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.

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.

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.

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.

Mammary extracellular matrix directs differentiation of testicular and embryonic stem cells to form functional mammary glands in vivo.

Previously, we demonstrated the ability of the normal mammary microenvironment (niche) to direct non-mammary cells including testicular and embryonic stem cells (ESCs) to adopt a mammary epithelial cell (MEC) fate. These studies relied upon the interaction of transplanted normal MECs with non-mammary cells within the mammary fat-pads of recipient mice that had their endogenous epithelium removed. Here, we tested whether acellular mammary extracellular matrix (mECM) preparations are sufficient to direct differentiation of testicular-derived cells and ESCs to form functional mammary epithelial trees in vivo. We found that mECMs isolated from adult mice and rats were sufficient to redirect testicular derived cells to produce normal mammary epithelial trees within epithelial divested mouse mammary fat-pads. Conversely, ECMs isolated from omental fat and lung did not redirect testicular cells to a MEC fate, indicating the necessity of tissue specific components of the mECM. mECM preparations also completely inhibited teratoma formation from ESC inoculations. Further, a phenotypically normal ductal outgrowth resulted from a single inoculation of ESCs and mECM. To the best of our knowledge, this is the first demonstration of a tissue specific ECM driving differentiation of cells to form a functional tissue in vivo.

Techniques for the Reprogramming of Exogenous Stem/Progenitor Cell Populations Towards a Mammary Epithelial Cell Fate.

This chapter considers the techniques necessary and required for the reprogramming of exogenous stem/progenitor cell populations towards a mammary epithelial cell fate. The protocols describe how to isolate cells from alternate mouse organs such as testicles of male mice and mix them with mammary cells to generate chimeric glands comprised of male and female epithelial cells that are fully competent. 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 are sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells. This therefore is a powerful technique for the redirection of cells from other organs/cancer cells to a normal mammary phenotype.

Telomerase and estrogen-sensing activities are essential for continued mammary growth in vivo but dispensable for "reprogramming" neural stem cells.

It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland in vivo, we serially transplanted mammary fragments from wild type (TER+/+), heterozygous (TER+/-), and homozygous (TER-/-) mammary tissues into the cleared mammary fat pads of immune-compromised nude mice. Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/- cells were able to direct neural stem cells to mammary cell fates.

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.

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.

Embryonic stem cells are redirected to non-tumorigenic epithelial cell fate by interaction with the mammary microenvironment.

Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1:5 and 1:50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+) progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. We demonstrate that these progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas.

Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors.

The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene expression in primary human breast tumors was interrogated. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors.

The mouse mammary microenvironment redirects mesoderm-derived bone marrow cells to a mammary epithelial progenitor cell fate.

Mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue leads to a reduced capacity of dispersed epithelial cells to recapitulate complete functional mammary structures. Previous studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the mammary stroma, nonmammary cells of ectodermal germ origin may be sequestered and reprogrammed to perform mammary epithelial cell (MEC) functions, including those ascribed to mammary stem/progenitor cells. To test whether tissue cells from organs derived from different germ layers could respond to mammary epithelial-specific signals, we utilized fluorescence-activated cell sorting-purified Lin(-) and Lin(-)/cKit+adult male bone marrow cells to mix with MECs. Our evidence shows that the signals provided by the mammary microenvironment are capable of redirecting mesoderm-derived adult progenitor cells to produce functional MEC progeny.

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.

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.

Reprogramming human cancer cells in the mouse mammary gland.

The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we showed that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescence in situ hybridization confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human-specific breast epithelial cytokeratins and human-specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66- to 660-fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results show that human embryonal carcinoma-derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.

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.

Reprogramming cell fates in the mammary microenvironment.

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 may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells.

Stem cells and the mammary microenvironment.

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct-limited and lobule-limited outgrowths were developed as well as complete, fully differentiated glands. Thus, transplantation has revealed three distinct mammary epithelial progenitors in the mouse. Recently, using cre-lox conditional activation of reporter genes, the lobule-limited progenitor was lineally marked by lacZ expression. In situ, these cells were shown to regenerate secretory lobules upon successive pregnancies. In transplant studies, they demonstrated the capacity for self- renewal and contributed to the new generation of all of the epithelial cell types among mammary secretory lobules. Using this conditional activation model, cells isolated from other tissues of the WAP-Cre/Rosa26/lacZReporter mice, co-mingled with normal wild type mammary epithelial cells and transplanted into epithelium-divested mammary fat pads, were shown to be amenable to redirection of their cell fate by interaction with the mammary microenvironment in vivo. This suggests the ascendancy of the microenvironment over the intrinsic nature of somatic stem cells.