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The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains extremely challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

The full-length mRNAs of the human immunodeficiency virus type-1 (HIV-1), the human T-cell lymphotropic virus type-1 (HTLV-1), and the mouse mammary tumor virus (MMTV) harbor IRESs. The activity of the retroviral-IRESs requires IRES-transacting factors (ITAFs), being hnRNP A1, a known ITAF for the HIV-1 IRES. In this study, we show that hnRNP A1 is also an ITAF for the HTLV-1 and MMTV IRESs. The MMTV IRES proved to be more responsive to hnRNP A1 than either the HTLV-1 or the HIV-1 IRESs. The impact of post-translational modifications of hnRNP A1 on HIV-1, HTLV-1 and MMTV IRES activity was also assessed. Results show that the HIV-1 and HTLV-1 IRESs were equally responsive to hnRNP A1 and its phosphorylation mutants S4A/S6A, S4D/S6D and S199A/D. However, the S4D/S6D mutant stimulated the activity from the MMTV-IRES to levels significantly higher than the wild type hnRNP A1. PRMT5-induced symmetrical di-methylation of arginine residues of hnRNP A1 enabled the ITAF to stimulate the HIV-1 and HTLV-1 IRESs while reducing the stimulatory ability of the ITAF over the MMTV IRES. We conclude that retroviral IRES activity is not only dependent on the recruited ITAFs but also relies on how these proteins are modified at the post-translational level.

Tetraspanin CD151 is increasingly implicated as a multifaceted mediator of cancer development and progression. Here we investigated the role of CD151 in breast cancer in the context of the Wnt oncogenic activation. Our data showed that removal of one or both of CD151 alleles in the MMTV-Wnt1 model significantly decreased the tumor-free survival of mice from 34 weeks on average to 22 weeks and 18 weeks, respectively. This effect coincided with an accelerated tumor growth and an increased number of Ki-67+ proliferative cells. Mechanistically, the CD151-deficient tumors were largely ER+, and exhibited hyperactivation of the Wnt pathway as reflected by a marked upregulation in β-catenin and Cyclin D1, and their target genes. In addition, E-cadherin displayed a cytosolic distribution and transcription factor Snail was markedly upregulated. Collectively, this data implies that CD151 suppresses the Wnt1-driven tumorigenesis, at least in part, via counteracting the epithelial-mesenchymal transition (EMT)-like program in luminal epithelial cells. Meanwhile, the proportion of tumor cells expressing CK5 or p63, the biomarkers of myoepithelial/basal cells, markedly decreased in the absence of CD151. This change was accompanied by a decreased invasiveness of tumors and their incompetence to form a long-term cell culture. Consistent with this basal cell-linked role, the CD151 downregulation impairs mammosphere formation in MCF-10A cells and the defect was rescued by re-expression of intact CD151 ORF, but not its integrin binding-defective mutant. Overall, our study suggests that CD151 is a key player in the Wnt oncogene-driven tumorigenesis and impacts breast cancer malignancy in a cell type-dependent manner.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

There are distinct cell subpopulations in normal epithelial tissue, including stem cells, progenitor cells, and more differentiated cells, all of which have been extensively studied for their susceptibility to tumorigenesis. However, normal cells usually have to progress through a precancerous lesion state before becoming a full-blown tumor. Precancerous early lesions are heterogeneous, and the cell subset that is the primary source of the eventual tumor remains largely unknown. By using mouse models that are tailored to address this question, we identified a keratin 6a-expressing precancerous stem cell (PcSC) subset and a more differentiated whey acidic protein-positive (WAP+) cell subset in mammary precancerous lesions initiated by the Wnt1 oncogene. Both cell subsets rapidly progressed to cancer upon introduction of constitutively active versions of either HRAS or BRAF. However, the resulting tumors were dramatically different in protein profiles and histopathology: keratin 6a+ precancerous cells gave rise to adenocarcinoma, whereas WAP+ cells yielded metaplastic carcinoma with severe squamous differentiation and more robust activation of MEK/ERK signaling. Therefore, both stem and non-stem cells in mammary precancerous lesions can contribute to the eventual cancers, but their differentiation status determines the resulting cancer phenotype. This work identifies a previously unknown player in cancer heterogeneity and suggests that cancer prevention should target precancerous cells broadly and not be limited to PcSC. Significance: This work uses a novel mouse mammary gland cancer model to show that tumors initiated from different precancerous mammary epithelial cells are distinct.

