Transcriptome analysis of mammary epithelial subpopulations ...

BMC Genomics 2008, 9:591http://www.biomedcentral.com/1471-2164/9/591BackgroundThe function of complex tissues, such as the mammaryepithelium, is a product of the interactions between theirconstituent cell types. In such tissues, disease states likecancer are essentially a failure of this cellular homeostasisand are characterised by insensitivity of cells to externalregulatory factors and aberrant cell fate choices. Understandingthe molecular regulation of the individual celltypes in complex tissues is, therefore, a prerequisite forunderstanding disease states. Furthermore, advances inmolecular pathology have demonstrated that differentdisease phenotypes correlate with different gene expressionprofiles [1]. In a complex tissue, composed of differentcell types with different molecular characteristics, thegene expression profiles of different diseases may reflectthe contribution of different cell types to that disease.Therefore, a detailed molecular characterisation of the celltypes in a complex tissue is essential for the interpretationof the molecular pathology of its diseases.The resting adult mammary epithelium consists of twomain structures, alveoli (which develop into milk-secretinglobulo-alveolar structures upon pregnancy) and ducts(which carry the milk from the lobulo-alveolar structuresto the nipple) [2]. These two structures are themselvescomposed of two main epithelial cell layers, basal cellsand luminal cells. The basal cell layer mainly consists ofmyoepithelial cells which contract in response to oxytocinrelease during lactation to force milk down the ducts tothe nipple. Recent work has demonstrated that the basalcell layer also contains the mammary epithelial stem cellcompartment [3-7]. The luminal cell layer has beenshown to be composed of two functionally distinct lineagesdefined by expression of the cell surface proteinsCD24 and Sca-1. CD24 +/High Sca-1 + luminal cells expressestrogen receptor alpha (ER), as well as receptors for prolactinand progesterone (the luminal ER + compartment),while CD24 +/High Sca-1 - luminal cells (the luminal ER -compartment) express genes (at low levels) for milk proteinseven in the virgin and likely include alveolar progenitors[5,7-9].Although it is known that the stem cells can generate allthe myoepithelial, luminal ER - and luminal ER + daughtercell types [5], the mechanisms which control cellularhomeostasis, fate determination and lineage commitmentin the mammary epithelium are poorly understood. Theyare likely to be a product of complex interactions betweencell extrinsic paracrine influences, cell intrinsic transcriptionalregulators and epigenomic modifications [10].Some progress has been made towards understandingsome of the cell intrinsic factors involved. For instance,Gata3 was recently identified as a transcription factorimportant in specifying commitment in the general luminallineage [11,12] and Elf5 was shown to be a specifier ofalveolar cell fate [13]. A number of the cell extrinsic (paracrine)factors operating within the mammary epitheliumhave also been characterised, such as Wnt-4, which actsdownstream of progesterone signalling in ductal sidebranching[14] and the EGF-family member Amphiregulin[15,16], which is produced by ER + cells in response toestrogen and stimulates mammary stem cell activity (mostlikely acting indirectly via non-epithelial cells and additionalparacrine factors) [17]. The Notch signalling pathwayhas also been shown to be an important determinantof luminal cell fate [18,19]. However, the full extent andnature of paracrine interactions in the mammary gland,and the degree to which the different lineages contributeto them, and are defined by them, is still not fully understood.Gene expression patterns have been previously examinedin the mouse mammary gland, either as changes in geneexpression across the whole tissue in developmental timecourses[20,21] or as comparisons between the total epitheliumand the mammary stroma [12]. In one report,gene expression patterns were examined in mouse luminaland myoepithelial cells purified by flow sorting as wellas in stem cell enriched basal cell populations [6]. However,these stem cell gene signatures were found to be notsignificantly different from the myoepithelial signatures,suggesting they were derived from cell populations dominatedby myoepithelial cells. The purity of the basal stemcell populations remains a persistent problem due the difficultyof isolating pure (as opposed to enriched) stem cellfractions. A number of gene expression studies have alsobeen carried out on human breast cells. The response ofhuman breast epithelium to estrogen has been analysed atthe gene expression level in breast cancer cell lines in vitroand as xenografts [22-24], in normal breast tissue maintainedas xenografts [25] and in normal human ER + breastcells isolated by transduction of primary breast epithelialcells with a virus carrying an estrogen response elementdriving GFP expression [26]. The comparative geneexpression profiles of normal human myoepithelial[27,28], basal non-myoepithelial (with a cell surface phenotypeCD10 - CD44 + ) [29] and luminal epithelial cells[27-29] have also been examined, as have the gene expressionprofiles of different in vitro progenitor (colony-forming)subpopulations of normal breast epithelial cells [18].However, to date no genome-wide transcriptome studyhas made a direct comparison between the two luminalepithelial populations (ER - and ER + ) and the basal/myoepithelial cells, confounding the molecular characterisationof the luminal cells and preventing the analysis ofthe lineage commitment of, and interactions between, thetwo luminal cell types and the other cell types in thegland.The aim of this study, therefore, was to carry out the firstcomprehensive gene expression study which examinedgene expression patterns in the three distinct mouse mam-Page 2 of 28(page number not for citation purposes)