment of the mammary lineage during embryogenesis..
Wansbury, O., Mackay, A., Kogata, N., Mitsopoulos, C., Kendrick, H., Davidson, K., Ruhrberg, C., Reis-Filho, J.S., Smalley, M.J., Zvelebil, M., et al.
(2011). Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment. Breast cancer research,
Rokicki, J., Das, P.M., Giltnane, J.M., Wansbury, O., Rimm, D.L., Howard, B.A. & Jones, F.E.
(2010). The ER alpha coactivator, HER4/4ICD, regulates progesterone receptor expression in normal and malignant breast epithelium. Mol cancer,
The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ER alpha) 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.-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 ERa 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 ERa and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERa coactivator and 4ICD cooperates with ERa 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..
Panchal, H., Wansbury, O. & Howard, B.A.
(2010). Embryonic mammary anlagen analysis using immunolabelling of whole mounts. Methods in molecular biology,
Schwarz, Q., Vieira, J.M., Howard, B., Eickholt, B.J. & Ruhrberg, C.
(2008). Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells. Development,
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..
Wansbury, O., Panchal, H., James, M., Parry, S., Ashworth, A. & Howard, B.
(2008). Dynamic expression of erbb pathway members during early mammary gland morphogenesis. J invest dermatol,
Similar to other epithelial appendages, mammary anlagen progress from stratified epithelium through placode and bud stages. Embryonic mammary morphogenesis is elicited by a combination of local cell migration, adhesion changes and proliferation, and these same developmental processes impact breast cancer etiology. The Erbb signaling network plays important roles in postnatal mammary gland morphogenesis and carcinogenesis. Neuregulin3 (Nrg3), an Erbb family ligand, has recently been shown to be involved in the specification of mammary glands in mice. To further examine the possible involvement of other Erbb family members and their ligands in early mammary morphogenesis, we have characterized their expression patterns during this process. We used whole mount in situ hybridization to analyze the expression patterns of these genes at stages prior to and during mammary placode formation. Immunohistochemistry was used to examine expression patterns at later bud stages. The Neuregulin ligands, Nrg1, Nrg2, Nrg3, Nrg4 and the receptors, Erbb1, Erbb2, Erbb3, Erbb4, were expressed either at stages prior to morphological appearance of the mammary placode or from the time that the placode is first morphologically distinct through to later bud stages. The expression patterns presented here suggest that multiple members of this signaling network are potential mediators of early mammary morphogenesis..
(2008). The role of NRG3 in mammary development. J mammary gland biol neoplasia,
The Neuregulin gene family encodes EGF-containing ligands which mediate their effects by binding to the ERBB receptor tyrosine kinases, a signalling network with important roles in both mammary gland development and breast cancer. Neuregulin3 (NRG3), a ligand for ERBB4, promotes early mammary morphogenesis and acts during specification of the mammary placode, an aggregate of epithelial cells that forms during mid-embryogenesis. Recent studies have shown that NRG3 can alter the cell fate of other epidermal progenitor populations when NRG3 is mis-expressed throughout the basal layer of the developing epidermis with the K14 promoter. Here evidence for a key function for NRG3 in promoting early mammary morphogenesis and the implication for the role of NRG3 in breast cancer and establishment of the mammary lineage are discussed..
Strizzi, L., Mancino, M., Bianco, C., Raafat, A., Gonzales, M., Booth, B.W., Watanabe, K., Nagaoka, T., Mack, D.L., Howard, B., et al.
(2008). Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland. J cell physiol,
Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-II mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland..
Panchal, H., Wansbury, O., Parry, S., Ashworth, A. & Howard, B.
(2007). Neuregulin3 alters cell fate in the epidermis and mammary gland. Bmc dev biol,
BACKGROUND: The 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. RESULTS: Transgenic 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, alpha6-integrin and beta1-integrin. CONCLUSION: These 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..
Howard, B. & Ashworth, A.
(2006). Signalling pathways implicated in early mammary gland morphogenesis and breast cancer. Plos genet,
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..
Howard, B., Panchal, H., McCarthy, A. & Ashworth, A.