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2 induced tumor initiation hasn’t been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2 induced tumorigenesis. miR-489 overexpression delayed HER2 induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2 induced tumorigenesis. The miR-489 overexpression reduced CD49fhiCD61hi populations in tumors that have stem- like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2 induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac–treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

The remodeling of the stromal extracellular matrix (ECM) plays a crucial, but incompletely understood role during tumor progression and metastasis. Hic-5, a focal adhesion scaffold protein, has previously been implicated in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in breast tumor progression in vivo, Hic-5−/− mice were generated and crossed with the Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) mouse. Tumors from the Hic-5−/−;PyMT mice exhibited increased latency and reduced growth, with fewer lung metastases, as compared to Hic-5+/−;PyMT mice. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs). Further analysis revealed that the Hic-5−/−;PyMT tumor stroma contains fewer CAFs and exhibits reduced ECM deposition. The remodeling of the stromal matrix by CAFs has been shown to increase tumor rigidity to indirectly regulate FAK Y397 phosphorylation in tumor cells to promote their growth and invasion. Accordingly, the Hic-5−/−;PyMT tumor cells exhibited a reduction in FAK Y397 phosphorylation. Isolated Hic-5−/−;PyMT CAFs were defective in stress fiber organization and exhibited reduced contractility. These cells also failed to efficiently deposit and organize the ECM in two and three dimensions. This, in turn, impacted three dimensional MDA-MB-231 tumor cell migration behavior. Thus, using a new knockout mouse model, we have identified Hic-5 expression in CAFs as a key requirement for deposition and remodeling of the stromal ECM to promote non-cell autonomous breast tumor progression.

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses1. Previous structural characterization of integrase–viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer2, 3, 4, 5, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase6, 7, 8, 9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain–carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.

Formate overflow coupled to mitochondrial oxidative metabolism\ has been observed in cancer cell lines, but whether that takes place in the tumor microenvironment is not known. Here we report the observation of serine catabolism to formate in normal murine tissues, with a relative rate correlating with serine levels and the tissue oxidative state. Yet, serine catabolism to formate is increased in the transformed tissue of in vivo models of intestinal adenomas and mammary carcinomas. The increased serine catabolism to formate is associated with increased serum formate levels. Finally, we show that inhibition of formate production by genetic interference reduces cancer cell invasion and this phenotype can be rescued by exogenous formate. We conclude that increased formate overflow is a hallmark of oxidative cancers and that high formate levels promote invasion via a yet unknown mechanism., Serine catabolism to formate supplies one-carbon units for biosynthesis. Here the authors show that formate production in murine cancers with high oxidative metabolism exceeds the biosynthetic demand and that high formate levels promotes invasion of cancer cells.

Breast cancer is the most common cancer among women worldwide. Roles of the Epstein-Barr, Merkel cell polyoma and mouse mammary tumor viruses in breast carcinogenesis are still controversial although any relationship would clearly be important for breast cancer etiology, early detection and prevention. In the present study associations between EBV, MMTV and Merkel cell polyoma virus and breast cancer in 100 Iranian patients were evaluated using paraffin-embedded tissues. EBER RNA and expression of p53 and large T antigen were evaluated by real time PCR and CD34, p63, HER2, PR and ER markers were studied by immunohistochemistry. EBV was detected in 8/100 (8%), MMTV in 12/100 (12%), MPy in 3/100 (3%) and EBER RNA in 18/100 (18%) cases. None of the control samples demonstrated any of the viruses. p53 was suppressed in EBV, MPy and MMTV positive samples. The large T antigen rate was raised in MPy positive samples. Our results showed that EBV, MMTV and the Merkel cell polyoma virus are foundwith some proportion of breast cancers in our patients, suggesting that these viruses might have a significant role in breast cancer in Kerman, southeast of Iran.

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

This short technical report describes the generation and characterization of a bioluminescence reporter mouse that is engineered to detect and longitudinally monitor the expression of doxycycline-induced constitutively active β-catenin. The new responder transgenic mouse contains the TetO-ΔN89β-CatTMILA transgene, which consists of the tet-operator followed by a bicistronic sequence encoding a stabilized form of active β-catenin (ΔN89β-catenin), an internal ribosome entry site, and the firefly luciferase gene. To confirm that the transgene operates as designed, TetO-ΔN89β-CatTMILA transgenic mouse lines were crossed with an effector mouse that harbors the mouse mammary tumor virus-reverse tetracycline transactivator (MMTV-rtTA) transgene (termed MTB hereon), which primarily targets rtTA expression to the mammary epithelium. Following doxycycline administration, the resultant MTB/CatTMILA bigenic reporter exhibited precocious lobuloalveologenesis, ductal hyperplasia, and mammary adenocarcinomas, which were visualized and monitored by in vivo bioluminescence detection. Therefore, we predict that the TetO-ΔN89β-CatTMILA transgenic responder mouse-when crossed with the appropriate effector transgenic-will have wide-applicability to non-invasively monitor the influence of constitutively active β-catenin expression on cell-fate specification, proliferation, differentiation, and neoplastic transformation in a broad spectrum of target tissues.

Background A nontoxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study was undertaken to determine the efficacy of an Ayurvedic medicine phytochemical (Withaferin A, [WA]) for chemoprevention of breast cancer and to elucidate its mode of action. Methods Chemopreventive efficacy of WA (4 and 8 mg/kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU; n = 14 for control group, n = 15 for 4 mg/kg group, and n = 18 for 8 mg/kg group). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by immunoblotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat (n = 8-12 for control group, n = 7-11 for 4 mg/kg group, and n = 8-12 for 8 mg/kg group) and/or mouse mammary tumor virus-neu (MMTV-neu) models (n = 4-11 for control group and n = 4-21 for 4 mg/kg group). Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and/or MDA-MB-231 cells. All statistical tests were two-sided. Results Incidence, multiplicity, and burden of breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg/kg group was lower by about 68% compared with controls (8 mg/kg vs control, mean = 2.76 vs 8.59, difference = -5.83, 95% confidence interval of difference = -9.89 to -1.76, P = .004). Mitotic arrest and apoptosis induction were some common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured breast cancer cells. Conclusions WA is a promising chemopreventative phytochemical with the ability to inhibit at least two different subtypes of breast cancer.

