Morphogenesis Of Mammary Epithelial Cells Research Articles

Species-specific control of hepatocyte growth factor expression and production in adipocytes in a differentiation-dependent manner

Hepatocyte growth factor (HGF) is a mesenchymal cell-derived factor that regulates cell growth, cell motility, and morphogenesis. Since there are conflicting reports on HGF-producing cells, we herein examined HGF activity in conditioned medium (CM) of bovine and mouse preadipocytes before and after adipogenic differentiation. CM of bovine adipocytes and mouse preadipocytes induced the morphogenesis of mammary epithelial cells that was inhibited by an NK4 HGF antagonist, whereas CM of bovine preadipocytes and mouse adipocytes did not. HGF mRNA expression was increased by a treatment with dexamethasone and isobutylmethylxanthine in bovine as well as human cells, whereas it was decreased in rodent cells. It was unfortunate that HGF gene promoter activity failed to reflect HGF mRNA expression in these cells. After actinomycin D treatment, expression of HGF mRNA remained stable in pre- and differentiated bovine adipocytes and mouse preadipocytes, whereas rapidly decreased in mouse-differentiated adipocytes. These results indicate that expression and production of HGF are regulated in a species-specific adipogenic differentiation-dependent manner and suggest that the decrease in HGF mRNA in mouse differentiated adipocytes is, at least in part, mediated by differentiation-dependent loss of its stability.

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Modular GAG-matrices to promote mammary epithelial morphogenesis invitro.

Matrix systems used to study complex three-dimensional (3D) cellular processes like mammary epithelial tissue morphogenesis and tumorigenesis exvivo often require ill-defined biological components, which lead to poor reproducibility and a lack of control over physical parameters. In this study, a well-defined, tunable synthetic biohybrid hydrogel composed of the glycosaminoglycan heparin, star-shaped poly(ethylene glycol) (starPEG), and matrix metalloproteinase- (MMP-) cleavable crosslinkers was applied to dissect the biophysical and biochemical signals promoting human mammary epithelial cell (MEC) morphogenesis. We show that compliant starPEG-heparin matrices promote the development of polarized MEC acini. Both the presence of heparin and MMP-cleavable crosslinks are essential in facilitating MEC morphogenesis without supplementation of exogenous adhesion ligands. In this system, MECs secrete and organize laminin in basement membrane-like assemblies to promote integrin signaling and drive acinar development. Therefore, starPEG-heparin hydrogels provide a versatile platform to study mammary epithelial tissue morphogenesis in a chemically defined and precisely tunable 3D invitro microenvironment. The system allows investigation of biophysical and biochemical aspects of mammary gland biology and potentially a variety of other organoid culture studies.

Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling.

Culturing mammary epithelial cells in laminin-rich extracellular matrices (three dimensional or 3D culture) offers significant advantages over that in the conventional two-dimensional (2D) tissue culture system in that it takes into considetation the impact of extracellular matrix (ECM) microenvironment on the proliferation, survival, and differentiation of mammary epithelial cells. When grown in the 3D culture, untransformed mammary epithelial cells undergo morphogenesis to form a multicellular and polarized acini-like structure that functionally mimics the differentiated alveoli in the pregnancy mammary gland. This process is subjected to regulation by many growth factors and cytokines. The transforming growth factor-ß (TGFß) is a multipotent cytokine that regulates multiple aspects of development and tumorigenesis. In addition to its effects on epithelial cell proliferation, survival, and differentiation, it is also a potent regulator of the cell-matrix interaction. Thus, the 3D culture model may recapitulate the complex in vivo epithelial cell microenvironment and allow us to fully evaluate the role of TGFß signaling in multiple aspects of normal and cancerous cell behavior. In this chapter we provide detailed protocols for growing mammary epithelial cells in the 3D Matrigel for analysis of signaling pathways.

A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells

We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function.

Loss of the BRCA1-Interacting Helicase BRIP1 Results in Abnormal Mammary Acinar Morphogenesis

BRIP1 is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1 and plays an important role in BRCA1-dependent DNA repair and DNA damage–induced checkpoint control. Recent studies implicate BRIP1 as a moderate/low-penetrance breast cancer susceptibility gene. However, the phenotypic effects of BRIP1 dysfunction and its role in breast cancer tumorigenesis remain unclear. To explore the function of BRIP1 in acinar morphogenesis of mammary epithelial cells, we generated BRIP1-knockdown MCF-10A cells by short hairpin RNA (shRNA)-mediated RNA interference and examined its effect in a three-dimensional culture model. Genome-wide gene expression profiling by microarray and quantitative RT-PCR were performed to identify alterations in gene expression in BRIP1-knockdown cells compared with control cells. The microarray data were further investigated using the pathway analysis and Gene Set Enrichment Analysis (GSEA) for pathway identification. BRIP1 knockdown in non-malignant MCF-10A mammary epithelial cells by RNA interference induced neoplastic-like changes such as abnormal cell adhesion, increased cell proliferation, large and irregular-shaped acini, invasive growth, and defective lumen formation. Differentially expressed genes, including MCAM, COL8A1, WIPF1, RICH2, PCSK5, GAS1, SATB1, and ELF3, in BRIP1-knockdown cells compared with control cells were categorized into several functional groups, such as cell adhesion, polarity, growth, signal transduction, and developmental process. Signaling-pathway analyses showed dysregulation of multiple cellular signaling pathways, involving LPA receptor, Myc, Wnt, PI3K, PTEN as well as DNA damage response, in BRIP1-knockdown cells. Loss of BRIP1 thus disrupts normal mammary morphogenesis and causes neoplastic-like changes, possibly via dysregulating multiple cellular signaling pathways functioning in the normal development of mammary glands.

P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.

PI3K regulates branch initiation and extension of cultured mammary epithelia via Akt and Rac1 respectively

The tree-like architecture of the mammary gland is generated by branching morphogenesis, which is regulated by many signals from the microenvironment. Here we examined how signaling downstream of phosphoinositide 3-kinase (PI3K) regulates different steps of mammary branching using three-dimensional culture models of the mammary epithelial duct. We found that PI3K was required for both branch initiation and elongation. Activated Akt was enhanced at branch initiation sites where its negative regulator, PTEN, was blocked by signaling via Sprouty2 (SPRY2); inhibiting Akt prevented branch initiation. The pattern of SPRY2 expression, and thus of Akt activation and branch initiation, was controlled by mechanical signaling from endogenous cytoskeletal contractility. In contrast, activated GTP-bound Rac1 localized to the leading edge of nascent branches and was required for branch elongation. These data suggest that the PI3K network integrates mechanical and biochemical signaling to control branching morphogenesis of mammary epithelial cells.

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.

The microRNA expression associated with morphogenesis of breast cancer cells in three-dimensional organotypic culture

Three-dimensional organotypic culture using reconstituted basement membrane matrix Matrigel (rBM 3-D) is an indispensable tool to characterize morphogenesis of mammary epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix (ECM). microRNAs (miRNAs) are a novel class of oncogenes and tumor suppressors. The majority of our current knowledge of miRNA expression and function in cancer cells is derived from monolayer 2-D culture on plastic substratum, which lacks consideration of the influence of ECM-mediated morphogenesis on miRNAs. In the present study, we compared the expression of miRNAs in rBM 3-D and 2-D cultures of the non-invasive MCF-7 and the invasive MDA-MB231 cells. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures within each cell type. Moreover, rBM 3-D culture exhibited greater discrimination in miRNA profiles between MCF-7 and MDA-MB231 cells than 2-D culture. The disparate miRNA profiles correlated with distinct mass morphogenesis of MCF-7 and invasive stellate morphogenesis of MDA-MB231 cells in rBM 3-D culture. Supplementation of the tumor promoting type I collagen in rBM 3-D culture substantially altered the miRNA signature of mass morphologenesis of MCF-7 cells in rBM 3-D culture. Overexpression of the differentially expressed miR-200 family member miR429 in MDA-MB231 cells attenuated their invasive stellate morphogenesis in rBM 3-D culture. In summary, we provide the first miRNA signatures of morphogenesis of human breast cancer cells in rBM 3-D culture and warrant further utilization of rBM 3-D culture in investigation of miRNAs in breast cancer.

P2-02-08: Int6 Regulates Both Proteasomal Degradation and Translation Initiation and Is Critical for Proper Formation of Acini by Human Mammary Epithelium.

Abstract The mammalian INT6/EIF3E gene has been implicated in breast tumor formation, but its functional activities remain poorly defined. Consistent with the fact that Int6 is frequently down-regulated in human breast tumors, we found that repressing INT6 expression induced transformed properties in normal human mammary epithelial cells (MCF10A) in both 2D and 3D cultures. Int6 contains a PCI domain, found in several components of both the proteasome and the translation initiation factor eIF3, suggesting that it can functionally interact with both. Indeed, our data show that Int6 associates with proteasomes in human cells, and that INT6 knockdown prevents proper assembly of active proteasomes. In addition, we show that down-regulation of INT6 reduces translation initiation (both polysome formation and global protein synthesis). However, Int6 regulates translation selectively, reducing translation from cap-dependent and Bcl2- IRES reporters, but stimulating translation from the CVB3 IRES. These data collectively suggest that Int6 controls both protein synthesis and protein degradation, and is thus capable of fine-tuning protein levels. Abnormal levels of regulatory proteins caused by Int6 abnormalities may thus be responsible for disrupting normal morphogenesis of mammary epithelial cells and causing transformed phenotypes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-08.

