Title: Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer
Abstract: Review11 January 2019Open Access Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer Davide Pellacani Davide Pellacani orcid.org/0000-0002-0522-8229 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Susanna Tan Susanna Tan Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Sylvain Lefort Sylvain Lefort orcid.org/0000-0001-7320-4256 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Connie J Eaves Corresponding Author Connie J Eaves [email protected] orcid.org/0000-0002-3452-1926 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Davide Pellacani Davide Pellacani orcid.org/0000-0002-0522-8229 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Susanna Tan Susanna Tan Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Sylvain Lefort Sylvain Lefort orcid.org/0000-0001-7320-4256 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Connie J Eaves Corresponding Author Connie J Eaves [email protected] orcid.org/0000-0002-3452-1926 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Author Information Davide Pellacani1, Susanna Tan1, Sylvain Lefort1 and Connie J Eaves *,1 1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada *Corresponding author. Tel: +1 604 675 8122; Fax: +1 604 877 0712; E-mail: [email protected] The EMBO Journal (2019)38:e100330https://doi.org/10.15252/embj.2018100330 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra-tumoral as well as inter-tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers. The normal adult human mammary gland The adult human female mammary gland is a continuous branching tree of ducts that extend radially from the nipple and terminate in expanded alveolar structures frequently called lobules (Fig 1A). This structure is encased in a basement membrane and an outer layer of fibroblasts, all of which are embedded in a collagen-rich stroma containing adipocytes, macrophages, lymphocytes, and blood and lymph vessels. The mammary gland, itself, consists of two layers of cells with different features and functions. The outer "basal" layer is made up of cells that are in direct contact with the basement membrane. These cells are also referred to as myoepithelial cells because they possess contractile, smooth muscle-like properties. The inner "luminal" layer of the gland contains cells with quite different, polarized epithelial features and an ability to produce and secrete milk upon hormone induction. Figure 1. Macro- and microscopic structure of the normal human breast(A) Diagram showing the macroscopic structure of the human breast and histological sections of ducts and alveoli (scale bar = 100 μm). (B) Effects of serum hormone levels on the human mammary epithelium during the menstrual cycle. Download figure Download PowerPoint The initial stages of development of the mammary gland that take place in humans before birth are not well documented, and hence, knowledge of these has had to rely on inferences drawn from studies of mice (Veltmaat et al, 2003; Spike et al, 2012; Makarem et al, 2013b). In that species, the mammary gland can be seen to originate in the embryo from cells in the ventral ectoderm that invade the underlying mesoderm to form a primitive branching structure. At this stage, the rudimentary gland is composed of cells with a mixture of properties that are associated with distinct cell types found in the adult mouse mammary gland. This primitive structure then expands rapidly after the onset of puberty. Thereafter, until menopause, the entire mammary gland in humans and mice alike undergoes continuous cyclical phases of expansion and involution under the control of changing levels of estrogen (E) and progesterone (P) (Fig 1B; Ramakrishnan et al, 2002). Current evidence indicates that the stimulatory effects of these hormones are exerted indirectly by activating paracrine signaling mechanisms that involve an upregulated production of amphiregulin by E, an induced secretion of RANKL by P, and an enhancing effect of hormonally controlled changes by WNT-producing macrophages (Wilson et al, 2006; Asselin-Labat et al, 2010; Brisken & O'Malley, 2010; Joshi et al, 2010; Roarty & Rosen, 2010; Visvader & Stingl, 2014; Arendt & Kuperwasser, 2015; Chakrabarti et al, 2018). Other growth factors implicated in regulating mammary gland development and homeostasis include members of the epidermal growth factor (EGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF) families (Hynes & Watson, 2010). The development of reproducible methods for isolating the different cell types that constitute the major components of the normal adult human