Catalytic oligonucleotides, known as DNAzymes, are a new class of nucleic acid-based gene therapy that have recently been used in preclinical animal studies to treat various cancers. In this study the systemic distribution, pharmacokinetics, and safety of intravenously administered anti-MMP (matrix metalloproteinase)-9 DNAzyme (AM9D) were determined in healthy FVB and in MMTV-polyoma virus middle T (PyMT) transgenic mice bearing mammary tumors. MMP-9 is known to be involved in tumor cell development, angiogenesis, invasion, and metastasis. Sulfur-35 (35S) labeled ([35S]-AM9D) administered intravenously, without the use of carrier molecules, to healthy and mammary tumor bearing MMTV-PyMT transgenic mice distributed to all major organs. The order of percentages of [35S]-AM9D accumulation in different organs of healthy and MMTV-PyMT mice were blood>liver>kidney>lung>spleen>heart and mammary tumor>blood≈liver>kidney>spleen>lung>heart, respectively. The amount of AM9D accumulated in mammary tumors 2 hours post injection was 0.6% and 0.2% higher than in either blood or liver, respectively, and its rate of initial clearance from mammary tissue was at least 50% slower than the other organs. Approximately 43% of the delivered dosage of [35S]-AM9D was cleared from the system via feces and urine over a period of 72 hours. No evidence of acute or chronic cytotoxicity, local or widespread, associated with AM9D treatment (up to 75 mg AM9D /kg of body weight) was observed in the organs examined. These data suggest that DNAzyme in general and AM9D in particular can be used systemically as a therapeutic agent to treat patients with breast cancer or other metastatic and surgically inaccessible tumors.

Separase, a protease encoded by the ESPL1 gene, cleaves the chromosomal cohesin during mitosis. Separase protein and transcripts are overexpressed in a wide range of human cancers (Meyer et al., Clin Cancer Res 2009; 15: 2703-2710). To investigate the physiological consequence of Separase overexpression in animals, we have generated a transgenic MMTVEspl1 mouse model that overexpresses Separase protein in the mammary glands. MMTV-Espl1 mice in a C57BL/6 genetic background develop aggressive, highly aneuploid, and estrogen receptor alpha positive (ERα+) mammary adenocarcinomas with an 80% penetrance. The mammary tumors caused by overexpression of Separase, alone or combined with p53 heterozygosity, in mammary epithelium mimic several aspects of the most aggressive forms of human breast cancer, including high levels of genetic instability, cell cycle defects, poor differentiation, distant metastasis, and metaplasia. Histopathologically, MMTV-Espl1 tumors are highly heterogeneous showing features of both luminal as well as basal subtypes of breast cancers, with aggressive disease phenotype. In addition to aneuploidy, Separase overexpression results in chromosomal instability (CIN) including premature chromatid separation (PCS), lagging chromosomes, anaphase bridges, micronuclei, centrosome amplification, multi nucleated cells, gradual accumulation of DNA damage, and progressive loss of tumor suppressors p53 and cadherin gene loci. These results suggest that Separase overexpressing mammary cells are not only susceptible to chromosomal missegregation-induced aneuploidy but also other genetic instabilities including DNA damage and loss of key tumor suppressor gene loci, which in combination can initiate tumorigenesis and disease progression.

HER2 (or ErbB2), a member of ErbB receptor tyrosine kinases, is overexpressed in approximately 20% of human breast cancer, and the ErbB2 signaling pathway is a critical therapeutic target for ErbB2-overexpressing breast cancer. We investigated the inhibitory effects of the Gemini vitamin D analog BXL0124, the synthetic triterpenoid CDDO-Im and the combination on the tumorigenesis of ErbB2-overexpressing breast cancer. MMTV-ErbB2/neu transgenic mice were treated with BXL0124, CDDO-Im, or the combination from three months of age until the end of the experiment. Formation and growth of MMTV-ErbB2/neu mammary tumors were monitored every week, and all three treatments delayed the development of mammary tumors without significant toxicity. Decreased activation of ErbB2 as well as other ErbB receptors, ErbB1 and ErbB3, in MMTV-ErbB2/neu mammary tumors was shown by all treatments. Protein levels of downstream targets of the ErbB2 signaling pathway, including activated-Erk1/2, activated-Akt, c-Myc, CycD1, and Bcl2, were repressed by all three treatments, with the combination treatment exhibiting the strongest effects. To investigate therapeutic efficacy, the combination of BXL0124 and CDDO-Im was given to MMTV-ErbB2/neu mice after mammary tumors were established between 23 and 30 weeks of age. Short-term treatment with the combination did not show effects on tumor growth nor the ErbB2 signaling pathway. The present study shows BXL0124, CDDO-Im, and the combination as potential agents for prevention, but not treatment, against the tumorigenesis of ErbB2-overexpressing breast cancer. Cancer Prev Res; 6(9); 959–70. ©2013 AACR.

There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines. Cancer Prev Res; 6(6); 540–7. ©2013 AACR.

Despite being the most evolutionarily conserved of the mammalian caspases, little is understood about the cellular function of caspase-2 in normal tissues or what role caspase-2 may have in the progression of human disease. It has been reported that deletion of the caspase-2 gene (Casp2), accelerates Eμ-myc lymphomagenesis in mice, and thus caspase-2 may act as a tumor suppressor in hematological malignancies. Here, we sought to extend these findings to epithelial cancers by examining the potential role of caspase-2 as a tumor suppressor in the mouse mammary carcinogenesis model; MMTV/c-neu. The rate of tumor acquisition was significantly higher in multiparous Casp2(-/-)/MMTV mice compared with Casp2(+/+)/MMTV and Casp2(+/-)/MMTV mice. Cells from Casp2(-/-)/MMTV tumors were often multinucleated and displayed bizarre mitoses and karyomegaly, while cells from Casp2(+/+)/MMTV and Casp2(+/-)/MMTV tumors never displayed this phenotype. Tumors from Casp2(-/-)/MMTV animals had a significantly higher mitotic index than tumors from Casp2(+/+)/MMTV and Casp2(+/-)/MMTV animals. Cell cycle analysis of Casp2(-/-) E1A/Ras-transformed mouse embryonic fibroblasts (MEF) also indicated a higher proliferative rate in the absence of caspase-2. In vitro assays further illustrated that MEF had increased genomic instability in the absence of caspase-2. This appears to be due to disruption of the p53 pathway because we observed a concomitant decrease in the induction of the p53 target genes, Pidd, p21 and Mdm2. Thus caspase-2 may function as a tumor suppressor, in part, through regulation of cell division and genomic stability.

Dipyridamole (DPM) is widely used to prevent strokes and vascular thrombosis. Combination therapy of DPM and antimetabolites has shown synergistic anticancer activity. This study investigated the chemopreventive effects of DPM in the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein metastatic breast cancer model. We also investigated the effects of DPM on gene and miRNA expression. Chemopreventive activity was assessed by comparing the time to onset of palpable lesions, primary tumor growth kinetics, and the number of lung metastases in transgenic mice treated with DPM or vehicle. Gene expression and miRNA expression profiles of mammary tumor tissues were then analyzed using the Affymetrix GeneChip or miRNA 2.0 arrays. Real-time quantitative PCR was used to confirm changes in gene expression. Treatment with DPM beginning at the age of 4 weeks delayed the onset of palpable lesions, delayed tumor progression, and suppressed lung metastasis. Microarray gene expression analysis identified 253 genes differentially expressed between DPM-treated and control mammary tumors. miRNA expression analysis revealed that 53 miRNAs were altered by DPM treatment. The results indicate that DPM has chemoprevention activity against breast cancer tumorigenesis and metastasis in mice. The array analyses provide insights into potential mechanisms of DPM's chemopreventive effects, involving upregulation of several genes and miRNAs known to suppress cancer growth and/or metastasis and downregulation of genes known to promote cancer. Some of these genes have not been previously studied in breast cancer and may serve as novel molecular targets for breast cancer chemoprevention. Cancer Prev Res; 6(5); 437–47. ©2013 AACR.

Previous studies have shown that retinoids and rexinoids can prevent breast cancer in animal models and in women with increased risk of developing the disease. The cellular effects of these vitamin A analogues have been primarily associated with induction of differentiation and inhibition of proliferation. In this study, we tested the hypothesis that bexarotene (LGD1069, Targretin), a rexinoid, can not only inhibit cell proliferation but also induce cellular senescence in mammary epithelial cells, premalignant lesions, and tumors of the MMTV-Neu model of mammary carcinogenesis, which develops estrogen receptor–negative tumors. Mice with palpable mammary tumors were treated for 4 weeks with bexarotene at 80 or 40 mg/kg body weight, and senescent cells were determined by SA-β-Gal assay. Bexarotene decreased in a dose-dependent manner the multiplicity of premalignant lesions and tumors, and this was associated with inhibition of cell proliferation and induction of cellular senescence and apoptosis. By double labeling of senescent cells, first by SA-β-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARβ, but not p53, were upregulated by bexarotene in mammary tumors and in breast cancer cell lines, suggesting involvement of multiple signaling pathways in mediating the senescence program of rexinoids. These findings indicate that, in addition to cell proliferation and apoptosis, cellular senescence could be used as a potential biomarker of response in breast cancer prevention and therapy studies with rexinoids and possibly with other antitumor agents. Cancer Prev Res; 6(4); 299–308. ©2013 AACR.

