Thirteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28-30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.

Intraductal Injections into the Mouse Mammary Gland.

The mammary intraductal xenografting technique has been established to inject cells or other substances directly into the mammary ducts of female mice. Using this refined xenografting method provides the possibility of mimicking the normal microenvironment of preinvasive breast lesions including, ductal carcinoma in situ (DCIS), to study of the progression of DCIS to invasive breast cancer in a more relevant manner than with other mammary xenografting methods. Xenografting into the mammary fat pad delivers cells directly into the stroma and bypasses the occurrence of invasive transition, during which cells invade through the basement membrane. Either breast cancer cell lines or patient-derived breast cancer cells can be injected into the mammary duct using this protocol to model breast cancer progression. This protocol will cover the procedures required to perform this technique.

Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1-deficient mouse mammary tumour cells.

Little is known about the role of Sox11 in the regulation of mammary progenitor cells. Sox11 is expressed by mammary bud epithelial cells during embryonic mammary gland development and is not detected in mammary epithelial cells after birth. As Sox11 is an oncofetal gene, we investigated the effects of reducing Sox11 levels in embryonic mammary progenitor cells and found that Sox11 regulates proliferative state, stem cell activity and lineage marker expression. We also investigated the effect of reducing Sox11 levels in two transplantable Brca1-deficient oestrogen receptor-negative mouse mammary tumour cell lines, to assess whether Sox11 regulates similar functions in tumour progenitor cells. When Sox11 levels were reduced in one Brca1-deficient mammary tumour cell line that expressed both epithelial and mesenchymal markers, similar effects on proliferation, stem cell activity and expression of lineage markers to those seen in the embryonic mammary progenitor cells were observed. Orthotopic grafting of mammary tumour cells with reduced Sox11 levels led to alterations in tumour-initiating capacity, latency, expression of lineage markers and metastatic burden. Our results support a model in which tumours expressing higher levels of Sox11 have more stem and tumour-initiating cells, and are less proliferative, whereas tumours expressing lower levels of Sox11 become more proliferative and capable of morphogenetic/metastatic growth, similar to what occurs during embryonic mammary developmental progression.

SOX11 promotes epithelial/mesenchymal hybrid state and alters tropism of invasive breast cancer cells.

SOX11 is an embryonic mammary epithelial marker that is normally silenced prior to birth. High SOX11 levels in breast tumours are significantly associated with distant metastasis and poor outcome in breast cancer patients. Here, we show that SOX11 confers distinct features to ER-negative DCIS.com breast cancer cells, leading to populations enriched with highly plastic hybrid epithelial/mesenchymal cells, which display invasive features and alterations in metastatic tropism when xenografted into mice. We found that SOX11+DCIS tumour cells metastasize to brain and bone at greater frequency and to lungs at lower frequency compared to cells with lower SOX11 levels. High levels of SOX11 leads to the expression of markers associated with mesenchymal state and embryonic cellular phenotypes. Our results suggest that SOX11 may be a potential biomarker for breast tumours with elevated risk of developing metastases and may require more aggressive therapies.

A mouse SWATH-mass spectrometry reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts.

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from 'bulk tumour' measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Regulatory roles for SOX11 in development, stem cells and cancer.

The transcription factor SOX11 (SRY-related high-mobility-group (HMG) box 11), a member of the SOXC group, is expressed during embryogenesis but largely absent in most adult differentiated tissues. SOX11 regulates progenitor and stem cell behavior, and often acts together with the other two SOXC group members, SOX4 and SOX12, in regulating developmental processes, including neurogenesis and skeletogenesis. Dysregulation of SOX11 has been implicated in a number of diseases including, neurodevelopmental disorders and osteoarthritis, and a wide variety of cancers. Functions of SOX11 during both development and disease could be attributed to its context-dependent post-transcriptional modifications or interaction with other co-factors. We review the molecular and functional roles of SOX11 during development where similar processes appear to be deregulated in cancers.

A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.

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.

European Network of Breast Development and Cancer turned 10 years: a growing family of mammary gland researchers.

The European Network for Breast Development and Cancer (ENBDC), a worldwide network ( http://www.enbdc.org/ ), celebrated its tenth anniversary with a fantastic meeting last March 15-17, 2018 in Weggis with 76 attendees.

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty.

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

SOX11 promotes invasive growth and ductal carcinoma in situ progression.

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.

Neuregulin-3 regulates epithelial progenitor cell positioning and specifies mammary phenotype.

Mutation of Neuregulin-3 (Nrg3) results in defective embryonic mammary gland development. Here, we investigate functions of Nrg3 signaling in embryonic mammary morphogenesis. Nrg3 regulates the distribution of epithelial progenitor cells within the presumptive mammary-forming region during early mammary morphogenesis. Basal and suprabasal epithelial cells are significantly smaller within the hypoplastic mammary primordium (MP) that forms in Nrg3 mutants, indicative of failure to acquire mammary epithelial cell (MEC) morphological phenotype. Activation of Erbb4 JM-a CYT-1, an Erbb4 isoform expressed in the developing MP, leads to MEC spreading and migration. Nrg3 promotes the accumulation of epithelial progenitor cells at the MP site in embryo explant cultures. Our results implicate Nrg3 signaling in mediating key events of mammary mesenchyme specification, including mesenchymal condensation, mitosis, and induction of mammary marker expression. Taken together, our results show Nrg3 has a major role in conferring specification of the mammary phenotype to both epithelial and mesenchymal progenitor cells.

Stromal regulation of embryonic and postnatal mammary epithelial development and differentiation.

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.

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.

A whole-mount immunofluorescence protocol for three-dimensional imaging of the embryonic mammary primordium.

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.

Neuregulin 3 and erbb signalling networks in embryonic mammary gland development.

We review the role of Neuregulin 3 (Nrg3) and Erbb receptor signalling in embryonic mammary gland development. Neuregulins are growth factors that bind and activate its cognate Erbb receptor tyrosine kinases, which form a signalling network with established roles in breast development and breast cancer. Studies have shown that Nrg3 expression profoundly impacts early stages of embryonic mammary development. Network analysis shows how Nrg/Erbb signals could integrate with other major regulators of embryonic mammary development to elicit the morphogenetic processes and cell fate decisions that occur as the mammary lineage is established.

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

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.

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

INTRODUCTION: The 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. METHODS: Here, 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. RESULTS: Several 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. CONCLUSIONS: These 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.

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

The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERalpha) 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 gamma-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 ERalpha 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 ERalpha and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERalpha coactivator and 4ICD cooperates with ERalpha 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 role of NRG3 in mammary development.

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.