The mammary gland microenvironment during postlactational involution shares similarities with inflammation, including high matrix metalloproteinase activity, fibrillar collagen deposition, and release of bioactive fragments of fibronectin and laminin. Because inflammation can promote tumorigenesis, we evaluated whether the tissue microenvironment of the involuting gland is also promotional. Extracellular matrix was isolated from mammary glands of nulliparous rats or rats with mammary glands undergoing weaning-induced involution. Using these matrices as substratum, nulliparous matrix was found to promote ductal organization of normal mammary epithelial MCF-12A cells in three-dimensional culture and to suppress invasion of mammary tumor MDA-MB-231 cells in transwell filter assays. Conversely, involution matrix failed to support ductal development in normal cells and promoted invasiveness in tumor cells. To evaluate the effects of these matrices on metastasis in vivo, MDA-MB-231 cells, premixed with Matrigel, nulliparous matrix, or involution matrix, were injected into mammary fat pads of nude mice. Metastases to lung, liver, and kidney were increased in the involution matrix group, and correlated with a twofold increase in tumor vascular endothelial growth factor expression and increased angiogenesis. These data suggest that the mammary gland microenvironment becomes promotional for tumor cell dissemination during involution, thus providing a plausible mechanism to explain the high rate of metastases that occur with pregnancy-associated breast cancer.

A GC-rich oligonucleotide containing an estrogen responsive element (ERE) half-site from the heat shock protein 27 (Hsp 27) gene promoter (-105 to -84) [ie. GGGCGGG(N)10GGTCA; Sp1(N)10ERE] forms a complex with the Sp1 and estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs containing this sequence (-108 to -84 or -108 to +23) are also estrogen-responsive. Mutation of the ERE half-site in the Hsp 27-derived oligonucleotides did not result in loss of estrogen responsiveness in transient transfection studies, suggesting that estrogen inducibility was mediated through the Sp1-DNA motif. Gel mobility shift assays using 32P-labeled wild type and ERE mutant Sp1(N)10ERE and consensus Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein complex with all three nucleotides, and the intensities of retarded bands were enhanced by coincubation with wild type ER and 11C-ER, which does not contain the DNA-binding domain. ER mutants in which N-terminal (19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance Sp1-DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas both wild type and 11C-ER restored inducibility. Immunoprecipitation studies also confirmed that the Sp1 and ER proteins physically interact. The interaction of the Sp1 and ER proteins and the resulting enhanced Sp1-DNA binding is observed in the presence or absence of estrogen (hormone-independent), whereas transactivation of promoter-reporter constructs is estrogen-dependent. Thus, the results illustrate a new estrogen-dependent transactivation pathway that involves ER-protein interactions and is ERE-independent.

17␤-Estradiol (E2) induces cathepsinThe aryl hydrocarbon (Ah) receptor protein is expressed in laboratory animals and humans and in mammalian cells in culture (54,65). Although the endogenous ligand(s) for this receptor has not been identified, several different classes of compounds reversibly bind the Ah receptor, and these include the polynuclear and polyhalogenated aromatic hydrocarbons (42,54,57,59,65,67). 3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are prototypical aromatic hydrocarbons which exhibit high-affinity binding (K d Յ 1 nM) for the Ah receptor (44, 58) and have been utilized for characterizing Ah receptor-mediated responses.TCDD induces a broad spectrum of biochemical and toxic responses including a wasting syndrome, immune suppression, hepatotoxicity, developmental and reproductive toxicity, carcinogenicity, dermal toxicity, alteration of endocrine response pathways, and modulation of diverse enzyme activities (26,29,59,73,82). The induction of CYP1A1 gene expression by TCDD and 3-methylcholanthrene has been extensively investigated, and the results indicate that the Ah receptor acts as a nuclear ligand-induced transcription factor (13,29,73,82).After treatment of animals or cells with TCDD, there is a rapid formation of a heterodimeric nuclear Ah receptor complex (19) which consists of the ligand-binding protein (7) and the Ah receptor nuclear translocator (Arnt) protein (31). The unbound Ah receptor is associated with heat shock protein 90 (48,55,60,61), and the subcellular distribution of these proteins in the absence of ligand is currently being investigated (60, 61). The nuclear Ah receptor complex interacts with cognate genomic sequences (dioxin or xenobiotic responsive elements [DREs and XREs, respectively]) in the 5Ј-promoter region of the CYP1A1 gene and transactivates CYP1A1 gene expression (15-17, 27, 34, 70, 83). There is evidence that this transactivation process is comparable for induction of CYP1A1 gene expression and induction of the expression of other members of the Ah gene battery, namely CYP1A2, glucuronosyl transferase, aldehyde-3-dehydrogenase, glutathione S-transferase Ya gene, and NAD(P)H:menadione oxidoreductase (1,35,53,56,62,64). Other studies also report that enhanced expression of other genes by TCDD is due to posttranscriptional processes (20).TCDD also decreases gene expression and/or the respective activities of the encoded proteins. Phosphoenolpyruvate carboxy kinase, glucose-6-phosphatase, and tryptophan 2,3-dioxygenase activities and mRNA levels are decreased in rats treated with an acutely toxic dose (125 g/kg of body weight) of TCDD (72). Rat uterine c-fos and epidermal growth factor * Corresponding author. Mailing address: Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466.

The short splice variant of the basic helix-loop-helix Per-Arnt-Sim transcription factor Singleminded-2, SIM2s, has been implicated in development and is frequently lost or reduced in primary breast tumors. Here, we show that loss of Sim2s causes aberrant mouse mammary gland ductal development with features suggestive of malignant transformation, including increased proliferation, loss of polarity, down-regulation of Ecadherin, and invasion of the surrounding stroma. Additionally, knockdown of SIM2s in MCF-7 breast cancer cells contributed to an epithelial-mesenchymal transition (EMT) and increased tumorigenesis. In both Sim2؊/؊ mammary glands and SIM2s-depleted MCF7 cells, these changes were associated with increased SLUG and MMP2 levels. SIM2s protein was detectable on the SLUG promoter, and overexpression of SIM2s repressed expression from a SLUG-controlled reporter in a dose-dependent manner. To our knowledge, SIM2s is the first protein shown to bind and repress the SLUG promoter, providing a plausible explanation for the development role and breast tumor-suppressive activity of SIM2s. Together, our results suggest that SIM2s is a key regulator of mammary-ductal development and that loss of SIM2s expression is associated with an invasive, EMT-like phenotype.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.

The epithelial-mesenchymal transition (EMT) bestows cancer cells with increased stem cell properties and metastatic potential. To date, multiple extracellular stimuli and transcription factors have been shown to regulate EMT. Many of them are not druggable and therefore it is necessary to identify targets, which can be inhibited using small molecules to prevent metastasis. Recently, we identified the ganglioside GD2 as a novel breast cancer stem cell marker. Moreover, we found that GD3 synthase (GD3S)—an enzyme involved in GD2 biosynthesis—is critical for GD2 production and could serve as a potential druggable target for inhibiting tumor initiation and metastasis. Indeed, there is a small-molecule known as triptolide that has been shown to inhibit GD3S function. Accordingly, in this manuscript, we demonstrate that the inhibition of GD3S using shRNA or triptolide compromises the initiation and maintenance of EMT instigated by various signaling pathways, including Snail, Twist and TGF-β1 as well as the mesenchymal characteristics of claudin-low breast cancer cell lines (SUM159 and MDA-MB-231). Moreover, GD3S is necessary for wound healing, migration, invasion and stem cell properties in vitro. Most importantly, inhibition of GD3S in vivo prevents metastasis in experimental as well as in spontaneous syngeneic wild-type mouse models. We also demonstrate that the transcription factor FOXC2, a central downstream mediator/effector of several EMT pathways, directly regulates GD3S expression by binding to its promoter. In clinical specimens, the expression of GD3S correlates with poor prognosis in triple negative human breast tumors. Moreover, GD3S expression correlates with activation of the c-Met signaling pathway leading to increased stem cell properties and metastatic competence. Collectively, these findings suggest that the GD3S-c-Met axis could serve as an effective target for the treatment of metastatic breast cancers.

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A GC-rich oligonucleotide containing an estrogen responsive element (ERE) half-site from the heat shock protein 27 (Hsp 27) gene promoter (-105 to -84) [ie. GGGCGGG(N)10GGTCA; Sp1(N)10ERE] forms a complex with the Sp1 and estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs containing this sequence (-108 to -84 or -108 to +23) are also estrogen-responsive. Mutation of the ERE half-site in the Hsp 27-derived oligonucleotides did not result in loss of estrogen responsiveness in transient transfection studies, suggesting that estrogen inducibility was mediated through the Sp1-DNA motif. Gel mobility shift assays using 32P-labeled wild type and ERE mutant Sp1(N)10ERE and consensus Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein complex with all three nucleotides, and the intensities of retarded bands were enhanced by coincubation with wild type ER and 11C-ER, which does not contain the DNA-binding domain. ER mutants in which N-terminal (19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance Sp1-DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas both wild type and 11C-ER restored inducibility. Immunoprecipitation studies also confirmed that the Sp1 and ER proteins physically interact. The interaction of the Sp1 and ER proteins and the resulting enhanced Sp1-DNA binding is observed in the presence or absence of estrogen (hormone-independent), whereas transactivation of promoter-reporter constructs is estrogen-dependent. Thus, the results illustrate a new estrogen-dependent transactivation pathway that involves ER-protein interactions and is ERE-independent.