Mammary Tumor Virus-long Terminal Repeat Research Articles

Mammary epithelium‐specific conditional expression of human mutant Ki‐Ras in mice generates estrogen receptor alpha (ERα)‐positive mammary adenocarcinomas

BackgroundSeveral genetically modified mouse models (GEMMs) have been generated for mechanistic and therapeutic studies of mammary tumors. Although a number of cancer GEMMs have been developed, an individual model may recapitulate only some aspects of this complex disease. Indeed, breast cancer features include at least five biologically and clinically distinct intrinsic subtypes, including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)‐enriched, basal‐like, and normal‐like, with luminal A and B subtypes representing ~ 70% of breast cancers, and thus being of major clinical interest. We have engineered a novel transgenic mouse line expressing human Ki‐Ras bearing an activating mutation (Ki‐Ras(G12V)) selectively in the mammary epithelium and characterized them.MethodsTo allow conditional Ki‐Ras(G12V) expression in mammary epithelium, we generated C‐Ki‐Ras(G12V) transgenic mice that express, after Cre‐mediated DNA recombination during lactation, Ki‐Ras(G12V) under the control of the mammary tumor virus long terminal repeat (MMTV‐LTR) promoter. Tumor development was followed in 48 females after the start of lactation. Tumor characterization was performed by histopathological analysis, immunohistochemistry and real time PCR. Two cell lines were isolated from mouse mammary tumors and their phenotypes were evaluated by immunoblotting, ‘in vitro’ cell growth assays and ‘in vivo’ tumor xenograft studies.ResultsAll mice developed homogenous invasive ductal adenocarcinomas with 100% incidence within 13–35 weeks after Ki‐Ras(G12V) expression. Immunohistochemical analysis revealed high levels of estrogen receptor (ER) α in more than 90% of epithelial cells and focal progesterone receptor (PR) staining in neoplastic epithelial cells, while no HER2 staining was observed. Mammary tumor cells exhibited less than 14% nuclei positive for the proliferation marker Ki‐67, a feature that can segregate the luminal A subtype. The cytokeratin profile, as well as quantification of transcript levels of specific markers in the tumor cells, also reflected a luminal cell phenotype. Cell lines isolated from such tumors have similar characteristics as the primary tumors, are estrogen‐responsive and transplantable. Importantly, palpable tumors were detected faster than those derived from the human luminal breast cancer MCF‐7 cells (one week versus one month).ConclusionsMG‐Ki‐Ras(G12V) mice develop within a couple of months at full penetrance mammary tumors that recapitulate the most common human breast cancer subtype, classified as ductal ER‐positive invasive adenocarcinoma. Thus, this animal model together with the derived primary cancer cell line represent valuable tools with high potential of translational relevance to investigate the biology of estrogen‐dependent mammary tumors and to test various therapeutic agents.Support or Funding InformationThis work was supported by: PRIN2015 #2015B7M39T (to SA), Fondazione Italiana per la Ricerca sul Cancro (AIRC) grants: IG no. 11595 (to SA), MFAG no. 16899 (to IB), PROGRAMMA ‘FUTURO IN RICERCA’ Anno 2012 no.RBFR12FI27 (to IB), AIRC Fellowships for abroad no. 19552 (to DR), EMBO ASTF no. 18‐2010 (to RM), European Social Fund operational programme of the Calabria region (to SP), Faculté de Médecine, Université de Strasbourg (to CE) and by the Centre Nationalpour la Recherche Scientifique (CNRS), the Institut National de la Santé et de laRecherche Médicale (INSERM), the Université de Strasbourg and French state funds through the Agence Nationale de la Recherche ANR‐10‐LABX‐0030‐INRT under the frame programme Investissements d'Avenir labelled ANR‐10‐IDEX‐0002‐02.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the hsp70 gene delays mammary tumor initiation and inhibits tumor cell metastasis

