Cathrin Brisken

The epithelial-mesenchymal transition generates cells with properties of stem cells

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Identification of molecular apocrine breast tumours by microarray analysis

Previous microarray studies on breast cancer identified multiple tumour classes, of which the most prominent, named luminal and basal, differ in expression of the oestrogen receptor α gene (ER). We report here the identification of a group of breast tumours with increased androgen signalling and a ‘molecular apocrine’ gene expression profile. Tumour samples from 49 patients with large operable or locally advanced breast cancers were tested on Affymetrix U133A gene expression microarrays. Principal components analysis and hierarchical clustering split the tumours into three groups: basal, luminal and a group we call molecular apocrine. All of the molecular apocrine tumours have strong apocrine features on histological examination (P=0.0002). The molecular apocrine group is androgen receptor (AR) positive and contains all of the ER-negative tumours outside the basal group. Kolmogorov–Smirnov testing indicates that oestrogen signalling is most active in the luminal group, and androgen signalling is most active in the molecular apocrine group. ERBB2 amplification is commoner in the molecular apocrine than the other groups. Genes that best split the three groups were identified by Wilcoxon test. Correlation of the average expression profile of these genes in our data with the expression profile of individual tumours in four published breast cancer studies suggest that molecular apocrine tumours represent 8–14% of tumours in these studies. Our data show that it is possible with microarray data to divide mammary tumour cells into three groups based on steroid receptor activity: luminal (ER+ AR+), basal (ER− AR−) and molecular apocrine (ER− AR+).

The Tumor Suppressor p53 Regulates Polarity of Self-Renewing Divisions in Mammary Stem Cells

Stem-like cells may be integral to the development and maintenance of human cancers. Direct proof is still lacking, mainly because of our poor understanding of the biological differences between normal and cancer stem cells (SCs). Using the ErbB2 transgenic model of breast cancer, we found that self-renewing divisions of cancer SCs are more frequent than their normal counterparts, unlimited and symmetric, thus contributing to increasing numbers of SCs in tumoral tissues. SCs with targeted mutation of the tumor suppressor p53 possess the same self-renewal properties as cancer SCs, and their number increases progressively in the p53 null premalignant mammary gland. Pharmacological reactivation of p53 correlates with restoration of asymmetric divisions in cancer SCs and tumor growth reduction, without significant effects on additional cancer cells. These data demonstrate that p53 regulates polarity of cell division in mammary SCs and suggest that loss of p53 favors symmetric divisions of cancer SCs, contributing to tumor growth.

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/β-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.

Hormone Action in the Mammary Gland

A womans breast cancer risk is affected by her reproductive history. The hormonal milieu also influences the course of the disease. The female reproductive hormones, estrogens, progesterone, and prolactin, have a major impact on breast cancer and control postnatal mammary gland development. Analysis of hormone receptor mutant mouse strains combined with tissue recombination techniques and proteomics revealed that sequential activation of hormone signaling in the mammary epithelium is required for progression of morphogenesis. Hormones impinge on a subset of luminal mammary epithelial cells (MECs) that express hormone receptors and act as sensor cells translating and amplifying systemic signals into local stimuli. Proliferation is induced by paracrine mechanisms mediated by distinct factors at different stages. Tissue and stage specificity of hormonal signaling is achieved at the molecular level by different chromatin contexts and differential recruitment of coactivators and corepressors.

Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development.

Most mammary gland development occurs after birth under the control of systemic hormones. Estrogens induce mammary epithelial cell proliferation during puberty via epithelial estrogen receptor α (ERα) by a paracrine mechanism. Epidermal growth factor receptor (EGFR) signaling has long been implicated downstream of ERα signaling, and several EGFR ligands have been described as estrogen-target genes in tumor cell lines. Here, we show that amphiregulin is the unique EGF family member to be transcriptionally induced by estrogen in the mammary glands of puberal mice at a time of exponential expansion of the ductal system. In fact, we find that estrogens induce amphiregulin through the ERα and require amphiregulin to induce proliferation of the mammary epithelium. Like ERα, amphiregulin is required in the epithelium of puberal mice for epithelial proliferation, terminal end buds formation, and ductal elongation. Subsequent stages, such as side-branching and alveologenesis, are not affected. When amphiregulin−/− mammary epithelial cells are in close vicinity to wild-type cells, they proliferate and contribute to all cell compartments of the ductal outgrowth. Thus, amphiregulin is an important paracrine mediator of estrogen function specifically required for puberty-induced ductal elongation, but not for any earlier or later developmental stages.

Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland

The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor α (ERα) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(−) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-κB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR−/− phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.

IGF-2 Is a Mediator of Prolactin-Induced Morphogenesis in the Breast

The mechanisms by which prolactin controls proliferation of mammary epithelial cells (MECs) and morphogenesis of the breast epithelium are poorly understood. We show that cyclin D1(-/-) MECs fail to proliferate in response to prolactin and identify IGF-2 as a downstream target of prolactin signaling that lies upstream of cyclin D1 transcription. Ectopic IGF-2 expression restores alveologenesis in prolactin receptor(-/-) epithelium. Alveologenesis is retarded in IGF-2-deficient MECs. IGF-2 and prolactin receptor mRNAs colocalize in the mammary epithelium. Prolactin induces IGF-2 mRNA and IGF-2 induces cyclin D1 protein in primary MECs. Thus, IGF-2 is a mediator of prolactin-induced alveologenesis; prolactin, IGF-2, and cyclin D1, all of which are overexpressed in breast cancers, are components of a developmental pathway in the mammary gland.

Progesterone signalling in breast cancer: a neglected hormone coming into the limelight

Understanding the biology of the breast and how ovarian hormones impinge on it is key to rational new approaches in breast cancer prevention and therapy. Because of the success of selective oestrogen receptor modulators (SERMs), such as tamoxifen, and aromatase inhibitors in breast cancer treatment, oestrogens have long received the most attention. Early progesterone receptor (PR) antagonists, however, were dismissed because of severe side effects, but awareness is now increasing that progesterone is an important hormone in breast cancer. Oestrogen receptor-α (ERα) signalling and PR signalling have distinct roles in normal mammary gland biology in mice; both ERα and PR delegate many of their biological functions to distinct paracrine mediators. If the findings in the mouse model translate to humans, new preventive and therapeutic perspectives might open up.

Progesterone/RANKL Is a Major Regulatory Axis in the Human Breast

Ex vivo model identifies the progesterone/RANKL axis as an important proliferative stimulus in the human breast. 3D Without the Glasses Biomedical research has the lofty goal of adding to our understanding of biology in the hope that we can improve human health. However, there are difficulties—both ethical and logistic—of performing experiments in humans. One way researchers have overcome these hurdles is through the use of cell culture or animal models. Yet, these models sometimes don’t accurately represent human biology, especially in complex tissues. Now, Tanos et al. develop an ex vivo three-dimensional model using fresh breast tissue microstructures to examine the role of the progesterone-mediator RANKL in human breast. They found that although progesterone failed to induce RANKL in cell lines and dissociated breast tissue, in their microstructures, RANKL expression responded to progesterone and was required for progesterone-induced breast tissue proliferation. They validated these findings in samples from human breast epithelium. These studies could have clinical relevance: The RANKL inhibitor denosumab is currently used in the clinic to treat bone disease and could be repurposed to block breast epithelial proliferation in breast cancer. Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR+ breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention.