Matthew J. Naylor

The Ets transcription factor Elf5 specifies mammary alveolar cell fate.

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial‐specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5+/− mammary glands. Mammary gland transplants into Rag−/− mice demonstrated that Elf5+/− mammary alveolar buds failed to develop in an Elf5+/+ mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5+/− glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Prolactin Regulation of Mammary Gland Development

Mammary morphogenesis is orchestrated with other reproductive events by pituitary-driven changes to the systemic hormone environment, initiating the formation of a mammary ductal network during puberty and the addition of secretory alveoli during pregnancy. Prolactin is the major driver of development during pregnancy via regulation of ovarian progesterone production (in many species) and direct effects on mammary epithelial cells (in all species). Together these hormones regulate two aspects of development that are the subject of intense interest: (1) a genomic regulatory network that integrates many additional spatial and temporal cues to control gene expression and (2), the activity of a stem and progenitor cell hierarchy. Amalgamation of these two aspects will increase our understanding of cell proliferation and differentiation within the mammary gland, with clear application to our attempts to control breast cancer. Here we focus on providing an over-view of prolactin action during development of the model murine mammary gland.

Prolactin regulates mammary epithelial cell proliferation via autocrine/paracrine mechanism.

Prolactin (PRL) is essential for a number of developmental events in the mammary gland. Work with PRL and PRL receptor knockout mice has shown that PRL indirectly regulates ductal side branching during puberty and directly controls lobuloalveolar development and lactogenesis during pregnancy. Anterior pituitary or placental PRL is thought to be responsible for these functions via an endocrine mechanism; however, PRL is also produced in a number of extrapituitary sites including the mammary gland. The physiologic relevance of mammary PRL remains unknown. In this study we utilized mammary recombination in Rag1−/− hosts, to determine whether mammary PRL plays a role in the regulation of mammary gland development. Mammary glands formed with the PRL gene deleted from either the epithelium, stroma, or both displayed normal development, on the basis of whole mount and hematoxylin and eosin histology, during puberty and lactation. At the end of pregnancy, a 2.8-fold decrease in bromodeoxyuridine incorporation was observed in the epithelial cells of mammary glands formed using PRL knockout epithelium compared with those formed using wildtype epithelium. No balancing alteration in the rates of apoptosis was detected. Thus, mammary-derived PRL influences mammary epithelial cell proliferation via an autocrine/paracrine mechanism, establishing a physiologic function for mammary PRL during mammopoiesis.

Mouse strain-specific patterns of mammary epithelial ductal side branching are elicited by stromal factors

Variations in mammary ductal side branching patterns are known to occur between different strains of mice and this is related to the rate of spontaneous mammary cancers, which are increased in those strains which show highly side‐branched mammary architecture. The cause of the variation in ductal side branching between mouse strains is unknown, but epithelial, stromal, and endocrine factors have been implicated. To define the mammary elements responsible for controlling strain‐specific ductal side branching patterns, we formed recombined mammary glands from epithelial and stroma elements taken from highly side‐branched 129 and poorly side‐branched C57BL/6J mammary glands and transplanted them to Rag1‐/‐ hosts on the inbred C57BL/6J background. When 129 epithelium was recombined with C57BL/6J stroma the poorly side‐branched C57BL/6J pattern was observed. C57BL/6J epithelium recombined with 129 stroma resulted in development of the highly side‐ branched pattern, as did 129 epithelium recombined with 129 stroma. All transplants used the same C57BL/6J endocrine background, demonstrating that strain differences in the mammary stroma are responsible for the strain‐specific ductal side branching patterns and that strain differences in epithelium or endocrine background play no part. Genes currently known to influence side branching by means of the stroma include activin/inhibin, epidermal growth factor receptor (EGFR), Wnt‐2, Wnt‐5a, and Wnt‐6. Of these, Wnt‐5a mRNA expression was decreased in 129 mammary glands compared with C57BL/6J mammary glands, but in F2 129:C57BL/6J animals Wnt‐5a mRNA expression level did not correlate with the highly variable side branching patterns observed. These experiments exclude variation in the expression level of known candidate genes as the mechanism responsible. Regardless of underlying mechanism, transplantation without regard to the genetic background of the stromal donor, whether inbred or mixed, will compromise experiments with side branching and associated gene expression endpoints.

Molecular dissection of integrin signalling proteins in the control of mammary epithelial development and differentiation.

Cell-matrix adhesion is essential for the development and tissue-specific functions of epithelia. For example, in the mammary gland, β1-integrin is necessary for the normal development of alveoli and for the activation of endocrine signalling pathways that determine cellular differentiation. However, the adhesion complex proteins linking integrins with downstream effectors of hormonal signalling pathways are not known. To understand the mechanisms involved in connecting adhesion with this aspect of cell phenotype, we examined the involvement of two proximal β1-integrin signalling intermediates, integrin-linked kinase (ILK) and focal adhesion kinase (FAK). By employing genetic analysis using the Cre-LoxP system, we provide evidence that ILK, but not FAK, has a key role in lactogenesis in vivo and in the differentiation of cultured luminal epithelial cells. Conditional deletion of ILK both in vivo and in primary cell cultures resulted in defective differentiation, by preventing phosphorylation and nuclear translocation of STAT5, a transcription factor required for lactation. Expression of an activated RAC (RAS-related C3 botulinum substrate) in ILK-null acini restored the lactation defect, indicating that RAC1 provides a mechanistic link between the integrin/ILK adhesion complex and the differentiation pathway. Thus, we have determined that ILK is an essential downstream component of integrin signalling involved in differentiation, and have identified a high degree of specificity within the integrin-based adhesome that links cell-matrix interactions with the tissue-specific function of epithelia.

c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial–mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2+ breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2+ breast cancer.

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer

The transcription factor ELF5 is responsible for gene expression patterning underlying molecular subtypes of breast cancer and may mediate acquired resistance to anti-estrogen therapy.

Breast cancer stem cells

Cancer metastasis, resistance to therapies and disease recurrence are significant hurdles to successful treatment of breast cancer. Identifying mechanisms by which cancer spreads, survives treatment regimes and regenerates more aggressive tumors are critical to improving patient survival. Substantial evidence gathered over the last 10 years suggests that breast cancer progression and recurrence is supported by cancer stem cells (CSCs). Understanding how CSCs form and how they contribute to the pathology of breast cancer will greatly aid the pursuit of novel therapies targeted at eliminating these cells. This review will summarize what is currently known about the origins of breast CSCs, their role in disease progression and ways in which they may be targeted therapeutically.

Runx2 Is a Novel Regulator of Mammary Epithelial Cell Fate in Development and Breast Cancer

Regulators of differentiated cell fate can offer targets for managing cancer development and progression. Here, we identify Runx2 as a new regulator of epithelial cell fate in mammary gland development and breast cancer. Runx2 is expressed in the epithelium of pregnant mice in a strict temporally and hormonally regulated manner. During pregnancy, Runx2 genetic deletion impaired alveolar differentiation in a manner that disrupted alveolar progenitor cell populations. Conversely, exogenous transgenic expression of Runx2 in mammary epithelial cells blocked milk production, suggesting that the decrease in endogenous Runx2 observed late in pregnancy is necessary for full differentiation. In addition, overexpression of Runx2 drove epithelial-to-mesenchymal transition-like changes in normal mammary epithelial cells, whereas Runx2 deletion in basal breast cancer cells inhibited cellular phenotypes associated with tumorigenesis. Notably, loss of Runx2 expression increased tumor latency and enhanced overall survival in a mouse model of breast cancer, with Runx2-deficient tumors exhibiting reduced cell proliferation. Together, our results establish a previously unreported function for Runx2 in breast cancer that may offer a novel generalized route for therapeutic interventions. Cancer Res; 74(18); 5277-86. ©2014 AACR.