Elevated levels of the inducible heat-shock protein 70 (Hsp72) have been implicated in mammary tumorigenesis in histological investigations of human breast cancer. We therefore examined the role of Hsp72 in mice, using animals in which the hsp70 gene was inactivated. We used a spontaneous tumor system with mice expressing the polyomavirus middle T (PyMT) oncogene under control of the mouse mammary tumor virus (MMTV) long-terminal repeat (MMT mice). These mice developed spontaneous, metastatic mammary cancer. We then showed Hsp72 to be upregulated in a fraction of mammary cancer initiating cells (CIC) within the MMT tumor cell population. These cells were characterized by elevated surface levels of stem cell markers CD44 and Sca1 and by rapid metastasis. Inactivation of the hsp70 gene delayed the initiation of mammary tumors. This delay in tumor initiation imposed by loss of hsp70 was correlated with a decreased pool of CIC. Interestingly, hsp70 knockout significantly reduced invasion and metastasis by mammary tumor cells and implicated its product Hsp72 in cell migration and formation of secondary neoplasms. Impaired tumorigenesis and metastasis in hsp70-knockout MMT mice was associated with downregulation of the met gene and reduced activition of the oncogenic c-Met protein. These experiments therefore showed Hsp72 to be involved in the growth and progression of mammary carcinoma and highlighted this protein as a potential target for anticancer drug development.

Response to fish specific reproductive hormones and endocrine disrupting chemicals of a Sertoli cell line expressing endogenous receptors from an endemic cyprinid Gnathopogon caerulescens

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20β-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.

Does Plasminogen Activator Inhibitor-1 Drive Lymphangiogenesis?

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.

Mouse Mammary Tumor Virus p75 and p110 CUX1 Transgenic Mice Develop Mammary Tumors of Various Histologic Types

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the beta-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA-mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/beta-catenin autocrine loop.

Abstract B70: A conditional aromatase over-expressing in the mammary gland transgenic mouse as a potential model in cancer prevention research

Abstract B70 Background Breast cancer develops as genetic changes accumulate in the ductal epithelium giving rise to precursor lesions such as atypical ductal hyperplasias, which may progress to ductal carcinoma in situ (DCIS) and eventually invasive breast cancer. This progress is promoted by estrogens. Estrogens are biosynthesized from androgens by the aromatase enzyme. Estrogen is required for the proliferation and morphogenesis of the normal mammary gland; however, studies suggest that estrogen and its metabolites may have mutagenic and carcinogenic potential in the mammary gland. Therefore, it is highly valuable to have mouse models with increased local estrogen production to use to develop optimal therapeutic and chemopreventive strategies to treat these lesions. Purpose The purpose of this work is to develop a conditional transgenic mouse model that would provide an in vivo method of controlling the spatial and temporal regulation of aromatase expression. Methodology The transgenic mouse model was developed by using two transgenes. One transgene is composed of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) linked to sequences encoding the tetracycline responsive reverse transactivator (rtTA). The other transgene has a tetracycline responsive promoter (tet-op) linked to sequences encoding the human aromatase (MMTV-rtTA/tet-op-Arom). Mammary glands were collected at 4 months of age for morphological, histological, and gene expression studies in both MMTV-rtTA/tet-op-Arom and wild-type mice. Results RT-PCR analysis of aromatase mRNA shows that the gene is conditionally expressed in the mammary gland as compared to control mice. The mammary glands were evaluated for morphological changes using confocal microscopy, whole-mount and hematoxylin and eosin staining. Preliminary data at 4 months of age shows that overexpressing aromatase in the mammary gland leads to developmental abnormalities, abnormal ductal growth and increased ductal density. Gene expression studies will be performed to evaluate additional markers of proliferation, survival, and differentiation. This model is being cross-bred to our conditional overexpression of estrogen receptor α (ERα) in the mammary gland (CERM) model. CERM mice are an established tool for investigation of mechanisms involved in progression and regression of ERα-induced mammary hyperplasia and DCIS. Conclusion The novel CERM/tet-op-aromatase mouse model (CERM/Arom) is being developed to model more closely the human breast with expression of both ERα and aromatase in mammary tissue. This model can be utilized to evaluate agents to prevent carcinogenesis in the presence of aromatase over-expression in the mammary gland at specific times during development. This research is supported by the Susan G. Komen for the Cure Postodoctoral Fellowship Award KG080359 and the NIH National Cancer Institute Grant R01CA112176. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B70.

