Mammary Ductal Growth Research Articles

Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth

BackgroundAmphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth.MethodsWe crossed AREG-null (AREG−/−) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG−/− PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors.ResultsIntriguingly, PyMT-induced lesions progress more rapidly in AREG−/− mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG−/− mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG−/− PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas.ConclusionsOur study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.

Abstract 534: Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation

Abstract Background: Breast cancer is a heterogeneous disease encompassing multiple subtypes that differ in their molecular profiles, clinical course of progression, and response to therapeutics. Interestingly, breast cancers fall broadly into luminal and basal subtypes, which resemble the normal luminal and basal epithelial cells of the breast. This correlation suggests that the molecular mechanisms controlling epithelial cell lineage identity during normal mammary development may also influence tumor subtype during cancer formation. Since many breast cancers are driven by oncogenes that are important for normal mammary development, we hypothesize that dysregulated oncogene signaling may alter mammary epithelial cell lineage identity at an early step of cancer initiation, priming the breast tumor subtype that will be formed upon full tumorigenesis. Methods: Human breast cell lines, MCF10a and MCF10f, and primary human breast tissues were infected with virus expressing single breast cancer oncogenes. Flow cytometry and quantitative RT-PCR were performed on breast cell lines to determine changes in the balance of luminal to basal cells and expression of luminal or basal gene sets. Primary tissues were grown in hydrogels and stained for luminal and basal markers to determine the effect of oncogene expression on the growth of mammary ductal structures. Mouse tumor models, MMTV-Myc and MMTV-Wnt1, were investigated for changes in their mammary epithelial architecture by histology and flow cytometry at ages prior to tumor formation. Results: Oncogene overexpression in human breast cell lines causes shifts in basal and luminal cell balance that correlate with changes seen in mammary ductal growth in hydrogel cultures of primary human breast epithelial cells. In mouse models, individual oncogenes alter mammary epithelial architecture and cell balance at early stages prior to tumor formation. In both models, individual oncogenes are able to regulate mammary epithelial cell lineage identity in the absence of other tumor-initiating signals, indicating that oncogenes may influence breast cancer subtype through mammary epithelial cell lineage regulation at early stages of cancer progression. Citation Format: Jerrica Breindel, Felix Nampanya, Aditya Datye, Daniel H. Miller, Piyush Gupta, Charlotte Kuperwasser. Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 534.

Genetic variation in sensitivity to estrogens and breast cancer risk.

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17β-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17β-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17β-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer.

The histone demethylase Kdm3a is required for normal epithelial proliferation, ductal elongation and tumor growth in the mouse mammary gland.

Histone modification alters chromatin architecture to regulate gene transcription. KDM3A is a histone demethylase in the JmjC domain-containing protein family. It removes di- and mono- methyl residues from di- or mono-methylated lysine 9 of histone H3 (H3K9me2/me1). Recent studies have shown that Kdm3a plays an important role in self-renewal of embryonic stem cells, spermatogenesis, metabolism, sex determination and tumor angiogenesis. However, its role in mammary gland development and breast carcinogenesis remains unclear. In this study, we found that Kdm3a is expressed in the mouse mammary gland epithelial cells. Knockout of Kdm3a significantly increased H3K9me2/me1 levels in these epithelial cells, which correlated with markedly decreased mammary gland ductal elongation and branching in the intact knockout virgin mice. Furthermore, estrogen replacement in the ovariectomized Kdm3a knockout mice couldn’t rescue the retarded ductal growth. Moreover, transplantation of KO mammary gland pieces to wild type recipient mice showed slower ductal growth compared with that of WT gland pieces. Consistently, knockout of Kdm3a also reduced the proliferation rates and cyclin D1 expression in the mammary gland epithelial cells. In addition, Kdm3a knockout did not significantly change the latency of the polyoma middle T oncogene-induced mammary gland tumorigenesis. Tumor growth, however, was slowed which might be due to the decrease in cyclin D1 expression and tumor cell proliferation. We also found that Kdm3a binds and activates the cyclin D1 promoter. These results demonstrate that Kdm3a plays an important intrinsic role in promoting mammary gland ductal growth and tumor growth probably through enhancing cyclin D1 expression and cell proliferation.

New insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen’s direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E)14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 β-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 μM BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.

Trans-Fatty Acid-Stimulated Mammary Gland Growth in Ovariectomized Mice is Fatty Acid Type and Isomer Specific.

We previously reported that the trans-18:2 fatty acid trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulates mammary gland development independent of estrogen and its receptor. Given the negative consequences of dietary trans-fatty acids on various aspects of human health, we sought to establish whether other trans-fatty acids could similarly induce ovary-independent mammary gland growth in mice. Prepubertal BALB/cJ mice were ovariectomized at 21 days of age then were fed diets enriched with cis-9, trans-11 CLA (c9,t11-CLA), or mixtures of trans-18:1 fatty acids supplied by partially hydrogenated sunflower, safflower, or linseed oil. The resultant mammary phenotype was evaluated 3 weeks later and compared to the growth response elicited by t10,c12-CLA, or the defined control diet. Whereas partially hydrogenated safflower oil increased mammary gland weight, none of the partially hydrogenated vegetable oils promoted mammary ductal growth. Similarly, the c9,t11-CLA supplemented diet was without effect on mammary development. Taken together, our data emphasize a unique effect of t10,c12-CLA in stimulating estrogen-independent mammary gland growth manifest as increased mammary ductal area and elongation that was not recapitulated by c9,t11-CLA or the partially hydrogenated vegetable oil diets.

Artificial Induction of Lactation in Bovines: Scope and Limitations

Estrogen, progesterone, prolactin and glucocorticoids work synergistically at the onset of lactogenesis. Estrogen stimulates mammary ductal growth, and the combination of estrogen and progesterone regulates lobular-alveolar development of the mammary gland. This development does not occur in infertile animal or animals suffering from reproductive disorders due to relative absence of sex steroid hormones. Artificial induction of lactation can be induced in such animals by the combination of steroid hormones estrogen and progesterone. Additionally reserpine (tranquilizer- increases prolactin level) and dexamethasone (a synthetic glucocorticoid) can be used in induction protocols to increase the success rate. Artificial induction reduces the involuntary culling rate as well as the genetic losses. To save such genetically superior bovines, induction of lactation is the most appropriate technology used under Indian conditions. The treatment is more effective in well-fed animals with good body condition score and having normal teat and udder. Hormonal induction of lactation should be practice only in infertile animals; it should not be followed as a routine practice, as an alternate to normal lactation. Estrogen and progesterone in 1:2.5 ratios is effective in exotic and cross bred while 1:1 ratio is effective in indigenous cows and buffaloes for artificial induction because of short luteal phase. These methods of artificial induction of lactation yield 60 to 70% milk of previous lactation.

Dysregulation of Notch and ERα signaling in AhR−/− male mice

The aryl hydrocarbon receptor (AhR) is now recognized as an important physiological regulator in the immune and reproductive systems, and in the development of the liver and vascular system. AhR regulates cell cycle, cell proliferation, and differentiation through interacting with other signaling pathways, like estrogen receptor α (ERα), androgen receptor (AR), and Notch signaling. In the present study, we investigated Notch and estrogen signaling in AhR-/- mice. We found low fertility with degenerative changes in the testes, germ cell apoptosis, and a reduced number of early spermatids. There was no change in aromatase, AR, ERα, or ERβ expression in the testis and no detectable change in serum estrogen levels. However, expression of Notch receptors (Notch1 and Notch3) and their target Hairy and Enhancer of Split homolog 1 (HES1) was reduced. In addition, the testosterone level was slightly reduced in the serum. In the mammary fat pad, AhR appeared to regulate estrogen signaling because, in AhR-/- males, there was significant growth of the mammary ducts with high expression of ERα in the ductal epithelium. The enhanced mammary ductal growth appears to be related to overexpression of ERα accompanied by a high proliferation index, whereas the reduced fertility appears to be related defects in Notch signaling that leads to reduced expression of HES1 and, consequently, early maturation of spermatocytes and a depletion of primary spermatids. Previous reports have indicated that AhR pathway is associated with infertility in men. Our results provide a mechanistic explanation for this defect.

Estetrol is a weak estrogen antagonizing estradiol-dependent mammary gland proliferation.

Estetrol (E4) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol and estradiol (E2), E4 has a minimal impact on liver cell activity and could provide a better safety profile in contraception or hormone therapy. The aim of this study was to delineate if E4 exhibits an activity profile distinct from that of E2 on mammary gland. Compared with E2, E4 acted as a low-affinity estrogen in both human in vitro and murine in vivo models. E4 was 100 times less potent than E2 to stimulate the proliferation of human breast epithelial (HBE) cells and murine mammary gland in vitro and in vivo respectively. This effect was prevented by fulvestrant and tamoxifen, supporting the notion that ERα (ESR1) is the main mediator of the estrogenic effect of E4 on the breast. Interestingly, when E4 was administered along with E2, it significantly antagonized the strong stimulatory effect of E2 on HBE cell proliferation and on the growth of mammary ducts. This study characterizes for the first time the impact of E4 on mammary gland. Our results highlight that E4 is less potent than E2 and exhibits antagonistic properties toward the proliferative effect of E2 on breast epithelial cells. These data support E4 as a potential new estrogen for clinical use with a reduced impact on breast proliferation.

Androgen receptor inactivation resulted in acceleration in pubertal mammary gland growth, upregulation of ERα expression, and Wnt/β-catenin signaling in female mice.

The androgen receptor (AR) is widely expressed in mammary cells of female mammals including humans and mice, indicating a possible role for AR-mediated androgen actions in breast development, function, and pathology, although the specific mechanisms remain unclear. To elucidate the mechanisms of androgen action in mammary gland physiology and development, we used AR-knockout (AR(Δex3)KO) female mice with a universally expressed, transcriptionally inactive AR protein harboring an in-frame deletion of its second zinc finger. Although in sexually mature wild-type (WT) and AR(ex3Δ)KO females, the mammary epithelial growth was fully extended to the edge of the fat pad, during puberty, AR(ex3Δ)KO females exhibit significantly accelerated mammary ductal growth and an increased number of terminal end buds compared with WT females. Accelerated AR(ex3Δ)KO female mammary growth was associated with significantly increased mammary epithelial ERα expression and activated Wnt/β-catenin signaling as shown by increased Wnt4 expression and accumulation of nuclear β-catenin. These findings are consistent with increased mammary estrogen exposure although ovarian estradiol content was unchanged compared with WT females. Furthermore, treatment with the potent pure androgen DHT markedly reduced ductal extension and terminal end bud numbers in WT but not in AR(Δex3)KO females, further supporting the concept that AR-mediated, androgen-induced suppression of murine mammary growth is a physiological characteristic of puberty. In summary, our findings reveal an inhibitory role of AR-mediated androgen actions in pubertal mammary gland development by reducing epithelial cell proliferation and could be mediated by regulation of Wnt/β-catenin signaling.

Genetic Control of Ductal Morphology, Estrogen-Induced Ductal Growth, and Gene Expression in Female Mouse Mammary Gland

The uterotropic response of the uterus to 17β-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high (C57BL6/J; B6) or low (C3H/HeJ; C3H) responders to E2 led to the identification of quantitative trait loci (QTL) associated with phenotypic variation in uterine growth and leukocyte infiltration. Like the uterus, phenotypic variation in the responsiveness of the mammary gland to E2 during both normal and pathologic conditions has been reported. In the current experiment, we utilized an E2-specific model of mammary ductal growth combined with a microarray approach to determine the degree to which genotype influences the responsiveness of the mammary gland to E2, including the associated transcriptional programs, in B6 and C3H mice. Our results reveal that E2-induced mammary ductal growth and ductal morphology are genetically controlled. In addition, we observed a paradoxical effect of mammary ductal growth in response to E2 compared with what has been reported for the uterus; B6 is a high responder for the uterus and was a low responder for mammary ductal growth, whereas the reverse was observed for C3H. In contrast, B6 was a high responder for mammary ductal side branching. The B6 phenotype was associated with increased mammary epithelial cell proliferation and apoptosis, and a distinct E2-induced transcriptional program. These findings lay the groundwork for future experiments designed to investigate the genes and mechanisms underlying phenotypic variation in tissue-specific sensitivity to systemic and environmental estrogens during various physiological and disease states.

Prenatal Bisphenol A exposure delays the development of the male rat mammary gland

Our aims were to evaluate whether exposure to Bisphenol A (BPA) modifies the development of the male rat mammary gland (MG) and to evaluate whether this modification is gender specific. From gestational day 9, pregnant rats were exposed either subcutaneously to 0, 25 or 250μg BPA/kgbw/day until parturition or orally to 0 and 64μg BPA/kgbw/day until weaning. MG development was analyzed on postnatal days (PND) 5, 15 and 30. On PND30, steroid hormone receptor expression and mammary growth were also evaluated. On PND30, the exposure to 64BPA and 250BPA induced a delay in male MG development, evidenced by reduced ductal growth, decreased number of terminal structures and lower expression of androgen receptor (AR). In contrast, female mammary ductal growth was altered only by 250BPA. Regardless of the administration route and length of the exposure period, BPA induced a delay in MG development and modified AR expression in prepubertal male rats.

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.

Characterization of WWOX inactivation in murine mammary gland development

The WW domain-containing oxidoreductase (WWOX) is commonly inactivated in multiple human cancers, including breast cancer. Wwox null mice die prematurely precluding adult tumor analysis. Nevertheless, aging Wwox-heterozygous mice at C3H genetic background develop higher incidence of mammary tumors. We recently generated a Wwox conditional knockout mouse in which loxp sites flank exon 1 in the Wwox allele and showed that total ablation of WWOX in these mice resembles that of conventional targeting of Wwox. Here, we report the characterization of WWOX ablation in mouse mammary gland using MMTV-Cre transgenic line. We demonstrated that WWOX ablation leads to impaired mammary ductal growth. Moreover, targeted deletion of WWOX is associated with increased levels of fibronectin, a component of the extracellular matrix. In addition, we showed that shRNA knockdown of WWOX in MCF10A breast epithelial cells dramatically increased fibronectin and is associated with enhanced cell survival and impaired growth in three-dimensional culture Matrigel assay. Taken together our results are consistent with a critical role for WWOX in normal breast development and tumorigenesis.

Effect of exogenous somatotropin and staged ovariectomy on mRNA expression of select ECM-related proteins in mammary tissue of prepubertal Holstein calves

Growth hormone (GH) and estrogen are essential stimulators of mammary cell proliferation and mammary development as mammals near puberty. Mammary ductal growth requires modifications of the extracellular matrix (ECM) for this tissue expansion to occur. Our purpose was to evaluate the effects of exogenous GH and ovariectomy (known to impact estrogen production) on gene expression of selected ECM proteins in the mammary parenchyma (PAR) and mammary fat pad (MFP) of prepubertal calves. Our hypothesis was that both GH and ovariectomy would alter the mRNA expression of multiple mammary ECM proteins. However, treatment with GH significantly reduced the expression of only fibronectin in PAR. However, the mRNA expression of all of the ECM proteins tested was numerically lower in PAR from GH treated calves. In contrast, staged ovariectomy decreased expression of fibronectin and heat shock protein 90 but increased expression of epimorphin in mammary PAR. In the MFP expression of Rac-1 and fascin were increased. These findings suggest that effects of exogenous GH on mammary gland composition are only marginally dependent on alterations in ECM proteins but the more pronounced effects of ovariectomy (reduced PAR mass and altered myoepithelial ontogeny) are more likely linked to changes in expression of ECM proteins.

P4-04-01: Identification of a Unique Mammary Cell Type Expressing Mesenchymal Markers, but Capable of Multilineage Epithelial Differentiation.