The differentiation of stem-like tumor cells may contribute to the cellular heterogeneity of breast cancers. We report the propagation of highly enriched mouse mammary cancer stem cells that retain the potential to differentiate both in vivo and in culture and their use to identify chemical compounds that influence both self-renewal and differentiation. We identify epithelial tumor-initiating cells (ETICs) that express lineage markers of both basal and luminal mammary cell lineages and retain the potential, from even single cells, to generate heterogeneous tumors similar to the tumor of origin. ETICs can progress through a Rho-associated coiled-coil containing protein kinase 1 dependent, epithelial to mesenchymal transition to generate mesenchymal tumor-initiating cells capable of initiating tumors of limited heterogeneity. The propagation of ETICs may allow for the identification of new therapeutic compounds that may inhibit or prevent progression of some types of breast cancer.

Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Background Breast cancer is considered the most common cancer in women worldwide and is the leading cause of cancer mortality. Sequences similar to Mouse Mammary Tumor Virus (MMTV) were detected in human breast cancer in several studies from different geographical areas. However, the role played by this virus in breast cancer tumorigenesis is not completely understood. These MMTV-like sequences were found to be associated with breast cancer of more malignant types. The aim of this study is to determine the prevalence of MMTV-like envelope gene (env) positivity in breast cancer and non-cancerous breast tissue from Saudi Arabia. Methods Detection of MMTV-like env proviral sequences was done using newly designed primers for conventional polymerase chain reaction (PCR). One hundred nighty four samples were collected from 103 females with breast cancer in addition to 51 control breast tissue obtained from individuals without cancer. We additionally investigated the association of proviral positivity with age of the patients, grade of breast cancer and presence of lymph node metastasis. The results were confirmed by sequencing. Results The prevalence of MMTV-like env proviral positivity was 8.7% (9/103). MMTV env proviral sequences were detected in 5.9% (6/101) of breast cancer tissues and 9.7% (9/93) of non-cancerous adjacent tissues obtained from the same patients. None of the 51 control sample showed positive result for the MMTV env gene. No significant association was found between detection of the virus and the age of the patient, grade of the cancer or presence of metastasis. Conclusion We document the presence of low frequency of MMTV env provirus sequence among breast cancer patients from Saudi Arabia. Further studies are needed to explore the role of the MMTV in breast cancer.

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

CD24 is a small, heavily glycosylated, GPI-linked membrane protein, whose expression has been associated with the tumorigenesis and progression of several types of cancer. Here, we studied the expression of CD24 in tumors of MMTV-PyMT, Apc(1572/T+) and TRAMP genetic mouse models that spontaneously develop mammary or prostate carcinoma, respectively. We found that CD24 is expressed during tumor development in all three models. In MMTV-PyMT and Apc(1572T/+) breast tumors, CD24 was strongly but heterogeneously expressed during early tumorigenesis, but decreased in more advanced stages, and accordingly was increased in poorly differentiated lesions compared with well differentiated lesions. In prostate tumors developing in TRAMP mice, CD24 expression was strong within hyperplastic lesions in comparison with non-hyperplastic regions, and heterogeneous CD24 expression was maintained in advanced prostate carcinomas. To investigate whether CD24 plays a functional role in tumorigenesis in these models, we crossed CD24 deficient mice with MMTV-PyMT, Apc(1572T/+) and TRAMP mice, and assessed the influence of CD24 deficiency on tumor onset and tumor burden. We found that mice negative or positive for CD24 did not significantly differ in terms of tumor initiation and burden in the genetic tumor models tested, with the exception of Apc(1572T/+) mice, in which lack of CD24 reduced the mammary tumor burden slightly but significantly. Together, our data suggest that while CD24 is distinctively expressed during the early development of murine mammary and prostate tumors, it is not essential for the formation of tumors developing in MMTV-PyMT, Apc(1572T/+) and TRAMP mice.

RANK expression is associated with poor prognosis in breast cancer even though its therapeutic potential remains unknown. RANKL and its receptor RANK are downstream effectors of the progesterone signaling pathway. However, RANK expression is enriched in hormone receptor negative adenocarcinomas, suggesting additional roles for RANK signaling beyond its hormone-dependent function. Here, to explore the role of RANK signaling once tumors have developed, we use the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT), which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacologic approaches demonstrate that therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, decreases tumor and metastasis initiation, and enhances sensitivity to chemotherapy. Mechanistically, genome-wide expression analyses show that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. Moreover, RANK signaling in tumor cells negatively regulates the expression of Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1-population, enriched in tumor-initiating cells. In addition, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with relapse-free tumors. These results support the use of RANKL inhibitors to reduce recurrence and metastasis in breast cancer patients based on its ability to induce tumor cell differentiation. Cancer Res; 76(19); 5857–69. ©2016 AACR.

The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene expression in primary human breast tumors was interrogated. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors.

Breast cancer stem cells, the root of tumor growth, present challenges to investigate: Primary human breast cancer cells are difficult to establish in culture and inconsistently yield tumors after transplantation into immune-deficient recipient mice. Furthermore, there is limited characterization of mammary cancer stem cells in mice, the ideal model for the study of breast cancer. We herein describe a pre-clinical breast cancer stem cell model, based on the properties of cancer stem cells, derived from transgenic MMTV-PyMT mice. Using a defined set of cell surface markers to identify cancer stem cells by flow cytometry, at least four cell populations were recovered from primary mammary cancers. Only two of the four populations, one epithelial and one mesenchymal, were able to survive and proliferate in vitro. The epithelial population exhibited tumor initiation potential with as few as 10 cells injected into syngeneic immune-competent recipients. Tumors initiated from injected cell lines recapitulated the morphological and physiological components of the primary tumor. To highlight the stemness potential of the putative cancer stem cells, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) expression was knocked down via shRNA targeting Bmi-1. Without Bmi-1 expression, putative cancer stem cells could no longer initiate tumors, but tumor initiation was rescued with the introduction of a Bmi-1 overexpression vector in the Bmi-1 knockdown cells. In conclusion, our data show that primary mammary cancers from MMTV-PyMT mice contain putative cancer stem cells that survive in culture and can be used to create a model for study of mammary cancer stem cells.

Novel approaches for chemoprevention of breast cancer are clinically attractive because many risk factors associated with this disease (eg, genetic predisposition, late-age at first full-term pregnancy, early menarche, and late menopause) are not easily modifiable (1–3). Breast cancer continues to be a leading cause of cancer-related death in women worldwide (4) despite tremendous advances towards targeted therapies and personalized medicine (5,6). Primary prevention of breast cancer is feasible considering successful clinical application of selective estrogen receptor modulators (eg, tamoxifen and raloxifene) (7,8). However, this strategy is ineffective against estrogen receptor–negative breast cancers, and it is associated with risk of rare but serious side effects, including increased risk of uterine cancer, thromboembolism, cataracts, and perimenopausal symptoms (7,8). Use of the aromatase inhibitor (AI) exemestane is now an effective strategy for prevention of breast cancer in postmenopausal women (9,10). Although long-term effects of AIs are still under study, their use raises concerns for potential for increased cardiovascular risk (10,11). For example, combined analysis of multiple randomized controlled trials comparing AIs with tamoxifen reveals higher incidence of grade 3 and 4 cardiovascular events associated with use of AIs (10,11). Increased risk of bone fracture is another potential adverse effect of AIs (10). Edible plants continue to gain traction in the ongoing search for novel chemopreventative agents (12,13). Phenethyl isothiocyanate (PEITC) is one such promising bioactive component of edible cruciferous vegetables (eg, watercress). PEITC occurs naturally as a thioglucoside conjugate, gluconasturtiin (14,15), and is produced by myrosinase-catalyzed hydrolytic breakdown of gluconasturtiin upon processing (cutting or chewing) of cruciferous vegetables (14). The anticancer potential of PEITC was initially recognized by Wattenberg, who showed that 7,12-dimethylbenz[a]- anthracene–induced mammary cancer development was inhibited in Sprague-Dawley rats that were administered PEITC 4 hours before challenge with the carcinogen (16). Likewise, administration of PEITC inhibited N-nitrosobenzylmethylamine–induced esophageal carcinogenesis in rats (17). More recent studies have now established the efficacy of PEITC as a chemopreventative agent in transgenic mouse models of colon or intestinal cancer or of prostate cancer (18,19). Successful clinical realization of a cancer chemopreventative strategy is contingent upon systematic investigations: from “flask”-based cellular studies to identify promising agents and to define the mechanisms of their action; to “fur”(animal)-based studies focusing not only on bioavailability, safety, and efficacy assessments but also on discovery of biomarker(s) associated with response; to “folks” in clinical trials, with a pilot biomarker modulation trial followed by larger studies with cancer incidence as the primary endpoint. The discovery of biomarkers associated with response is especially desirable for cancer chemopreventative agents because cancer incidence is too rigorous an endpoint for cancers with long latency, such as breast cancer. Previous studies from our laboratory have shown that PEITC treatment, in association with apoptosis induction, decreases viability of cancer cells, including BRI-JM04, a breast cancer cell line derived from a mammary tumor in a mouse mammary tumor virus–neu (MMTV-neu) transgenic mouse (20,21). By contrast, MCF-10A cells, a spontaneously immortalized and nontumorigenic human mammary epithelial cell line derived from a patient with fibrocystic breast disease, are substantially more resistant than breast cancer cells to PEITC-induced apoptosis (21). PEITC-induced apoptosis in BRI-JM04 cells is regulated by B-cell lymphoma 2 interacting mediator of cell death (Bim) (21). These in vitro observations prompted us to test PEITC-mediated mammary cancer chemoprevention in mice, with two objectives: 1) to determine in vivo efficacy of dietary PEITC administration for chemoprevention of breast cancer using MMTV-neu model, which not only faithfully recapitulates aspects of human disease progression but also mimics human epidermal growth factor receptor-2 (HER-2/neu) overexpression and amplification in a subset of human breast cancers (22–24), and 2) to identify biomarker(s) associated with PEITC exposure that could be potentially useful in future clinical investigations.

The ability to transplant mammary cancer stem cells, identified by the phenotype CD24(+)CD29(+)CD49f(+)Sca-1(low), is dependent on the microenvironment in which the cells are placed. Using the MMTV-PyMT mouse model of mammary cancer, we now report two methods of tumor growth enhancement: contributions of tumor stroma in the form of tumor-derived mesenchymal stem cells and orthotopic vs. heterotopic transplantation sites. To support evidence of stem cell function, tumor-derived mesenchymal stem cells differentiated into adipocyte- and osteocyte-like cells after culture in specific medium. Co-injection of tumor-initiating cells with tumor-derived mesenchymal stem cells significantly increased tumor initiation compared to subcutaneous injection of TICs alone; co-injection also allowed tumor initiation with a single TIC. Interestingly, we observed the formation of sarcomas after co-injections of tumor-derived mesenchymal stem cells or mouse embryonic fibroblasts with TICs; sarcomas are not observed in spontaneous MMTV-PyMT tumors and rarely observed in injections of TICs alone. Tumor initiation was also significantly increased in the orthotopic injection site compared to heterotopic injections. We conclude that tumor stroma and orthotopic sites both enhance tumor initiation by mammary cancer stem cells.

Introduction. The initial clinical experience from targeted therapy for breast cancer has been mixed. While important progress has been made in the care of a subset of patients characterized by amplification of HER2 through the use of trastuzumab, other targeted therapies have failed to improve the outcome for large, unselected groups of patients. Thus, efforts to find prognostic or predictive biomarkers to enable tailored therapy are highly warranted. Genetically engineered mouse models of human cancer provide a convenient setting in which to perform explorative studies. However, there is a paucity of comparative studies between mouse and human tumours in order to validate the use of mouse models as discovery tools. Materials and methods. Here, we have compared the localization of markers for cancer-associated fibroblasts in the MMTV-PyMT mouse model of mammary carcinoma with that of human breast cancer. The expression of α-smooth muscle actin, platelet-derived growth factor receptor-α, and fibroblast-specific protein-1 was assessed by immunostaining of sections from tumours of MMTV-PyMT mice. Information about the distribution of the same markers in human breast cancer was derived from the publicly available database the Human Protein Atlas. Results. Both mouse and human mammary carcinomas were infused by a rich fibrotic stroma. While no marker was capable of identifying all stromal fibroblasts, the expression pattern of each marker was remarkably similar in mouse and human. Discussion. We conclude that the MMTV-PyMT mouse model of breast cancer will have utility as a discovery tool for biomarkers of cancer-associated fibroblasts during malignant conversion.

Selenium is an essential micronutrient in the diet of humans and other mammals. Based largely on animal studies and epidemiological evidence, selenium is purported to be a promising cancer chemopreventive agent. However, the biological mechanisms by which chemopreventive activity takes place are poorly understood. It remains unclear whether selenium acts in its elemental form, through incorporation into organic compounds, through selenoproteins or any combination of these. The purpose of this study was to determine whether selenoproteins mitigate the risk of developing chemically induced mammary cancer. Selenoprotein expression was ablated in mouse mammary epithelial cells through genetic deletion of the selenocysteine (Sec) tRNA gene (Trsp), whose product, designated selenocysteine tRNA, is required for selenoprotein translation. Trsp floxed and mouse mammary tumor virus (MMTV)-cre mice were crossed to achieve tissue-specific excision of Trsp in targeted mammary glands. Eight- to twelve-week-old second generation Trsp(fl/+);wt, Trsp(fl/+);MMTV-cre, Trsp(fl/fl);wt and Trsp(fl/fl);MMTV-cre female mice were administered standard doses of the carcinogen, 7,12-dimethylbenzylbenz[a]antracene. Our results revealed that heterozygous, Trsp(fl/+);MMTV-cre mice showed no difference in tumor incidence, tumor rate and survival compared with the Trsp(fl/+);wt mice. However, 54.8% of homozygous Trsp(fl/f)(l);MMTV-cre mice developed mammary tumors and exhibited significantly shorter survival than the corresponding Trsp(fl/fl);wt mice, where only 36.4% developed tumors. Loss of the homozygous Trsp alleles was associated with the reduction of selenoprotein expression. The results suggest that mice with reduced selenoprotein expression have increased susceptibility to developing carcinogen-induced mammary tumors and that a major protective mechanism against carcinogen-induced mammary cancer requires the expression of these selenoproteins.

Wnt ligand–driven tumor growth is inhibited by the soluble Wnt inhibitor Fzd8CRD, but the mechanism through which this effect is mediated is unknown. In the MMTV-Wnt1 mouse model, regression of mammary tumors by Fzd8CRD treatment coincides with an acute and strong induction of insulin-like growth factor (IGF)–binding protein IGFBP5, an antagonist of IGF signaling that mediates involution of mammary gland in females after offspring are weaned. In this study, we show that repression of this IGF inhibitory pathway is crucial for Wnt-driven growth of mammary tumors. We found that IGFBP5 regulation was mediated by the β-catenin–dependent Wnt pathway. Wnt, in addition to IGF ligands, facilitated tumor growth by paracrine communication among tumor cells. In addition, Fzd8CRD caused precocious induction of IGFBP5 in normal mammary glands undergoing involution, implying an acceleration of the involution process by inhibition of Wnt signaling. The molecular and phenotypic parallel between tumor regression and mammary gland involution suggests that Wnt-driven mammary tumors use the same growth mechanism as proliferating normal mammary glands. Cancer Res; 72(6); 1568–78. ©2012 AACR.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/β-catenin pathway, or short hairpin RNA targeting β-catenin expression demonstrated that the invasive activity was not mediated by β-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the β-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

