In vivo reprogramming of non-mammary cells to an epithelial cell fate is independent of amphiregulin signaling.

Amphiregulin (AREG)-/- mice demonstrate impaired mammary development and form only rudimentary ductal epithelial trees; however, AREG-/- glands are still capable of undergoing alveologenesis and lactogenesis during pregnancy. Transplantation of AREG-/- mammary epithelial cells into cleared mouse mammary fat pads results in a diminished capacity for epithelial growth (∼15%) as compared to that of wild-type mammary epithelial cells. To determine whether estrogen receptor α (ERα, also known as ESR1) and/or AREG signaling were necessary for non-mammary cell redirection, we inoculated either ERα-/- or AREG-/- mammary cells with non-mammary progenitor cells (WAP-Cre/Rosa26LacZ+ male testicular cells or GFP-positive embryonic neuronal stem cells). ERα-/- cells possessed a limited ability to grow or reprogram non-mammary cells in transplanted mammary fat pads. AREG-/- mammary cells were capable of redirecting both types of non-mammary cell populations to mammary phenotypes in regenerating mammary outgrowths. Transplantation of fragments from AREG-reprogrammed chimeric outgrowths resulted in secondary outgrowths in six out of ten fat pads, demonstrating the self-renewing capacity of the redirected non-mammary cells to contribute new progeny to chimeric outgrowths. Nestin was detected at the leading edges of developing alveoli, suggesting that its expression may be essential for lobular expansion.

Does the Mouse Mammary Gland Arise from Unipotent or Multipotent Mammary Stem/Progenitor Cells?

The presence of long-lived lineage restricted progenitor and multipotent progenitor cells in adult mouse mammary gland for cancer development is compelling. Mammary cancers are phenotypically diverse This might be explained by transformation of long-lived, lineage-limited progenitor subpopulations. Mammary multipotent epithelial stem cells and their environmental niches must be considered, since their niche(s), once empty might be occupied by lineage-limited progenitors that are proximal. The existence of premalignant mammary populationst that manifest characteristics of lineage limitation argues strongly for this proposition.

Binuclear Cells in the Lactating Mammary Gland: New Insights on an Old Concept?

In a recent paper (Rios et al. Nat Commun. 7:11400, 2016), it was reported that polyploid cells are frequent in lactating mammary tissues. This phenomenon was observed in mammary tissue sampled from five separate mammalian species. According to that report, these binucleated cells occur late in pregnancy and early in lactation. Unfortunately, this paper did not mention a number of earlier observations and findings that remain pertinent to this day (Banerjee et al. Life sciences Pt 2: Biochemistry, general and molecular biology. 10(15):867-77, 1971; Banerjee MR, Wagner JE. Biochem. Biophys. Res. Commun. 49(2):480-7, 1972). In these classical experiments, the authors demonstrated in vivo that DNA synthesis continued without commensurate cell division during late pregnancy and lactation, and that this DNA synthesis was imperative for functional differentiation of the mammary epithelium. Later studies showed that DNA synthesis was indispensable to the induction of milk protein production in explant cultures of mammary tissue from unprimed, nulliparous mice. This dependence on DNA synthesis in mammary explant cultures stimulated by lactogenic hormones was found to be dispensable following a single pregnancy. The absolute requirement for DNA synthesis in nulliparous mouse mammary explants stimulated to synthesize milk protein in vitro has remained unexplained, as has the need for DNA synthesis prior to the onset of lactation. From a historical perspective, it is more likely that binuclear secretory cells in the lactating mammary gland are a consequence of the DNA synthesis requirement for lactation, rather than an essential element.

Paracrine-rescued lobulogenesis in chimeric outgrowths comprising progesterone-receptor-null mammary epithelium and redirected wild-type testicular cells.

We have previously shown that non-mammary and tumorigenic cells can respond to the signals of the mammary niche and alter their cell fate to that of mammary epithelial progenitor cells. Here we tested the hypothesis that paracrine signals from mammary epithelial cells expressing progesterone receptor (PR) are dispensable for redirection of testicular cells, and that re-directed wild-type testicular-derived mammary cells can rescue lobulogenesis of PR-null mammary epithelium by paracrine signaling during pregnancy. We injected PR-null epithelial cells mixed with testicular cells from wild-type adult male mice into cleared fat-pads of recipient mice. The testicular cells were redirected in vivo to mammary epithelial cell fate during regeneration of the mammary epithelium, and persisted in second-generation outgrowths. In the process, the redirected testicular cells rescued the developmentally deficient PR-null cells, signaling them through the paracrine factor RANKL to produce alveolar secretory structures during pregnancy. This is the first demonstration that paracrine signaling required for alveolar development is not required for cellular reprogramming in the mammary gland, and that reprogrammed testicular cells can provide paracrine signals to the surrounding mammary epithelium.

Hormone signaling requirements for the conversion of non-mammary mouse cells to mammary cell fate(s) in vivo.

Mammotropic hormones and growth factors play a very important role in mammary growth and differentiation. Here, hormones including Estrogen, Progesterone, Prolactin, their cognate receptors, and the growth factor Amphiregulin, are tested with respect to their roles in signaling non-mammary cells from the mouse to redirect to mammary epithelial cell fate(s). This was done in the context of glandular regeneration in pubertal athymic female mice. Our previous studies demonstrated that mammary stem cell niches are recapitulated during gland regeneration in vivo. During this process, cells of exogenous origin cooperate with mammary epithelial cells to form mammary stem cell niches and thus respond to normal developmental signals. In all cases tested with the possible exception of estrogen receptor alpha (ER-α), hormone signaling is dispensable for non-mammary cells to undertake mammary epithelial cell fate(s), proliferate, and contribute progeny to chimeric mammary outgrowths. Importantly, redirected non-mammary cell progeny, regardless of their source, have the ability to self-renew and contribute offspring to secondary mammary outgrowths derived from transplanted chimeric mammary fragments; thus suggesting that some of these cells are capable of mammary stem cell/progenitor functions.

Reprogramming non-mammary and cancer cells in the developing mouse mammary gland.

The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the "niche" and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary cells, including mouse and human cancer cells, may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells.

A potential mechanism for extracellular matrix induction of breast cancer cell normality.

Extracellular matrix proteins from embryonic mesenchyme have a normalizing effect on cancer cells in vitro and slow tumor growth in vivo. This concept is suggestive of a new method for controlling the growth and spread of existing cancer cells in situ and indicates the possibility that extracellular proteins and/or embryonic mesenchymal fibroblasts may represent a fertile subject for study of new anti-cancer treatments.

Late developing mammary tumors and hyperplasia induced by a low-oncogenic variant of mouse mammary tumor virus (MMTV) express genes identical to those induced by canonical MMTV.

BACKGROUND: The canonical milk-transmitted mouse mammary tumor virus (MMTV) of C3H mice (C3H-MMTV) rapidly induces tumors in 90% of infected animals by 8 months of age. Pro-viral insertions of C3H-MMTV into genomic DNA results in the overexpression of common core insertion site (CIS) genes, including Wnt1/10b, Rspo2, and Fgf3. Conversely, infection by either the endogenous Mtv-1 virus (in C3Hf) or the exogenous nodule-inducing virus (NIV) (in Balb/c NIV) induces premalignant mammary lesions and tumors with reduced incidence and longer latency than C3H-MMTV. Here, we asked whether Mtv-1/NIV affected the expression of core CIS genes. FINDINGS: We confirmed the presence of active virus in Mtv-1/NIV infected tissues and using quantitative reverse transcription PCR (qRT-PCR) found that Mtv-1/NIV induced neoplasms (tumors and hyperplasia) commonly expressed the core CIS genes Wnt1, Wnt10b, Rspo2, Fgf3. CONCLUSIONS: These results underscore the importance of core CIS gene expression in the early events leading to MMTV-induced mammary tumor initiation regardless of the viral variant.

Stem cell marker prominin-1 regulates branching morphogenesis, but not regenerative capacity, in the mammary gland.

Prominin-1 (Prom1) is recognized as a stem cell marker in several tissues, including blood, neuroepithelium, and gut, and in human and mouse embryos and many cancers. Although Prom1 is routinely used as a marker for isolating stem cells, its biological function remains unclear. Here we use a knockout model to investigate the role of Prom1 in the mammary gland. We demonstrate that complete loss of Prom1 does not affect the regenerative capacity of the mammary epithelium. Surprisingly, we also show that in the absence of Prom1, mammary glands have reduced ductal branching, and an increased ratio of luminal to basal cells. The effects of Prom1 loss in the mammary gland are associated with decreased expression of prolactin receptor and matrix metalloproteinase-3. These experiments reveal a novel, functional role for Prom1 that is not related to stem cell activity, and demonstrate the importance of tissue-specific characterization of putative stem cell markers.

Role of epithelial stem/progenitor cells in mammary cancer.

Both mouse and human mammary glands contain stem/progenitor functional hierarchies that are maintained through the entire life span of the animal. Cells with such functional capacities are potential candidates for tumorigenesis as they are long lived, multipotent, and self-renewing. Using the mouse as a model, this review will discuss what is known about the mammary stem/progenitor hierarchy, the evidence that particular progenitor functions are susceptible to tumorigenic stimuli, how these findings in mice are relevant to the disease in humans, and the role of the local microenvironment in controlling tumorigenesis.

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics.

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG(-/-) mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

The mammary microenvironment alters the differentiation repertoire of neural stem cells.

A fundamental issue in stem cell biology is whether adult somatic stem cells are capable of accessing alternate tissue sites and continue functioning as stem cells in the new microenvironment. To address this issue relative to neurogenic stem cells in the mouse mammary gland microenvironment, we mixed wild-type mammary epithelial cells (MECs) with bona fide neural stem cells (NSCs) isolated from WAP-Cre/Rosa26R mice and inoculated them into cleared fat pads of immunocompromised females. Hosts were bred 6-8 weeks later and examined postinvolution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination, and constitutive expression of LacZ. The NSCs and their progeny contributed to mammary epithelial growth during ductal morphogenesis, and the Rosa26-LacZ reporter gene was activated by WAP-Cre expression during pregnancy. Some NSC-derived LacZ(+) cells expressed mammary-specific functions, including milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSCs and their progeny enter mammary epithelium-specific niches and adopt the function of similarly endowed mammary cells. This result supports the conclusion that tissue-specific signals emanating from the stroma and from the differentiated somatic cells of the mouse mammary gland can redirect the NSCs to produce cellular progeny committed to MEC fates.

Immortalized, pre-malignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA.

INTRODUCTION: During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents', simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy. METHODS: The glands of 3-week old immune competent Balb/C female mice were utilized intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells which retain their template DNA. Nulliparous mice were then either injected with [(3)H]-thymidine ((3)H-TdR) to distinguish 5-BrdU-label retaining cells that enter the cell cycle and euthanized, or mated, injected with (3)H-TdR, and euthanized at various days post-coitus. Sections were stained for estrogen receptor-α(ER-α) or progesterone receptor (PR) via immunohistochemistry. Cells labelled with both 5-BrdU and (3)H-TdR were indicative of label-retaining epithelial cells (LREC). RESULTS: Cells that retained a 5-BrdU label and cells labelled with [(3)H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labelled nuclei (LREC) were found in the intact mammary gland of both pregnant and nulliparous mice, and in mammary glands implanted with pre-malignant cells. Double-labelled cells ((3)H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label ((3)H-TdR) to daughter cells; and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary implant groups. CONCLUSIONS: The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.

Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice.

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA "de novo" SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA "de novo" SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious "de novo" SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to "heteroplasmic-shifting". A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Induction of phenotypic changes in HER2-postive breast cancer cells in vivo and in vitro.

The influence of breast cancer cells on normal cells of the microenvironment, such as fibroblasts and macrophages, has been heavily studied but the influence of normal epithelial cells on breast cancer cells has not. Here using in vivo and in vitro models we demonstrate the impact epithelial cells and the mammary microenvironment can exert on breast cancer cells. Under specific conditions, signals that originate in epithelial cells can induce phenotypic and genotypic changes in cancer cells. We have termed this phenomenon "cancer cell redirection." Once breast cancer cells are redirected, either in vivo or in vitro, they lose their tumor forming capacity and undergo a genetic expression profile shift away from one that supports a cancer profile towards one that supports a non-tumorigenic epithelial profile. These findings indicate that epithelial cells and the normal microenvironment influence breast cancer cells and that under certain circumstances restrict proliferation of tumorigenic cells.

Common integration sites for MMTV in viral induced mouse mammary tumors.

The paradigm of mammary cancer induction by the mouse mammary tumor virus (MMTV) is used to illustrate the body of evidence that supports the hypothesis that mammary epithelial stem/progenitor cells represent targets for oncogenic transformation. It is argued that this is not a special case applicable only to MMTV-induced mammary cancer, because MMTV acts as an environmental mutagen producing random interruptions in the somatic DNA of infected cells by insertion of proviral DNA copies. In addition to disrupting the host genome, the proviral DNA also influences gene expression through its associated enhancer sequences over significant inter-genomic distances. Genes commonly affected by MMTV insertion in multiple individual tumors include, the Wnt, FGF, RSpo gene families as well as eIF3e and Notch4. All of these gene families are known to play essential roles in stem cell maintenance and behavior in a variety of organs. The MMTV-induced mutations accumulate in cells that are long-lived and possess the properties of stem cells, namely, self-renewal and the capacity to produce divergent epithelial progeny through asymmetric division. The evidence shows that epithelial cells with these properties are present in normal mammary glands, may be infected with MMTV, become transformed to produce epithelial hyperplasia through MMTV-induced mutagenesis and progress to frank mammary malignancy. Retroviral marking via MMTV proviral insertion demonstrates that this process progresses from a single mammary epithelial cell that possesses all of the features ascribed to tissue-specific stem cells.

Long-label-retaining mammary epithelial cells are created early in ductal development and distributed throughout the branching ducts.

Long-label retention has been used by many to prove Cairns' immortal strand hypothesis and to identify potential stem cells. Here, we describe two strategies using 5-ethynl-2'-deoxyuridine (EdU) to identify and understand the distribution of long-label-retaining mammary epithelial cells during formation of the mouse mammary ductal system. First, EdU was given upon two consecutive days per week during weeks 4 through 10 and analyzed for label retention at 13 weeks of age. Alternatively, EdU was given for 14 consecutive days beginning at 28 days of age and ending at 42 days of age. Analyses were conducted at >91 days of age (13 weeks). Many more LREC were detected following the second labeling method and their distribution among the subsequently developed ducts. This finding indicated that the early-labeled cells that retained their label were distributed into portions of the gland that developed after the ending of EdU treatment (i.e. 42->91 days). These observations may have important meaning with respect to the previously demonstrated retention of regenerative capacity throughout the mouse mammary gland despite age or reproductive history. These results suggest LREC may represent long-lived progenitor cells that are responsible for mammary gland homeostasis. Additionally, these cells may act as multipotent stem cells capable of mammary gland regeneration upon random fragment transplantation into epithelium-denuded mammary fat pads.

Microarray and pathway analysis of two COMMA-Dß derived clones reveal important differences relevant to their developmental capacity in-vivo.

Microarray technologies were used to analyze transcriptomes from Comma-Dß and clonal derivatives, SP3 (Lobule-competent) and NSP2 (Lobule-incompetent), during different mouse mammary growth phases: in-vitro, in-vivo 5-weeks, and in-vivo 12-weeks. A differentially expressed gene (DEG) algorithm was used to enrich for genes associated with cellular proliferation, differentiation, cell cycle regulation, and carcinogenesis. A pairwise comparison analysis, of SP3 vs. NSP2 in-vitro, revealed a total of 45 DEGs significantly up-regulated in SP3. Of the 45 DEGs, only Ccnd1 (Cyclin D1), Id2 (Inhibitor of DNA binding 2) and Sox9 (SRY Box 9) were identified to be associated with cellular proliferation, regulation of G1/S mitotic cell cycle, mammary gland and alveolar development in SP3. During the regenerative growth phase, in-vivo 5-weeks, we identified a total of 545 DEGs. 308 DEGs, of the 545 DEGs, were significantly up-regulated and 237 DEGs were significantly down-regulated in SP3 vs. NSP2. In addition, we identified 9 DEGs significantly up-regulated, within SP3's cell cycle pathway and a persistent overexpression of Cyclin D1, Id2, and Sox9, consistent with our in-vitro study. During the maintenance phase, in-vivo 12-weeks, we identified 407 DEGs. Of these, 336 DEGs were up-regulated, and 71 were down-regulated in SP3 vs. NSP2. Our data shows 15 DEGs significantly up-regulated, simultaneously, affecting 8 signal transducing carcinogenic pathways. In conclusion, increased expression of Cyclin D1, Id2 and Sox9 appear to be important for lobular genesis in SP3. Also, in-vivo 12 week displays increase expression of genes and pathways, involved in tumorigenesis.

Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland.

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.