Human Breast Progenitor Cell Numbers Are Regulated by WNT and TBX3

Abstract

BackgroundAlthough human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties.MethodsTo examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone.ResultsGrowth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response.ConclusionsWe identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3.

Hormone-Sensing Mammary Epithelial Progenitors: Emerging Identity and Hormonal Regulation

“…In both human and mouse MECs, TBX3 was shown to be expressed by both basal and HS-MECs, with HS-MECs having the greatest level of expression [35,164]. Lentiviral knockdown of mouse TBX3 in mammary reconstitution assays prevents formation of HSMECs [35], a process that may involve a loss of autocrine WNT signalling, normally functioning as a hormoneindependent mechanism of regulating the HS-MEC population [164]. It is conceivable that the same TBX3-driven promotion of WNT signalling in the developing mammary epithelium applies to paracrine regulation at later stages of mammary development.…”

Form and Function: how Estrogen and Progesterone Regulate the Mammary Epithelial Hierarchy

“…Moreover, the effects of estrogen are modulated by interactions at the PR promoter, which may be cell-type specific [15]. Recent evidence suggests that ERα and PR are also expressed independently within separate cell populations in the breast, and these cell populations may have divergent regulation and growth potential [16–19]. Thus, being able to isolate and study a pure population of cells that are only sensitive to one hormone or the other while only expressing one receptor is difficult.…”

Impact of Progesterone on Stem/Progenitor Cells in the Human Breast

“…This was demonstrated by a P-mediated increase in the proportion of cells that formed mammospheres in suspension (an assay which preferentially selects for progenitors able to survive in suspension culture [47]), as well as the proportion of cells with stem-like ALDH activity [46]. This was supported more recently by the demonstration that P enhanced secondary mammosphere formation and increased progenitor activity in primary normal human breast epithelial cells [48]. Also using the 3D culture method of primary normal human breast epithelial cells, P stimulated the numbers of acini composed of both luminal and myoepithelial cells, concordant with P stimulating bipotent progenitor cells in the normal human breast [49], as well as increasing the proportion of CD10+ cells [49].…”

“…As discussed above, P stimulates progenitor cells in normal human breast epithelial cells grown in 3D culture, and PR is expressed in a subset of basal cells enriched for progenitor characteristics [46,48,49,89]. Therefore, one could postulate that a small number of PR+ progenitor cells within the breast lobule are able to Fig.…”

“…Recent cell fractionation studies have also detected PR transcripts in EpCAM-CD10+ cells which were enriched for basal progenitor characteristics, and IF analysis revealed strong PR expression in basally located cells which also expressed the luminal marker, cytokeratin-8 (CK8), suggested to be putative progenitor cells [48]. It is not possible to definitively determine whether these basal cells are in fact progenitor cells from these Fig.…”