TRIM29 Functions as a Tumor Suppressor in Nontumorigenic Breast Cells and Invasive ER+ Breast Cancer

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women.

The Rho GTPase Cdc42 is required for primary mammary epithelial cell morphogenesis in vitro

The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is known about the role of Cdc42 during normal mammary epithelial cell (MEC) morphogenesis. Here we aimed to define the precise role for Cdc42 during primary mammary acinus formation in vitro. For these studies, MECs were isolated from Cdc42fl/fl conditional knockout mice, transduced with Adeno-cre-GFP virus to delete Cdc42 or Adeno-GFP control virus, and effects on morphogenesis were investigated using a three-dimensional (3D) culture assay. Interestingly, markedly fewer mammary acini developed in Cdc42 deficient cultures, and the acini that formed were significantly smaller and disorganized. Cellular proliferation and survival were reduced in the Cdc42 deficient acini. However, control and knockout MECs cultured as monolayers displayed similar cell cycle profiles, suggesting that Cdc42 is important for MEC proliferation in the context of 3D polarity. Overexpression of cyclin D1, which promotes cell cycle progression downstream of Cdc42, failed to rescue the defect in acinus size. Furthermore, lumen formation and apical-basal polarity were disrupted, and mitotic spindle orientation and Cdc42/aPKC polarity complex defects likely contributed to these phenotypes. Studies using dominant negative Cdc42 and siRNA to knockdown Cdc42 in MDCK and Caco-2 cell lines undergoing cystogenesis in 3D cultures revealed critical roles for Cdc42 in spindle orientation, polarity, and lumen formation. Our studies, using complete knockout in primary epithelial cells, demonstrate that Cdc42 is not only an important regulator of polarity and lumen formation; it is also essential for proliferation and survival, which are key cellular processes that drive MEC morphogenesis in vitro and in vivo.

Epithelial Cell Organization Suppresses Myc Function by Attenuating Myc Expression

c-Myc is an oncogene transcription factor that causes cancer in many settings, including solid tumors that arise in the context of organized tissue structures. Given that disruption of tissue architecture frequently occurs in cancer, there is considerable interest in how cell organization impacts oncogene function. A previous report found that organization of mammary epithelial cells into defined 3-dimensional structures renders them insensitive to the effects of retrovirus-mediated overexpression of Myc, leading to the notion that organization tempers the sensitivity of individual cells to Myc activity. In this article, we report that epithelial cell organization does not profoundly alter Myc activity but, instead, suppresses Myc by modulating its expression. We show that the morphogenesis of mammary epithelial cells into organized acinar structures in vitro is accompanied by widespread changes in gene expression patterns, including a substantial decrease in the expression of Myc. Concomitant with the decrease in endogenous Myc expression, we observe a decrease in transcription from retroviral vectors during morphogenesis and find that Myc transgene expression in acini is much lower than in unorganized cells. This decrease in Myc transgene activity is responsible for the apparent recalcitrance of organized cells to ectopic Myc, as adenovirus-mediated expression of Myc in organized structures potently induces apoptosis. These observations reveal that organization does not alter the inherent response of epithelial cells to Myc and suggest that other tumor suppression mechanisms, apart from structure, antagonize Myc in the development of solid tumors.

Adipose Stroma Induces Branching Morphogenesis of Engineered Epithelial Tubules

The mammary gland and other treelike organs develop their characteristic fractal geometries through branching morphogenesis, a process in which the epithelium bifurcates and invades into the surrounding stroma. Controlling the pattern of branching is critical for engineering these organs. In vivo, the branching process is instructed by stromal-epithelial interactions and adipocytes form the largest component of the fatty stroma that surrounds the mammary epithelium. Here, we used microlithographic approaches to engineer a three-dimensional culture model that enables analysis of the effect of adipocytes on the pattern of branching morphogenesis of mammary epithelial cells. We found that adipocyte-rich stroma induces branching through paracrine signals, including hepatocyte growth factor, but does not affect the branching pattern per se. This tissue engineering approach can be expanded to other organs, and should enable piecemeal analysis of the cellular populations that control patterning during normal development.