Abstract We have identified a previously uncharacterized cell population within the epithelial compartment of the post-pubertal mouse mammary gland. These cells can be found as singlets or as like pairs in both the cap and body cell layer of terminal end buds (TEB) of developing ducts, and are ultimately localized in a suprabasal position distributed throughout the mature mammary ductal tree. In differentiated ducts, these cells typically extend long processes that contact multiple surrounding luminal epithelial and myoepithelial cells suggesting a function in cell-cell communication. In younger animals, identified cells do not express epithelial markers including the major cytokeratins (CK) 5, 6, 8, and 14, or E-cadherin, nor do they express myoepithelial cell markers such as smooth muscle actin or p63. However, in 20 week old non-parous females, these cells can give rise to epithelial cell types expressing CK 5, 6, 8, or 14. In transplantation assays, epithelial fragment and whole mammary gland transplantation did not indicate a major role in regenerative growth of mammary ducts. Transplantation of dissociated epithelial cells and wound-healing assays are in progress. Taken together, these observations suggest that this unique cell population possess stem cell-like features in that they are capable of self-renewal, and of giving rise to all major differentiated cell types within the mouse mammary gland. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-04-01.

Mammary Ductal Growth Is Impaired in Mice Lacking Leptin-Dependent Signal Transducer and Activator of Transcription 3 Signaling

Mice lacking leptin (ob/ob) or its full-length receptor (db/db) are obese and reproductively incompetent. Fertility, pregnancy, and lactation are restored, respectively, in ob/ob mice treated with leptin through mating, d 6.5 post coitum, and pregnancy. Therefore, leptin signaling is needed for lactation, but the timing of its action and the affected mammary process remain unknown. To address this issue, we used s/s mice lacking only leptin-dependent signal transducer and activator of transcription (STAT)3 signaling. These mice share many features with db/db mice, including obesity, but differ by retaining sufficient activity of the hypothalamic-pituitary-ovarian axis to support reproduction. The s/s mammary epithelium was normal at 3 wk of age but failed to expand through the mammary fat pad (MFP) during the subsequent pubertal period. Ductal growth failure was not corrected by estrogen therapy and did not relate to inadequate IGF-I production by the MFP or to the need for epithelial or stromal leptin-STAT3 signaling. Ductal growth failure coincided with adipocyte hypertrophy and increased MFP production of leptin, TNFalpha, and IL6. These cytokines, however, were unable to inhibit the proliferation of a collection of mouse mammary epithelial cell lines. In conclusion, the very first step of postnatal mammary development fails in s/s mice despite sufficient estrogen IGF-I and an hypothalamic-pituitary-ovarian axis capable of supporting reproduction. This failure is not caused by mammary loss of leptin-dependent STAT3 signaling or by the development of inflammation. These data imply the existence of an unknown mechanism whereby leptin-dependent STAT3 signaling and obesity alter mammary ductal development.

Tissue‐Specific Roles of MED1/TRAP220 Nuclear Receptor‐Interacting LxxLL Motifs in vivo

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand‐dependent interactions of the MED1 (a.k.a. TRAP220) subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is no information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Towards this end, we have generated MED1 LxxLL motif‐mutant knockin mice. Interestingly, these mice are both viable and fertile, and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. This was manifested as a significant impairment in duct elongation and branching morphogenesis and a decrease in mammary epithelial cell proliferation in the mutant mice. Consistent with this phenotype, as well as a loss of the strong ligand‐dependent estrogen receptor (ER)α‐Mediator interaction, expression of a number of known ERα‐regulated genes was down‐regulated in MED1‐mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen‐stimulated mammary duct growth in MED1‐mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor‐ and cell‐ specific in vivo role for MED1 LxxLL motifs, through Mediator‐ERα interactions, in mammary gland development.

Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)alpha-Mediator interaction, expression of a number of known ERalpha-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERalpha interactions, in mammary gland development.

Profile of estrogen‐responsive genes in an estrogen‐specific mammary gland outgrowth model

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen-induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17beta-estradiol from two to 28 days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: (a) gene expression changes in our estrogen-only model mimic those changes that occur in normal pubertal development in intact mice, (b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and (c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol-responsive genes that are also co-expressed with estrogen receptor alpha in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development.