(2005). Identification of the scaramanga gene implicates Neuregulin3 in mammary gland specification. Genes dev,
The mouse scaramanga (ska) mutation impairs mammary gland development such that both abrogation and stimulation of gland formation occurs. We used positional cloning to narrow the interval containing scaramanga (ska) to a 75.6-kb interval containing the distal part of the Neuregulin3 (Nrg3) gene. Within this region the only sequence difference between ska and wild-type mice is in a microsatellite repeat within intron 7. This alteration correlates with variations in Nrg3 expression profiles both at the whole embryo level and locally in the presumptive mammary region in ska mice. Localized expression of Nrg3 and its receptor, Erbb4, in the presumptive mammary region around the future bud site prior to morphological appearance of buds and the expression of bud epithelial markers further support an inductive role. Finally, Neuregulin3 (Nrg3)-soaked beads can induce expression of the early bud marker Lef1 in mouse embryo explant cultures, and epithelial bud formation can be observed histologically, suggesting that initiation of mammary bud development occurs. Taken together, these results indicate that a Neuregulin signaling pathway is involved in specification of mammary gland morphogenesis and support the long-held view that mesenchymal signal(s) are responsible for mammary gland inductive/initiating events..
Howard, B.A. & Gusterson, B.A.
(2000). Mammary gland patterning in the AXB/BXA recombinant inbred strains of mouse. Mech develop,
Howard, B.A. & Gusterson, B.A.
(2000). The characterization of a mouse mutant that displays abnormal mammary gland development. Mamm genome,
Mallon, E., Osin, P., Nasiri, N., Blain, I., Howard, B. & Gusterson, B.
(2000). The basic pathology of human breast cancer. J mammary gland biol,
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..
Howard, B.A. & Gusterson, B.A.
(2000). Human breast development. J mammary gland biol,
This review presents an atlas of the histology of the normal physiological states of the human breast including prenatal, prepubertal, and pubertal development, adult resting gland, pregnancy, lactation, and postinvolution. The aim is to produce a pictorial overview of the main stages in development and the common findings in the adult that are considered to be within the range of normality. Unlike inbred strains of animals, in humans it is clear that the chronology of ductal and lobular development is not predictable, either in the fetus, the infant, the peripubertal breast, or the adult. This is probably due to the individual variation in hormone levels both in utero and after birth. For many of the developmental time points there are very little data available. In this review we indicate the current state of knowledge of human breast development and some of the main similarities and differences with the rodent, the main animal model. The major phases of growth and development are described and accompanied by photographs that are representative of each stage. Stress is placed on terminology as there is confusion in the literature. This article is written as an accessory to the companion review on breast cancer..
Koledova, Z., Howard, B., Englund, J., Bach, K., Bentires-Alj, M. & Gonzalez-Suarez, E.
ENBDC turned 10 years, a growing family of mammary gland researchers. Breast cancer research,
Bland, P. & Howard, B.
Mammary lineage restriction in development. Nature cell biology,
Howard, B., elo, T., lindfors, P., lan, Q., voutilainen, M., trela, E., ohlsson, C., ornitz, D., huh, S., poutanen, M., et al.
Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty. Scientific reports,
Zvelebil, M.J., Wansbury, O., Gao, Q., Mackay, A., Kendrick, H., Liemuller, E., Smalley, M.J., Reis-Filho, J.S. & Howard, B.A.
Embryonic Mammary Signature Subsets are Activated in Brca1-/- and Basal-like Breast Cancers. Breast cancer research,
Rakic, S., Kanatani, S., Hunt, D., Faux, C., Cariboni, C., Chiara, F., Khan, S., Wansbury, O., Howard, B.A., Nakajima, K., et al.
Cdk5 phosphorylation of ErbB4 is crucial for cortical interneuron migration. Under review at development,
Interneuron dysfunction is often associated with neurological and psychiatric disorders, such
as epilepsy, schizophrenia and autism. Some of these disorders arise during brain formation, at the
time of interneuron specification, migration and synapse formation. Here, we showed that the
signalling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the migration of
interneurons towards and within the cerebral cortex by modulating the motogenic
ErbB4/phosphatidylinositol (PI) 3-kinase signalling pathway. We first identified ErbB4 as a novel
p35/Cdk5 kinase substrate. We then demonstrated that Cdk5-dependent ErbB4/PI3-kinase signalling
cascade regulates interneuron leading process dynamics (morphology) and directionality. Finally, we
showed that lack of Cdk5 activity in p35 mutants leads to permanent reduction in the final number of a
subtype of interneurons that may affect formation of neuronal circuits, thus increasing the risk of
neurodevelopmental disorders. Together, these findings identify Cdk5 as a crucial signalling factor in
cortical interneuron development..
Kogata, N., Oliemuller, E., Wansbury, O. & Howard, B.A.
Neuregulin-3 regulates epithelial progenitor cell positioning and specifies mammary phenotype. Stem cells and development,