Prognostic Breast Cancer Signature Identified from 3D Culture Model Accurately Predicts Clinical Outcome across Independent Datasets

BackgroundOne of the major tenets in breast cancer research is that early detection is vital for patient survival by increasing treatment options. To that end, we have previously used a novel unsupervised approach to identify a set of genes whose expression predicts prognosis of breast cancer patients. The predictive genes were selected in a well-defined three dimensional (3D) cell culture model of non-malignant human mammary epithelial cell morphogenesis as down-regulated during breast epithelial cell acinar formation and cell cycle arrest. Here we examine the ability of this gene signature (3D-signature) to predict prognosis in three independent breast cancer microarray datasets having 295, 286, and 118 samples, respectively.Methods and FindingsOur results show that the 3D-signature accurately predicts prognosis in three unrelated patient datasets. At 10 years, the probability of positive outcome was 52, 51, and 47 percent in the group with a poor-prognosis signature and 91, 75, and 71 percent in the group with a good-prognosis signature for the three datasets, respectively (Kaplan-Meier survival analysis, p<0.05). Hazard ratios for poor outcome were 5.5 (95% CI 3.0 to 12.2, p<0.0001), 2.4 (95% CI 1.6 to 3.6, p<0.0001) and 1.9 (95% CI 1.1 to 3.2, p = 0.016) and remained significant for the two larger datasets when corrected for estrogen receptor (ER) status. Hence the 3D-signature accurately predicts breast cancer outcome in both ER-positive and ER-negative tumors, though individual genes differed in their prognostic ability in the two subtypes. Genes that were prognostic in ER+ patients are AURKA, CEP55, RRM2, EPHA2, FGFBP1, and VRK1, while genes prognostic in ER− patients include ACTB, FOXM1 and SERPINE2 (Kaplan-Meier p<0.05). Multivariable Cox regression analysis in the largest dataset showed that the 3D-signature was a strong independent factor in predicting breast cancer outcome.ConclusionsThe 3D-signature accurately predicts breast cancer outcome across multiple datasets and holds prognostic value for both ER-positive and ER-negative breast cancer. The signature was selected using a novel biological approach and hence holds promise to represent the key biological processes of breast cancer.

Overexpression of Activated Murine Notch1 and Notch3 in Transgenic Mice Blocks Mammary Gland Development and Induces Mammary Tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.

Regulation of Milk Protein Gene Expression in Normal Mammary Epithelial Cells by Tumor Necrosis Factor

Tumor necrosis factor-α (TNF) is a physiologically significant regulator of mammary gland development, stimulating growth and branching morphogenesis of mammary epithelial cells (MEC) and modulating functional differentiation. The present studies were performed to determine the mechanism by which TNF modulated functional differentiation. In rat MEC in primary culture, TNF inhibited accumulation of whey acidic protein and β-casein messenger RNAs in a time- and concentration-dependent manner. In contrast, levels of transferrin messenger RNA, the product of another milk protein gene, were not inhibited by TNF, suggesting selectivity. Using a nuclear run-on assay in the immortalized HC11 mammary epithelial cell line and the transcriptional inhibitor actinomycin D in MEC in primary culture, the effects of TNF were shown to be mediated by both a decrease in transcription and a decrease in the stability of the whey acidic protein and β-casein transcripts. Additionally, TNF stimulated the binding of nuclear factor-κB to a consensus κB-oligonucleotide, increased the stability of matrix metalloproteinase-9 (MMP-9) transcripts, and increased MMP-9 activity. Together, these data suggest that TNF may exert its effects on milk protein gene expression either directly via nuclear factor-κB modulation of transcription, or indirectly via MMP-9-induced remodeling of the architectural or hormonal environment surrounding the MEC.

Regulation of milk protein gene expression in normal mammary epithelial cells by tumor necrosis factor.

Tumor necrosis factor-alpha (TNF) is a physiologically significant regulator of mammary gland development, stimulating growth and branching morphogenesis of mammary epithelial cells (MEC) and modulating functional differentiation. The present studies were performed to determine the mechanism by which TNF modulated functional differentiation. In rat MEC in primary culture, TNF inhibited accumulation of whey acidic protein and beta-casein messenger RNAs in a time- and concentration-dependent manner. In contrast, levels of transferrin messenger RNA, the product of another milk protein gene, were not inhibited by TNF, suggesting selectivity. Using a nuclear run-on assay in the immortalized HC11 mammary epithelial cell line and the transcriptional inhibitor actinomycin D in MEC in primary culture, the effects of TNF were shown to be mediated by both a decrease in transcription and a decrease in the stability of the whey acidic protein and beta-casein transcripts. Additionally, TNF stimulated the binding of nuclear factor-kappaB to a consensus kappaB-oligonucleotide, increased the stability of matrix metalloproteinase-9 (MMP-9) transcripts, and increased MMP-9 activity. Together, these data suggest that TNF may exert its effects on milk protein gene expression either directly via nuclear factor-kappaB modulation of transcription, or indirectly via MMP-9-induced remodeling of the architectural or hormonal environment surrounding the MEC.

Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells.

Signals generated by the interaction of beta1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of beta1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly altered mouse mammary epithelial cell morphogenesis but it did not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibited both morphogenesis and differentiation. Overexpression of wild-type ILK also caused the cells to lose the cell-cell adhesion molecule E-cadherin, become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescued epithelial cytokeratin expression and it partially restored the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions.