Mouse Mammary Tumor Virus Promoter Research Articles

Abstract B14: Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models

Abstract The hotspot Akt1-E17K mutation that results in a glutamic acid to lysine substitution in the seventeenth amino acid position in the pleckstrin homology domain of Akt1 occurs in approximately 0.6-2% of human lung cancers and in 6-8% of human breast cancer. To determine whether this Akt1 variant is a bona-fide activating mutation and to investigate the mechanistic basis of the Akt1 gain of function dependent mechanisms in lung and breast tumorigenesis we generated a transgenic Cre-inducible murine strain in the ROSA26 (R26) locus (R26-Akt1-E17K mice). To analyse the effects of mutant Akt1 in lung epithelium, we made use of two different systems to activate the transgene into pulmonary cells: intranasal instillation of an Adenovirus carrying the Cre recombinase (Ad-Cre) and the mating of R26-Akt1-E17K mice with Thyroid transcription factor-1 (TTF-1)-Cre mice that allow expression of the transgenes in lung epithelium. We report that in murine lung epithelium, mutant Akt1-E17K induces bronchial and/or bronchiolar hyperplastic lesions that at low frequency progress to frank carcinoma and accelerates tumor formation induced by chemical carcinogens. To assess the potential role of Akt1 as an oncogene in mammary epithelium, we examined the consequences of the mutant Akt1-E17K in the mammary gland of mice by mating of R26-Akt1-E17K mice with Mouse mammary tumor virus (MMTV)-Cre mice. We provide strong evidences that the expression of mutant Akt1-E17K in the mammary gland, driven by the MMTV promoter, was sufficient to produce a tumoral response in mice starting from 5-6 months of age. Transgenic mice developed invasive and in situ carcinoma in 45% of the cases and, in agreement with these results, mice that express activated Akt1 in the mammary gland showed poor survival Citation Format: Donatella Malanga, Stefania Belmonte, Stefania Belmonte, Carmela De Marco, Orlando Paciello, Alfonso Baldi, Giuseppe Viglietto. Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B14.

Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

Un parcours de santé innovant en terre isolée

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure–activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

A Novel Point Mutation of the Human Glucocorticoid Receptor Gene Causes Primary Generalized Glucocorticoid Resistance Through Impaired Interaction With the LXXLL Motif of the p160 Coactivators: Dissociation of the Transactivating and Transreppressive Activities

Primary generalized glucocorticoid resistance is a rare genetic disorder characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. The molecular basis of the condition has been ascribed to inactivating mutations in the human glucocorticoid receptor (hGR) gene. The objective of the study was to present three new cases caused by a novel mutation in the hGR gene and to delineate the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. The index case (father) and his two daughters presented with increased urinary free cortisol excretion and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression in the absence of clinical manifestations suggestive of Cushing syndrome. All subjects harbored a novel, heterozygous, point mutation (T→G) at nucleotide position 1724 of the hGR gene, which resulted in substitution of valine by glycine at amino acid 575 of the receptor. Compared with the wild-type receptor, the hGRαV575G demonstrated a significant (33%) reduction in its ability to transactivate the mouse mammary tumor virus promoter in response to dexamethasone, a 50% decrease in its affinity for the ligand, and a 2.5-fold delay in nuclear translocation. Although it did not exert a dominant negative effect on the wild-type receptor and preserved its ability to bind to DNA, hGRαV575G displayed significantly enhanced (∼80%) ability to transrepress the nuclear factor-κΒ signaling pathway. Finally, the mutant receptor hGRαV575G demonstrated impaired interaction with the LXXLL motif of the glucocorticoid receptor-interacting protein 1 coactivator in vitro and in computer-based structural simulation via its defective activation function-2 (AF-2) domain. The natural mutant receptor hGRαV575G causes primary generalized glucocorticoid resistance by affecting multiple steps in the glucocorticoid signaling cascade, including the affinity for the ligand, the time required for nuclear translocation, and the interaction with the glucocorticoid-interacting protein-1 coactivator.

Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.

Class I Lysine Deacetylases Facilitate Glucocorticoid-induced Transcription

Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi exposure has a significant impact on GR signaling and thus has ramifications for the clinical use of these drugs.

Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure–activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Improving mouse models

Knight et al. have developed a mouse model that resembles the claudin-low subtype of triple-negative breast cancer (TNBC) by expressing a weakly oncogenic MET receptor tyrosine kinase under the control of the mouse mammary tumour virus promoter concomitant with conditionally deleting Trp53 in the mammary gland. Tumours in these mice have a similar molecular signature to human claudin-low TNBC, and require MET for proliferation and for maintaining the claudin-low morphological phenotype. Therefore, MET inhibitors may be effective against TNBC.

A Novel Point Mutation in the DNA-Binding Domain (DBD) of the Human Glucocorticoid Receptor Causes Primary Generalized Glucocorticoid Resistance by Disrupting the Hydrophobic Structure of its DBD

Primary generalized glucocorticoid resistance is a rare genetic condition characterized by partial end-organ insensitivity to glucocorticoids. Most affected subjects present with clinical manifestations of mineralocorticoid and androgen excess. The condition has been associated with inactivating mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGRα action, thereby reducing tissue sensitivity to glucocorticoids. ΤHE aim of our study was to investigate the molecular mechanisms through which one previously described natural heterozygous V423A mutation, the second mutation detected in the DNA-binding domain (DBD) of the hGRα, affects glucocorticoid signal transduction. Compared with the wild-type receptor, hGRαV423A demonstrated a 72% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone. The hGRαV423A receptor showed a significant reduction in its ability to bind to glucocorticoid-response elements of glucocorticoid-responsive genes, owing to structural alterations of the DBD confirmed by computer-based structural analysis. In addition, hGRαV423A demonstrated a 2.6-fold delay in nuclear translocation following exposure to the ligand, although it did not exert a dominant negative effect on the wild-type hGRα, had a similar affinity to the ligand with the wild-type receptor, and displayed a normal interaction with the GRIP1 coactivator in vitro. The natural mutant receptor hGRαV423A causes primary generalized glucocorticoid resistance by affecting multiple steps in the cascade of glucocorticoid receptor action, which primarily involve decreased ability to bind to target glucocorticoid response elements and delayed translocation into the nucleus.

Abstract B78: Rebastinib, a small molecule TIE2 kinase inhibitor, prevents primary tumor growth and lung metastasis in the PyMT breast cancer model

Abstract Rebastinib is an inhibitor of tyrosine kinases TIE2 and ABL1. A clinical trial in chronic myeloid leukemia (CML) to inhibit BCR-ABL was previously performed but efficacy was modest and development in CML is not continuing. Rebastinib is approximately 50 times more potent as an inhibitor of TIE2 than BCR-ABL. Biomarker analysis of patients from the CML trial of rebastinib revealed that a majority displayed significant increases in circulating angiopoietin 2 (ANG2) levels. Increases in ANG2, a TIE2 ligand, have been previously reported to correlate with inhibition of the TIE2 pathway. The hypoxic tumor environment engendered by radiation therapy, cytotoxic chemotherapy, or anti-angiogenic treatments (such as anti-VEGF therapies) leads to rebound tumor vascularization by the recruitment of pro-vasculogenic TIE2 expressing monocytes (TEMs) from the bone marrow to these hypoxic tumor sites. TEMs are believed to play an important role in the revascularization of tumors after these treatments, leading to progression of residual tumor. Thus, inhibition of TEM recruitment and activity could lead to better patient outcomes. The polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model utilizes the mouse mammary tumor virus (MMTV) promoter, a breast specific promoter, to express PyMT in mouse breast tissue. In this model, primary breast cancers spontaneously occur, proliferate, and metastasize (mainly to the lungs) and lead to the death of the mice. Unlike xenografts, the PyMT model utilizes fully immunocompetent mice and metastasis in this model is known to be modulated by TEMs. Rebastinib has been evaluated in a PyMT mouse where primary tumors were allowed to reach 800 mg in size before starting treatment. Rebastinib therapy resulted in a significant decrease in the growth rate of the primary breast tumor (75%), a 71% reduction in lung metastases, a decrease in the levels of tumoral TIE2 staining by immunohistochemical analysis (IHC) and caused the remaining tumor to become necrotic. Combining rebastinib with paclitaxel resulted in a synergistic response with 90% inhibition of tumor growth and a 93% reduction of lung metastases. This presentation will focus on the biochemical, cellular and in vivo activity of rebastinib as a novel small molecule inhibitor of TIE2 kinase and its potential for prevention of primary tumor rebound and metastasis in combination with first line interventional therapy. A clinical trial exploring this activity is planned for 2013. Citation Format: Bryan D. Smith, Molly M. Hood, Michael D. Kaufman, Mark Berger, Daniel L. Flynn, Scott C. Wise. Rebastinib, a small molecule TIE2 kinase inhibitor, prevents primary tumor growth and lung metastasis in the PyMT breast cancer model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B78.

Interactive Chromatin Folding, Nucleosome (Mis)Positioning and Chromatin Structure

In the absence of the myriad influences that exist in a cell nucleus, the positioning and stability of nucleosomes on a short segment of DNA are governed by the material properties of the DNA. In the simplest view sequences of DNA that are intrinsically flexible or that are intrinsically curved lead to nucleosome positioning. Nucleosome positioning is thus an intra-nucleosome based effect. In vivo or for sequences of DNA that support multiple nucleosomes, nucleosome-nucleosome interactions and extra-nucleosomal (e.g. chromatin remodeling factors) interactions must also be considered.In case of intra-nucleosomal based positioning, DNA material defects (defined as variations in flexibility or conformation) are hidden because the histones force the DNA to assume a well defined super helical fold. Except for specifically constructed sequences of DNA, chromatin structures determined by intra-nucleosome interactions are irregular because the nucleosome positions will be irregular.In case inter- or extra- nucleosomal interactions determine the nucleosome positions some DNA defects will be exposed in linker DNA regions. Exposure of the defects impacts the global structure of chromatin, e.g. a bent linker yields bent chromatin.We propose that chromatin structure is biased toward one method of positioning or the other depending on environmental conditions. Thus for a given sequence of DNA both irregular and regular structures can be obtained. Our Interactive Chromatin Modeling Web Server http://www.latech.edu/∼bishop captures these ideas. Here we present a case study of how ICM Web can be used to study the folding of the mouse mammary tumor virus promoter complex(MMTV). ICM properly predicts the locations of six positioned nucleosomes in the MMTV and shows how mispositioning reveals sequence specific material defects that bend chromatin.

Manganese superoxide dismutase is dispensable for post-natal development and lactation in the murine mammary gland

Mammary gland development is a multistage process requiring tightly regulated spatial and temporal signalling pathways. Many of these pathways have been shown to be sensitive to oxidative stress. Understanding that the loss of manganese superoxide dismutase (Sod2) leads to increased cellular oxidative stress, and that the loss or silencing of this enzyme has been implicated in numerous pathologies including those of the mammary gland, we sought to examine the role of Sod2 in mammary gland development and function in situ in the mouse mammary gland. Using Cre-recombination driven by the mouse mammary tumor virus (MMTV) promoter, we created a mammary-specific post-natal conditional Sod2 knock-out mouse model. Surprisingly, while substantial decreases in Sod2 were noted throughout both virgin and lactating adult mammary glands, no significant changes in developmental structures either pre- or post-pregnancy were observed histologically. Moreover, mothers lacking mammary gland expression of Sod2 were able to sustain equal numbers of litters, equal pups per litter, and equal pup weights as were control animals. Overall, our results demonstrate that loss of Sod2 expression is not universally toxic to all cell types and that excess mitochondrial superoxide can apparently be tolerated during the development and function of post-natal mammary glands.

Overexpression of Follistatin in the Mouse Epididymis Disrupts Fluid Resorption and Sperm Transit in Testicular Excurrent Ducts1

Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.

Abstract 3416: Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis

Abstract Morbidity and mortality in breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, increased molecular understanding to identify biomarkers and targets is needed. We have previously shown that breast cancer metastasis suppressor 1 (Brms1) can suppress development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Our lab has developed two transgenic Brms1 mouse models, one which expresses murine Brms1 cDNA specifically in mammary tissue (expression by the mouse mammary tumor virus (MMTV) promoter) and a ubiquitous Brms1 expression model (expression by the chicken beta actin promoter). The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using a polyoma middle T (PyMT) oncogene-induced model. Brms1 expression, either ubiquitously or predominantly in the mammary gland, did not significantly alter growth of the primary tumor, confirming earlier studies. When expressed ubiquitously, Brms1 suppressed pulmonary metastasis and promoted tumor cell apoptosis in the lung but not in the mammary gland. However, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in mammary glands or lungs despite increased expression within primary tumors and the lungs. These results suggest tissue- or cell-type specific expression of Brms1 is a critical determinant for Brms1-mediated metastasis suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3416. doi:1538-7445.AM2012-3416