mammary gland as separate suspensions of single viable cells was a key advance because it then enabled the further biological and molecular characterization of these different cell types. Most studies of normal human mammary cells have made use of discarded tissue obtained from women without known breast disease undergoing reduction mammoplasties. The pieces of tissue obtained are then subjected to a series of enzymatic dissociation and filtration steps, followed by removal of prevalent blood and endothelial cells using antibodies against CD45 and CD31. The three major cell types that constitute the mammary gland, plus remaining stromal fibroblasts, can then be separately isolated using flow cytometry according to their differential staining with antibodies to CD49f and EpCAM (Fig 2A). The three subpopulations of mammary cells obtained are typically referred to as basal cells (BCs), luminal progenitors (LPs), and luminal cells (LCs). Other antibody cocktails have also been used to obtain highly overlapping phenotypes with very similar biological and molecular properties (Raouf et al, 2008; Bachelard-Cascales et al, 2010; Keller et al, 2012; Kannan et al, 2014; Nguyen et al, 2014; Fridriksdottir et al, 2015; Lawson et al, 2015; Britschgi et al, 2017), and additional markers have proven useful to subdivide these three subpopulations of human mammary cells even further (Eirew et al, 2012; Shehata et al, 2012; Knapp et al, 2017; Morel et al, 2017). However, the combination of antibodies to CD49f and EpCAM has generally been the most widely utilized. Figure 2. Subpopulations of cells within the normal adult human mammary gland(A) Diagram showing the workflow for separating the four main cell populations present in the breast in addition to blood cells and endothelial cells (scale bar = 400 μm). (B) Examples of typical Giemsa-stained colonies derived from BCs and LPs and assessed after 7–9 days in vitro (scale bar = 400 μm). Download figure Download PowerPoint BCs are defined by their CD49f+EpCAMlow phenotype and are so-named because they express numerous markers (e.g., KRT14, TP63, ACTA2/SMA, MME/CD10, and THY1/CD90) that distinguish cells of the basal layer from those of the luminal layer in histological preparations of normal human mammary tissue. In culture media containing insulin and EGF, as well as other supplements and a feeder layer of fibroblasts, ~ 10–20% of freshly isolated BCs plated at low density will produce readily visualized adherent colonies within 8–10 days (Fig 2B; Eirew et al, 2008; Kannan et al, 2013). Many of the individual colonies produced from BCs under these conditions will contain a mixture of cells expressing either basal or luminal markers (Stingl et al, 2001). A smaller fraction of the BCs (~ 0.1%) will produce bilayered epithelial structures that resemble the normal human mammary gland when injected directly into "humanized" fat pads (Kuperwasser et al, 2004; Proia & Kuperwasser, 2006; Lim et al, 2009) or when transplanted in collagen gels that are then inserted either under the kidney capsule (Eirew et al, 2008, 2010; Nguyen et al, 2015) or subcutaneously (Pellacani D and Eaves C, unpublished) in immunocompromised mice. In both of these sites, the regenerated human mammary gland-like structures contain the same spectrum of EGF-dependent in vitro mammary colony-forming cells (CFCs) that are present in the normal human mammary gland, as well as rarer cells that can regenerate similar bilayered mammary gland structures and mammary CFCs upon transplantation into secondary hosts (Eirew et al, 2008; Lim et al, 2009; Nguyen et al, 2014). In addition, the regenerated human gland-like structures will produce human milk proteins when appropriately hormonally stimulated (Eirew et al, 2008). LPs and LCs are defined by their shared high expression of EpCAM, a well-established marker of cells that constitute the luminal layer of mammary glands. Both LPs and LCs also express other markers histologically associated with the luminal layer (e.g., KRT8, KRT18, and MUC1). However, these EpCAM+ mammary cells can be readily subdivided according to their differential expression of CD49f (and CD117, c-KIT). LC is the term assigned to the CD49f− cells within the EpCAM+ fraction, and they include most of the cells that express E and P receptors (ER/ESR1 and PR/PGR) and express low to undetectable levels of EGFR (Lim et al, 2009). Not surprisingly, LCs do not mount a significant direct signaling response to EGF (Knapp et al, 2017) and do not proliferate when exposed to EGF in vitro (Kannan et al, 2013, 2014). They are also incapable of reconstituting epithelial structures in vivo that contain clonogenic progeny (Eirew et al, 2008). However, it was recently reported that a small proportion (~ 0.4%) of EpCAM+CD271−CD166highCD117low human mammary cells, a phenotype expected to overlap with CD49f−EpCAMhi LCs, will form colonies in cultures containing inhibitors to the TGF-β pathway (Fridriksdottir et al, 2015). Interestingly, cultures established from these cells could be expanded for 15 population doublings and their progeny continued to express ER and respond to E stimulation. In mice, similar evidence of the proliferative activity in vivo of non-clonogenic LCs has also been obtained from BrdU incorporation studies (Giraddi et al, 2015). Together, these findings raise the possibility that at least some human mammary cells with a LC phenotype can proliferate when appropriately stimulated. Nevertheless, the relevance of these in vitro findings to events that underpin the cellular dynamics within the mammary gland of normal adult women remains obscure as, in situ, very few ER+ or PR+ mammary cells appear to be proliferating (Clarke et al, 1997; Stingl, 2011). LPs are defined as the EpCAM+ cells that co-express CD49f, suggesting that they might be an intermediate stage between BCs and LCs. However, these cells express other markers specific to the luminal layer of the epithelium assessed histologically, although only a minority express ER or PR (Lim et al, 2009). LPs are also distinct in their expression of high levels of CD117, a marker often used for their differential isolation (Fridriksdottir et al, 2015; Lawson et al, 2015). Approximately 50% of LPs also express KRT5/6 (Lim et al, 2009), a type of cytokeratin known to be expressed by cells in the basal layer of many types of epithelia (Purkis et al, 1990; Böcker et al, 1992). On average, 20–30% of LPs will generate colonies in vitro under the same conditions as BCs (Fig 2B). But, in this case, only cells with luminal features are produced (Stingl et al, 2005). A small proportion of LPs have also been reported to regenerate epithelial structures in vivo (Shehata et al, 2012), but the structures produced do not contain CFCs (Eirew et al, 2008). Most LPs have very short telomeres and display a pronounced telomere-associated DNA damage response, even in mammary cells obtained from women in their twenties (Kannan et al, 2013). Interestingly, some LPs expressing activated caspase-3 will still show considerable subsequent proliferative activity in vitro (Knapp et al, 2017). LPs are also distinguished by elevated levels of reactive oxygen species (ROS) compared to LCs and BCs. In addition, they display an innately greater resistance to oxidative stress and a higher level of associated DNA damage (Kannan et al, 2014), two processes that have been proposed to accelerate telomere shortening (von Zglinicki, 2002; Richter & von Zglinicki, 2007), and predispose cells to transformation. More recently, single-cell mass cytometry (Knapp et al, 2017) and RNA sequencing methodologies (Nguyen et al, 2018) have provided further support for the segregation of normal human mammary epithelial cells into the same three main cell types. On the other hand, these studies have also highlighted their extensive molecular heterogeneity and the possible existence of new subsets within each (Shehata et al, 2012; Knapp et al, 2017; Nguyen et al, 2018). Nevertheless, pseudo-temporal ordering of the available single-cell transcriptional data produces a differentiation trajectory profile that separates into three main branches corresponding to the historically visualized distinction of cells produced in the normal adult human mammary gland (Nguyen et al, 2018). Taken together, these findings are consistent with a hierarchically organized sequence of changes initiated in bipotent BCs that are able to generate progeny with either luminal or basal features. Cells with luminal features can then be phenotypically and biologically segregated into an intermediate, luminal-restricted but EGF-responsive state, and a state in which the capacity to proliferate in response to EGF has been lost. However, this model of a hierarchical differentiation process should not be viewed as necessarily reflecting a series of tightly co-ordinated events and may also not reflect the operation of mechanisms that maintain these subpopulations under normal homeostatic conditions. Indeed, in the mouse, where analogous populations of BCs, LPs, and LCs have been identified, some luminal cells possess or can acquire the regenerative activity originally thought to be restricted to BCs (Shehata et al, 2012; Makarem et al, 2013a). In addition, in mice, in situ lineage-tracing experiments suggest that both myoepithelial and luminal lineages can display self-sustaining dynamics (Van Keymeulen et al, 2011), despite the continued presence and activity of transplantable cells with the bipotent regenerative properties of "stem cells" (Rios et al, 2014). Such findings are consistent with increasing evidence of an incomplete overlap of mechanisms that control mammary cell proliferative potential and those that determine whether their differentiated state will change (or not) with sequential divisions. At the same time, it is important to recognize the caveats and assumptions inherent in available methods for associating functional and molecular properties of individual human mammary cells or the history of their acquisition and display. Deriving these associations is necessarily limited by an inability to undertake the requisite prospective lineage-tracing experiments in humans. Accordingly, direct measurements of normal human mammary cell outputs in situ cannot be compared with the outputs that can be elicited from the same cells when they are exposed to highly stimulatory conditions in vitro or following their transplantation into mice. In addition, both flow cytometry and clonal assays have technical limitations of efficiency and specificity. They may also be compromised by the use of markers that are not co-ordinately controlled by mechanisms that regulate their functional properties. However, these caveats may be partially reduced by the use of index-sorting strategies to link molecular and functional properties more directly (Wilson et al, 2015), thereby circumventing the problem of assigning functions of rare cells present in bulk isolates. Transcriptional differences between human mammary cell subsets A variety of technologies have been used over the past 10 years to characterize the transcriptomes of BCs, LPs, and LCs isolated from normal adult female breast tissue (Bloushtain-Qimron et al, 2008; Raouf et al, 2008; Lim et al, 2009, 2010; Maruyama et al, 2011; Shehata et al, 2012; Kannan et al, 2013; Gascard et al, 2015; Pellacani et al, 2016). These studies have revealed consistent differences in the activity of hundreds of genes in each of these phenotypically defined subsets. In turn, these studies have pointed to a number of differentially activated pathways that may regulate their different biological properties (Liu et al, 2005). For example, many components of the NOTCH pathway are expressed at different levels in BCs, LPs, and LCs, with some evidence of corresponding functional consequences (Dontu et al, 2004; Raouf et al, 2008). WNT pathway components also show differential patterns of expression, with biological evidence of their importance in maintaining a mammary stem cell state, at least as inferred from studies of the mouse mammary gland (Teulière et al, 2005; Roarty & Rosen, 2010; Zeng & Nusse, 2010; van Amerongen et al, 2012; Gu et al, 2013) with more limited, but consistent data for human cells (Arendt et al, 2014). Other pathways similarly implicated are the TGF-β (Moses & Barcellos-Hoff, 2011; Kahata et al, 2017) and the Hippo pathways (Chen et al, 2014; Pelissier et al, 2014; Skibinski et al, 2014; Shi et al, 2015; Britschgi et al, 2017). Importantly, all of these are variably deregulated in breast cancers (Howard & Ashworth, 2006). Human mammary cell epigenomes reflect their transcriptional profiles Several studies have now characterized the epigenomic features of human as well as mouse mammary cells (Maruyama et al, 2011; Choudhury et al, 2013; Dos Santos et al, 2015; Gascard et al, 2015; Huh et al, 2015; Pellacani et al, 2016; Shin et al, 2016; Lee et al, 2017). Early studies reported an association of differences in the H3K27me3 and DNA methylation of genes that are differently expressed in luminal and basal subsets (Maruyama et al, 2011). These genes include several that encode transcriptional regulators and/or other members of pathways of reported activity in the mammary gland. Subsequent analyses revealed DNA methylation to be a stable mark of exonic and intronic usage, with evidence of intron retention events specific to each subpopulation and linked to differences in protein expression (Gascard et al, 2015). The latter study also found many more hypo-methylated enhancer elements in luminal cells (LPs + LCs) than in BCs and these were commonly associated with binding sites for FOXA1, GATA3, and ZNF217. These studies also indicated a higher overall transcriptional activity in the luminal cells. More extensive epigenomic characterization of highly purified human BCs, LPs, LCs and their