A 5′ distal palindrome within the mouse mammary tumor virus-long terminal repeat recruits a mammary gland-specific complex and is required for a synergistic response to progesterone plus prolactin

Progesterone (P) and prolactin (PRL) fulfill crucial roles during growth and differentiation of the mammary epithelium, and each has been implicated in the pathogenesis of mammary cancer. We previously identified that these hormones synergistically stimulate the proliferation of mouse mammary epithelial cells in vivo, although the mechanism(s) underlying their cooperative effect are unknown. We now report a novel pathway by which P and PRL synergize to activate transcription from the long terminal repeat (LTR) of the mouse mammary tumor virus-LTR (MMTV-LTR) in T47D breast cancer cells. Using serial 5' and 3' deletions of the MMTV-LTR, in addition to selective mutations, we identified that a previously uncharacterized inverted palindrome on the distal enhancer (-941/-930), in addition to a signal transducer and activator of transcription 5 site, was essential for the synergistic activation of transcription by P and PRL. Notably, hormone synergy occurred via a mechanism that was independent of the P receptor DNA-binding elements found in the proximal MMTV-LTR hormone-response element. The palindrome specifically recruited a protein complex (herein termed mammary gland-specific complex) that was almost exclusive to normal and cancerous mammary cells. The synergy between P and PRL occurred via a Janus kinase 2 and c-Src/Fyn-dependent signaling cascade downstream of P and PRL receptors. Combined, our data outline a novel pathway in T47D cells that may facilitate the action(s) of P and PRL during mammary development and breast cancer.

Transgenic Mice Expressing the p75 CCAAT-Displacement Protein/Cut Homeobox Isoform Develop a Myeloproliferative Disease–Like Myeloid Leukemia

The p75 CCAAT-displacement protein/Cut homeobox (CDP/Cux) isoform was previously reported to be overexpressed in human breast cancers. To investigate its oncogenic potential, we engineered two transgenic mouse lines expressing p75 CDP/Cux under the control of the mouse mammary tumor virus-long terminal repeat. The FVB strain of mouse is generally used in the generation of mouse models for breast cancer. The transgene was introduced into the hprt locus of 129/Ola embryonic stem cells and, following germ line passage, was backcrossed onto the FVB and C57BL/6 mouse strains. Here, we describe the phenotype of p75 CDP/Cux transgenic virgin female mice of the first backcross generations. We report that after a long latency period, approximately 33% of mice from two independent transgenic lines and from backcrosses into either the FVB or the C57BL/6 strains succumbed to a similar disease characterized by splenomegaly, hepatomegaly, and frequent infiltration of leukocytes into nonhematopoietic organs like the kidneys and lungs. Although an excess of B or T cells was observed in three diseased mice, in 17 other cases, histologic and flow cytometry analyses revealed the expansion of a population of neutrophils in the blood, spleen, and bone marrow. The increase in neutrophils correlated with signs of anemia and thrombocytopenia, whereas there was no indication of a reactive process. Therefore, p75 CDP/Cux transgenic mice displayed heightened susceptibility to a disease defined as a myeloproliferative disease-like myeloid leukemia. These results indicate that the overexpression of p75 CDP/Cux could alter homeostasis in the hematopoietic compartment.

Epithelial-Specific and Stage-Specific Functions of Insulin-Like Growth Factor-I during Postnatal Mammary Development

Postnatal development of the mammary gland requires interactions between the epithelial and stromal compartments, which regulate actions of hormones and growth factors. IGF-I is expressed in both epithelial and stromal compartments during postnatal development of the mammary gland. However, little is known about how local expression of IGF-I in epithelium or stroma regulates mammary growth and differentiation during puberty and pregnancy-induced alveolar development. The goal of this study was to investigate the mechanisms of IGF-I actions in the postnatal mammary gland and test the hypothesis that IGF-I expressed in stromal and epithelial compartments has distinct functions. We established mouse lines with inactivation of the igf1 gene in mammary epithelium by crossing igf1/loxP mice with mouse lines expressing Cre recombinase under the control of either the mouse mammary tumor virus long-terminal repeat or the whey acidic protein gene promoter. Epithelial-specific loss of IGF-I during pubertal growth resulted in deficits in ductal branching. In contrast, heterozygous reduction of IGF-I throughout the gland decreased expression of cyclins A2 and B1 during pubertal growth and resulted in alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF-I at either of these stages had no effect on these indices. Taken together, our results support distinct roles for IGF-I expressed in epithelial and stromal compartments in mediating growth of the postnatal mammary gland.

Kit and PDGFR-α activities are necessary for Notch4/Int3-induced tumorigenesis

Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.

A Role of the Amino-Terminal (N) and Carboxyl-Terminal (C) Interaction in Binding of Androgen Receptor to Chromatin

The N-terminal domain of AR is known to engage a hormone-dependent interaction with its C-terminal ligand-binding domain, and this N/C interaction is known to modulate AR transcriptional activity. Using Xenopus oocytes as a model system to study transcriptional regulation in chromatin, we found that two previously reported N/C interaction-defective AR mutants, one with deletion of 23FQNLF27(ARDeltaF) and one with a Gly 21 to Glu mutation (ARG21E), were surprisingly inactive in activating transcription from various reporters assembled into chromatin. Further study using chromatin immunoprecipitation assay revealed that these mutants failed to bind both mouse mammary tumor virus-long terminal repeat and prostate-specific antigen enhancer assembled into chromatin. This defect is specific to chromatin because both mutants could bind to a consensus AR response element in vitro and activate transcription driven by mouse mammary tumor virus-long terminal repeat in transient transfection as effective as the wild-type AR. To further substantiate this novel finding, we established 293 cell lines that stably expressed either AR or ARDeltaF mutant in an inducible manner. Using these cell lines, we confirmed by using chromatin immunoprecipitation assay that AR but not ARDeltaF could bind to the endogenous prostate-specific antigen enhancer. Furthermore, we found that the ARDeltaF mutant interacts poorly with Brg1, the ATPase subunit of the chromatin-remodeling factor SWI/SNF. Taken together, our study reveals a novel role of AR N/C interaction in control of AR chromatin binding and suggests a working model that the proper N/C interaction is required for AR to recruit SWI/SNF complex, which in turn remodels chromatin to allow AR to bind to AR response elements in chromatin.

Kinase-Inactive Glycogen Synthase Kinase 3β Promotes Wnt Signaling and Mammary Tumorigenesis

Recent studies have implicated ectopic activation of the Wnt pathway in many human cancers, including breast cancer. beta-catenin is a critical coactivator in this signaling pathway and is regulated in a complex fashion by phosphorylation, degradation, and nuclear translocation. Glycogen synthase kinase 3beta (GSK3beta) phosphorylation of the NH2-terminal domain of beta-catenin targets it for ubiquitination and proteosomal degradation. We hypothesized that expression of kinase-inactive GSK3beta (KI-GSK3beta) in mammary glands would function in a dominant-negative fashion by antagonizing the endogenous activity of GSK3beta and promoting breast cancer development. Consistent with this, we find that KI-GSK3beta stabilizes beta-catenin expression, catalyzes its localization to the nucleus, and up-regulates the downstream target gene, cyclin D1, in vitro. In vivo, transgenic mice overexpressing the KI-GSK3beta under the control of the mouse mammary tumor virus-long terminal repeat develop mammary tumors with overexpression of beta-catenin and cyclin D1. Thus, antagonism of GSK3beta activity is oncogenic in the mammary epithelium; mutation or pharmacologic down-regulation of GSK3beta could promote mammary tumors.

276 Phase I trial of low dose interferon-alpha (IFN), thalidomide with gemcitabine and capecitabine in patients with progressive metastatic renal cell carcinoma (RCC)

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29–32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditioned by MAC-T cells. Des(l-3)IGF-I added to CM from both MAC-T and MD-IGF-I cells respectively, stimulated a 10.2 and 6.9-fold increase in [3H]thymidine incorporation into DNA of MAC-T cells. We suggest that expression of IGF-I-induced IGFBP-3 is an important component of an autocrine loop which potentiates the local mitogenic actions of IGF-I in bovine mammary epithelial cells.

Unliganded thyroid hormone receptor-beta1 represses liver X receptor alpha/oxysterol-dependent transactivation.

The thyroid hormone receptor (TR) and liver X receptor (LXR)-alpha are members of the nuclear hormone receptor family and are ligand-dependent transcription factors. Among the promoter target genes, TR and LXR recognize the T3 response element and LXR response element (LXRE), respectively. Because T3 response elements and LXREs have similar configurations, referred to as direct repeat 4, we investigated the possibility of cross-talk between the two ligand-dependent signal transduction pathways. We found that TRbeta1, a major isoform of TR in the liver, binds and transactivates LXREs derived from the mouse mammary tumor virus long-terminal repeat and the promoter of the sterol regulatory element binding protein 1c. Moreover, unliganded TRbeta1 suppresses promoter activity driven by LXRalpha and its ligand, whereas transactivation by T3-bound TRbeta1 is not affected by LXRalpha in the presence or absence of oxysterols. Gel shift, mammalian two-hybrid, and glutathione S-transferase pull-down assays demonstrated the direct binding of TRbeta1 to these LXREs and revealed that the interaction between TRbeta1 and corepressors is important to the unliganded TR-mediated suppression of LXRalpha-transactivation. Our findings suggest that T3 and TR influence lipid metabolism regulated by oxysterol/LXRalpha at the transcriptional level.

Long-term exposure to elevated levels of circulating TIMP-1 but not mammary TIMP-1 suppresses growth of mammary carcinomas in transgenic mice

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates matrix metalloproteinase activity, acts as a growth stimulator and inhibits apoptosis. We developed transgenic mice to evaluate the relevance of circulating versus mammary TIMP-1 in mammary carcinogenesis. The transgene was placed under the control of the albumin (Alb) promoter for the production of large amounts of TIMP-1 in the liver and release into the systemic circulation to achieve chronically elevated blood levels. The initial 7,12-dimethylbenz[a]anthracene (DMBA) mammary carcinogenesis study showed greatly decreased tumor incidence in heterozygous Alb-TIMP-1 mice (25%), compared with their wild-type (wt) littermates (83.3%). Metastatic mammary carcinomas were induced in the Alb-TIMP-1 mice through breeding with mice expressing the polyomavirus Middle T antigen (MT) under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Both the mammary tumor burden and the incidence of lung metastases were lower in the Alb-TIMP-1/MMTV-MT mice than their MMTV-MT littermates. Analysis of the Alb-TIMP-1/MMTV-MT tumors showed evidence of decreased proliferative activity and inhibition of apoptosis, whereas microvascular density was not affected. Transgenic expression of TIMP-1 in mammary epithelial cells was accomplished by using MMTV-LTR. In contrast to the Alb-TIMP-1 mice, there was insignificant difference in the growth of both DMBA- and MT-induced mammary tumors between heterozygous MMTV-TIMP-1 mice and their wt littermates. The MT-induced mammary tumors of the MMTV-TIMP-1 mice were separated into 'low' and 'high' TIMP-1 expressing groups. The 'high' TIMP-1 expressing tumors exhibited significantly higher proliferative activity than the tumors of the MMTV-MT only mice, whereas the number of apoptotic cells and microvascular density were not different. The findings of this study show that circulating TIMP-1, but not mammary-derived TIMP-1, has growth suppressive effects on DMBA and MT-induced mammary carcinomas.

Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation.

The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

Quantitative characterization of specific genomic promoters using agarose gel electrophoresis

Over the past decade a large number of studies have focused attention on the role of nucleosomes as negative and positive regulators of specific nuclear functions. Due to the lack of an analytical method to determine the higher order conformation of the nucleosomal arrays that encompass specific genetic loci (e.g., promoters, enhancers), research emphasis has mostly been centered on chromatin remodeling and histone posttranslational modifications. We have recently developed an agarose gel electrophoresis method that permits us to analyze the higher order structure of specific in vivo assembled chromatin fragments. After calibration using a well-defined in vitro system, we have been able to experimentally determine the size, shape, and conformational flexibility of the Mouse Mammary Tumor Virus long-terminal repeat promoter region in its repressed and activated states. These studies pave the way for widespread analyses of the higher order structure of specific, functionally important chromosomal loci, and in so doing enhance our understanding of the roles that the higher order structure of chromatin play in genome regulations.

Heparin-binding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function.

Peptide growth factors have been implicated in progression of prostate cancer (PCa) to the androgen-independent state; however, much of the evidence linking diffusible mitogens and survival factors to this process remains circumstantial. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate stroma-derived factor, promotes survival, proliferation, and neuroendocrine differentiation of androgen-dependent LNCaP PCa cells in vitro. To test whether sustained exposure to HB-EGF can confer an androgen-independent phenotype, we generated stable populations of LNCaP cells that express constitutively a secreted form of HB-EGF (LNCaP/sHB). LNCaP/sHB cells proliferated more rapidly under androgen-depleted conditions in vitro and formed larger tumors with higher frequency in intact and castrated severe combined immunodeficient mice, in comparison to control cells. LNCaP/sHB tumors also expressed higher levels of the neuroendocrine marker, neuron-specific enolase, compared with control tumors. In castrates, increased neuron-specific enolase expression in LNCaP/sHB tumors was associated with reduced androgen receptor (AR) levels. In vitro, AR protein levels were reduced in LNCaP/sHB cells, and in transient transfection assays using an androgen-responsive promoter (mouse mammary tumor virus-long terminal repeat), LNCaP/sHB cells showed reduced sensitivity to dihydrotestosterone compared with controls. This is the first demonstration that continuous exposure of AR-positive PCa cells to a single growth factor can promote an androgen-independent phenotype in vivo. These findings also emphasize the potential role of pathways other than the AR axis in acquisition of androgen independence.

TEF-1 transcription factors regulate activity of the mouse mammary tumor virus LTR

The mouse mammary tumor virus long terminal repeat (LTR) is a potent transcriptional enhancer. We identified several putative binding sites for the TEF-1 family of transcription factors (TEF-1, RTEF-1, DTEF-1, and ETF) in the proximal negative regulatory element of the LTR. Gel mobility shift assays revealed strong TEF-1 factor binding to one site using nuclear extracts from CV-1 cells and from the human breast cancer cell line MCF-7. Mutation of this site increased basal activity of the LTR. In transient transfection assays, TEF-1 squelched the basal LTR activity and completely abrogated the response to the glucocorticoid dexamethasone. RTEF-1 and DTEF-1 had little effect on the basal activity, whereas ETF activated the LTR. These TEF-1 factors also interfered with the response to dexamethasone. Taken together, our results reveal an important new role for TEF-1 factors in regulating MMTV LTR activity and suggest that TEF-1 factors might participate in mammary tumorigenesis.

CDP binding to multiple sites in the mouse mammary tumor virus long terminal repeat suppresses basal and glucocorticoid-induced transcription.

Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.