Abstract 3289: Tissue specific expression of the PI 3-kinase mutation Pik3caH1047R induces hyperplasia and tumorigenesis in a mouse model

Abstract Mutations in the p110α subunit of phosphoinositide 3-kinase (PI3K) (encoded by the gene PIK3CA) are frequent in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis in vivo, we generated a conditional (Cre recombinase-inducible) knock-in mouse model of the common activating mutation, Pik3caH1047R, expressed from the endogenous locus. We have then targeted the mutation to specific tissues by crossing our mouse to mice expressing Cre under the control of tissue-specific promoters. The mouse mammary gland was targeted using a transgenic mouse expressing Cre under the control of the mouse mammary tumor virus promoter (MMTV-Cre). To target the gastrointestinal tract, we used the A33CrePR2 mouse, expressing Ru486-inducible version of Cre under the control of the gpa33 promoter. Tumors were formed in both these models but only following a long latency (>15 months) suggesting that a ‘second-hit’ may be necessary for tumor development. Similarly, activation of this mutation in the mouse ovary, by injection of an adenovirus expressing Cre into the ovarian bursa, revealed that Pik3caH1047R alone is a weak activator of AKT capable of inducing premalignant hyperplasia in ovarian surface epithelium. Interestingly, we found that combining Pik3caH1047R mutation with deletion of Pten in the mouse ovary resulted in robust epithelial AKT signaling and progression to ovarian serous adenocarcinomas and granulosa cell tumors within 6 months. Therapeutic inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. This novel mouse is proving to be a fascinating model with which to explore the biological consequences and clinical significance of Pik3caH1047R mutation in diverse tissue types and a valuable tool for the preclinical evaluation of PI3K pathway inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3289. doi:1538-7445.AM2012-3289

Abstract 941: Suppression of the androgen receptor function by c-Jun in prostate cancer via a dual mechanism

Abstract Androgen receptor (AR) signaling plays a pivotal role in growth and survival of prostate cancer cells. Upon ligand binding, AR translocates into the nucleus and regulates expression of multiple genes through binding their promoter regions. Thus, altered expression and activity of AR contribute to disease progression. Consequently, significant effort has been undertaken to identify and characterize numerous coactivators and corepressors of AR. Here we report that the transcription factor c-Jun, a basic region leucine zipper (bZIP) transcription factor and is an important member of the AP-1 family, acts as a transcriptional repressor to inhibit the AR transcriptional activity. Furthermore, we have found that c-Jun also represses the transcription of AR. Overexpression of c-Jun in LNCaP prostate cancer cells inhibited luciferase reporters driven by the androgen-induced prostate-specific antigen (PSA) promoter, probasin promoter, mouse mammary tumor virus (MMTV) promoter and tandem repeats of androgen response elements in a dose-dependent manner. Conversely, knockdown of c-Jun enhanced androgen-stimulated AR activity in LNCaP cells. To further confirm the results, we generated a doxycycline-inducible c-Jun overexpression LNCaP stable cell line using lentivrial system. Consistent with the results of promoter activity assay, induction of c-Jun by doxycycline inhibited both steady state and androgen-induced PSA mRNA and protein levels. Interestingly, the protein and mRNA of AR were also downregulated when c-Jun was induced, suggesting that c-Jun suppresses AR expression at the transcriptional level. Moreover, c-Jun induction resulted in a significant reduction in cell number, suggesting that c-Jun suppresses LNCaP cell proliferation by antagonizing AR signaling. In summary, our results suggest that c-Jun negatively regulates the AR signaling by a dual mechanism. Future identification of negative regulators of c-Jun expression will provide a new opportunity to develop novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 941. doi:1538-7445.AM2012-941

Abstract 5622: Effect of novel RhoC inhibitor on breast cancer progression and metastasis in vivo

Abstract Introduction: The oncogene RhoC GTPase is overexpressed in highly aggressive inflammatory breast cancer (IBC) and increases cellular motility and invasiveness in mammary tissue by interacting with downstream effectors to reorganize cytoskeletal elements in the cell. A small molecule inhibitor of RhoC-effector interaction has been identified. This antagonist decreases proliferation and inhibits motility of IBC cells in vitro. Here we evaluate the in vivo effects of the novel RhoC antagonist in a transgenic mouse model of breast cancer. Methods: The PyMT-RhoC transgenic mouse model was used in this study. This double transgenic strain was created by crossing the polyoma middle T antigen (PyMT) breast cancer model to a RhoC-overexpressing strain driven by the mouse mammary tumor virus (MMTV) promoter. The RhoC inhibitor was administered by intraperitoneal injection beginning at 50 days of age. Tumor size was monitored throughout treatment. At the end of the treatment, mammary glands, tumors, and lungs were extracted, fixed, sectioned, and stained with H&E. Results: PyMT-RhoC mice treated with the RhoC inhibitor showed a statistically significant delay in time of onset of the primary tumor and exhibited slower growth of the primary tumor compared to the control group. Western blot showed decreased RhoC protein levels in the treated PyMT-RhoC group relative to control, suggesting that the drug's in vivo activity includes reducing RhoC protein expression. Finally, as determined through H&E staining of lung sections, tumor metastasis to the lungs was decreased in the treated group compared to age-matched controls. Conclusions: We conclude that this novel RhoC antagonist shows promise in vivo as an inhibitor of mammary tumor proliferation and metastasis, and has the potential to be a clinically relevant treatment for inflammatory breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5622. doi:1538-7445.AM2012-5622

Abstract 311: HER2/neu oncogene induced tumors in mammary epithelial cell COX-2 deficient mice have disease delaying modifications of tumor microenvironment

Abstract Cyclooxygenase 2 (COX-2) enzyme, which catalyzes the synthesis of several pro- and anti-tumorigenic prostanoids, is associated with more than 40% of breast cancers. COX-2 inhibitors decrease the risk of tumorigenesis and breast cancer recurrence, however, systemic COX-2 inhibition is associated with a serious cardiovascular hazard that is unrelated to the anti-tumorigenic benefits. Targeting the COX-2 pathway in cancer chemoprevention or therapy, therefore, requires refined mechanistic understanding of how COX-2 and its main pro-tumorigenic product PGE2, act in the complex tumor microenvironment to support tumor growth. To that end, we engineered mice on a pure FVB/N background that lack COX-2 only in mammary epithelial cells (MECs). Mice transgenic for COX-2 exons 5 and 6, flanked by loxP sites (COX-2flox/flox (wild type; WT)), were crossed with mice expressing cre recombinase under control of the mouse mammary tumor virus (mmtv) promoter (Cremmtv) to achieve MEC-targeted deletion of COX-2 (COX-2MECKO). Unlike global COX-2 deficiency, mice lacking COX-2 in MECs were viable, fertile and disease-free making them highly suitable for cancer studies. WT and COX-2MECKO mice were crossed with mice that express activated HER2/neu oncogene also under control of the mmtv promoter (HER2/neummtv), to drive mammary tumor development. This is a valid molecular model for human breast cancer as amplification of HER2/neu proto-oncogene occurs in 10-30% of breast cancers. Tumor onset was significantly delayed in HER2/neummtvCOX-2MECKO mice compared to their HER2/neummtv WT littermates (median disease free survival 23 and 29 weeks, respectively (p=0.006, n=12-17)). Tumor multiplicity was also decreased (p=0.043) in HER2/neummtv COX-2MEC KO mice (3.857 ± 1.388; n=7) vs. HER2/neummtv WT (6.929 ± 0.9804; n=14). Tumors from HER2/neummtv WT showed higher expression of mRNA for the vascular endothelial growth factor (VEGF) receptors VEGFR2 and VEGFR3 by real time PCR (Q-PCR). Elevated CD31 mRNA and protein levels were evident in HER2/neummtv WT tumors by Q-PCR and immunohistochemistry, indicating limited vascularization in the HER2/neummtvCOX-2MECKO mice. In addition, cell sorting by flow cytometry revealed increased infiltration of HER2/neummtv COX-2MECKO tumors by CD3+ CD4+ lymphocytes as well as CD3+ CD8+ cytotoxic T cells. These data suggest a paracrine role for mediators, most likely PGE2, derived from COX-2 in mammary epithelium in promoting and tumor supportive microenvironment including angiogenesis and immune escape. Targeted approaches to restrain COX-2/PGE2-dependent promotion of microenvironmental permissiveness may be useful in chemoprevention or treatment of breast and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 311. doi:1538-7445.AM2012-311