associated stromal cells has now been derived from ChIP-seq analyses of H3K4me1, H3K4me3, H3K27me3, H3K27ac, H3K36me3, and H3K9me3 marks on histones and accompanying whole-genome bisulfite sequencing, with matching mRNA-seq and miRNA-seq data for the same cells (Pellacani et al, 2016). From these datasets, the chromatin landscape at putative enhancer sites of these different mammary cell types has been derived. Comparisons of these have also shown LPs to be intermediate between BCs and LCs, consistent with their different biological properties. Analysis of transcription factor binding sites (TFBS) and derived TF networks for each subpopulation has also enabled novel TFs to be identified as potential regulators of each subpopulation, in addition to others previously reported. Analysis of our more recently accrued epigenomic data has also provided new evidence of a bipartite TF network in LPs that includes elements of those operative in BCs and LCs (Fig 3A). In addition, this study showed that the epigenomic and transcriptional profiles of primary sources of normal human mammary cells are very different from those of established lines of immortalized but non-tumorigenic mammary cells (Fig 3B; Pellacani et al, 2016). This latter finding highlights the caveats of relying on data from such immortalized cell lines to infer mechanisms controlling the biological properties of normal human mammary cells, and, conversely, the importance of analyzing primary isolates for this purpose. Figure 3. Transcriptional regulation of normal human mammary cell subpopulations(A) TF regulatory networks constructed from the chromatin profiles at enhancers of BCs, LPs, and LCs. (B) Genome browser plots showing the differences in chromatin states defined for normal human mammary cell subpopulations and non-tumorigenic mammary cell lines around the PROM1 and the NT5E genes. Download figure Download PowerPoint Epigenomic and transcriptional changes related to aging and reproductive history Aging and pregnancy are associated, respectively, with an increase and decrease in breast cancer risk. Several groups have therefore started dissecting the molecular changes evident in mammary cells obtained from donors of different ages or different reproductive histories. These include a report of an expansion with aging of defective multipotent progenitors that show altered interactions with extracellular matrix elements and in KRT14+ and CD49f+ luminal cells (Garbe et al, 2012; Pelissier et al, 2014). Accompanying transcriptome changes suggested an aging-associated epigenomic deregulation, potentially mediated by changes in the microenvironment of the mammary gland (Miyano et al, 2017). Comparison of the transcriptomes of purified mammary cell subsets isolated from breast tissue of parous and nulliparous women has shown differences between the CD44+ cells from these two sources, with CDKN1B (p27) as one of the most differentially expressed genes (Choudhury et al, 2013). More extensive studies in mice have shown pregnancy to be associated with long-lasting alterations in DNA methylation profiles at sequences enriched for STAT5 binding sites (Dos Santos et al, 2015). Transcription factors regulating normal mammary cells Epigenomic and transcriptional profiling of primary human mammary cells has also led to the identification of many candidate TFs that show subpopulation specificity. For example, several TFs are significantly elevated in only one of the three major subpopulations of normal human mammary cells (Fig 4A–C). In silico predictions further identify a differential enrichment of associated TFBSs at epigenetically defined promoter and enhancer regions in these cell types (Lim et al, 2010; Kannan et al, 2013; Gascard et al, 2015; Pellacani et al, 2016). Several studies in mice or human cell lines have also implicated a multitude of TFs to be involved in mammary cell development and differentiation. However, similar analyses of primary human cells are still very limited, although the strong correlations found between in silico predictions and results obtained from mice justify a brief overview of these. Figure 4. Transcriptional regulators active in the normal human mammary gland and in human breast cancer(A) Ternary plot of relative expression of all transcriptional regulators in normal human mammary cell subpopulations from a re-analysis of the RNA-seq data presented in Pellacani et al (2016). Transcriptional regulators discussed in the text are highlighted. (B) List of the top 20 transcriptional regulators most specific to each cell type highlighted in (A). (C) Clustering of the tumors profiled by RNA-seq in Nik-Zainal et al (2016) using the genes shown in (B). Download figure Download PowerPoint One of the TFs implicated in modulating mouse mammary stem cell activity by acting directly on BCs is ∆Np63, a known regulator of normal stem cell maintenance in multiple epithelial tissues (Senoo et al, 2007). ∆Np63 appears to act by modulating several key pathways. These include enhancing WNT signaling by upregulating Fzd7 expression (Chakrabarti et al, 2014), activating Hedgehog signaling (Li et al, 2008; Memmi et al, 2015), and partially counteracting the effects of Notch signaling (Yalcin-Ozuysal et al, 2010). TP63 expression in basal cells is also necessary during pregnancy and lactation: Genetic deletion of Trp63 in keratin 14-expressing cells of the adult mouse leads to defects in luminal cell proliferation and differentiation, and failure to produce milk, due to lack of expression of the EGF family ligand NRG1 in basal cells which is required for ERBB4/STAT5A activation in luminal cells (Forster et al, 2014). Several SOX family TFs have likewise been implicated. For example, modulation of SOX9 expression was found to directly influence the ability of mouse mammary cells to produce organoid structures in vitro (Guo et al, 2012) and its conditional knockout impaired postnatal development of the gland (Malhotra et al, 2014). SOX10 is expressed specifically in mammary cells exhibiting the highest levels of stem/progenitor activity (Dravis et al, 2015) and SOX2 has also been implicated, albeit less directly, as its expression was induced by LGR4 downstream of WNT signaling (Wang et al, 2013). Many of these studies in mice have associated expression of SOX TFs with the acquisition of features characteristic of mesenchymal cells in a process resembling an embryonic epithelial–mesenchymal transition (EMT). In fact, the possession of mesenchymal features has been frequently associated with mammary stem cell activity, both during development and subsequently throughout adulthood (Mani et al, 2008; Guen et al, 2017), although this is still controversial (Sikandar et al, 2017). Nevertheless, many other TFs associated with EMT have been directly linked to changes in the clonogenic or repopulating activity of mouse mammary cells. Of these, SNAI2 (SLUG) has been reported to cooperate with SOX9 (Guo et al, 2012) in regulating the transition of mouse mammary stem cells to short-term progenitors (Phillips et al, 2014). SNAI1 (SNAIL) is another member of this group, and it was found to regulate the spindle orientation machinery in mammary stem cells responding to SLIT2/ROBO1 signaling (Ballard et al, 2015). OVOL2, a transcriptional repressor, was likewise reported to restrict activation of EMT (Watanabe et al, 2014). More recently, another transcription factor, ZEB1, was shown to be expressed at high levels in a fraction of mammary BCs (Nguyen et al, 2018) and associated with cells expressing protein C receptor (ProCR; Wang et al, 2015). ZEB1 was also recently reported to have a protective role against oncogene-induced DNA damage in normal human mammary epithelial cells (Morel et al, 2017). Other TFs involved in mammary stem cell function include FOXO1 (Sreekumar et al, 2017), RUNX2 (Ferrari et al, 2015), MYC (Hynes & Stoelzle, 2009; Moumen et al, 2012), CEBPB (C/EBPβ; LaMarca et al, 2010), BCL11A (Khaled et al, 2015), and BCL11B (Miller et al, 2018). TFs implicated in regulating luminal cell production and maintenance have also been identified. Of these, GATA3 was found to have an essential role in controlling the morphogenesis of the mammary gland in the mouse embryo, during puberty, and in adult life (Kouros-Mehr et al, 2006; Asselin-Labat et al, 2007). In addition, GATA3 promoted differentiation of cells within the luminal lineage in mice, potentially through a positive regulatory loop with ESR1 (Eeckhoute et al, 2007). FOXA1 was found to be involved in hormone-induced mammary ductal invasion (Bernardo et al, 2010), but did not affect lobulo-alveolar maturation and milk production. ELF5 was shown to be necessary for alveologenesis during pregnancy (Choi et al, 2009), and its deletion led to an accumulation of cells with mixed basal/luminal molecular phenotypes (Chakrabarti et al, 2012b). ELF5 was found to suppress EMT by down-regulating transcription of SNAI2 (Chakrabarti et al, 2012a). ELF5 also acted directly in LPs (Yamaji et al, 2009) to influence expression of STAT5A (Choi et al, 2009), another TF involved in alveologenesis (Liu et al, 1997). Contrary to the effects of RUNX2, RUNX1 was shown to induce the appearance of ER+ luminal cells at least partially through the modul