TA2 mice have a high incidence of spontaneous breast cancer without chemical stimulus. There are two proposed explanations for this phenomenon: one is gravidity and the frequency of pregnancies, and the other is related to the presence of the mouse mammary tumor virus (MMTV). MMTV is hormonally regulated and indirectly promotes tumor formation by leading to the activation of Wnt oncogenes through insertional mutagenesis. In order to clarify the relationship between estrogen, progesterone, MMTV, Wnt oncogenes and breast cancer, ovaries from virgin female TA2 mice were removed and the mice were treated with exogenous estradiol and progesterone in different patterns. This study found that the combination of exogenous estradiol and progesterone induced breast cancer formation in TA2 mice without ovaries. MMTV-LTR mRNA exhibited the highest expression in tumor tissue from the combination treatment group (CT). Mammary tissue from mice in the CT group had the highest Wnt1, Wnt5a, Wnt5b and Wnt10b mRNA expression levels. These results indicate that estradiol and progesterone act in a synergistic manner to upregulate MMTV, which subsequently induces breast cancer in TA2 mice. Various members of the Wnt gene family may play specific roles in different stages of carcinogenesis in TA2 mice.

Lysyl oxidase (LOX) is the enzyme that facilitates the cross-linking of collagen and elastin, although other functions for this enzyme have been indicated. Of these other functions, we describe herein the ability of LOX to regulate several gene promoters, like collagen III, elastin, and cyclin D1. We have previously demonstrated a specific binding between LOX and histone H1, in vitro. Therefore, we investigated whether LOX would affect the mouse mammary tumor virus (MMTV) promoter and its glucocorticoid regulation, which depends on the phophorylation status of histone H1. Our results show that the over-expression of recombinant human LOX was able to trigger MMTV activity, both in the presence and absence of glucocorticoids. Moreover, we demonstrated that histone H1 from cells expressing recombinant LOX contained isodesmosine and desmosine, indicating specific lysyl-oxidase-dependent lysine modifications. Finally, we were able to co-immunoprecipitate the exogenous LOX and histone H1 from the LOX transfected cells. The data are compatible with a decreased positive charge of histone H1, owing to deamination by LOX of its lysine residues. This event would favor H1 detachment from the target DNA, and consequent opening of the MMTV promoter structure to the activating transcription factors. The presented data, therefore, suggest a possible histone-H1-dependent mechanism for the modulation of MMTV promoter by LOX.

The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of "luminal-like" cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.

The authors present a protocol for the in vivo evaluation, using different imaging techniques, of lymph node (LN) homing of tumor-specific dendritic cells (DCs) in a murine breast cancer model.Bone marrow DCs were labeled with paramagnetic nanoparticles (MNPs) or (111)In-oxine. Antigen loading was performed using tumor lysate. Mature DCs were injected into the footpads of transgenic tumor-bearing mice (MMTV-Ras) and DC migration was tracked by magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). Ex vivo analyses were performed to validate the imaging data.DC labeling, both with MNPs and with (111)In-oxine, did not affect DC phenotype or functionality. MRI and SPECT allowed the detection of iron and (111)In in both axillary and popliteal LNs. Immunohistochemistry and γ-counting revealed the presence of DCs in LNs.MRI and SPECT imaging, by allowing in vivo dynamic monitoring of DC migration, could further the development and optimization of efficient anti-cancer vaccines.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN–expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN–expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When mouse mammary tumor virus (MMTV)-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of non-transformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu-transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells show the capacity to differentiate into normal mammary cell types, including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals, combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and it has been hypothesized that companion animals might have a role in viral transmission. The aim of our study was to investigate the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline (FMCs) and canine mammary carcinomas (CMCs) by nested PCR and immunohistochemistry. Results showed that the presence of MMTV-like env sequence in FMCs was 7% (6/86), while all the CMCs and canine dysplastic lesions scored negative. All PCR-positive FMCs scored positive for the MMTV p14 signal peptide of the envelope precursor protein of the virus. In contrast, all PCR-negative FMCs and canine mammary lesions were also negative for immunohistochemistry analysis. Canine and feline normal mammary gland tissues scored negative for both PCR and MMTV-p14 protein. Multiple nucleotide alignment of MMTV-like env gene sequences isolated from cat showed 97% and 99% similarity with HMTV and MMTV, respectively, while the others two presented some polimorphisms. Particularly the sequences of one of these two tumors showed a polymorphism (c.7575 A> G), that causes a previously unreported amino acid substitution (Thr > Ala). In conclusion, the results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus does play a role in the development of FMCs, if MMTV-like is an exogenous virus as these data suggest and, in such a case, how and from whom this virus was acquired.

Fibroblast growth factor (FGF) cooperates with the Wnt/β-catenin pathway to promote mammary tumorigenesis. To investigate the mechanisms involved in FGF/Wnt cooperation, we genetically engineered a model of inducible FGF receptor (iFGFR) signaling in the context of the well-established mouse mammary tumor virus–Wnt-1 transgenic mouse. In the bigenic mice, iFGFR1 activation dramatically enhanced mammary tumorigenesis. Expression microarray analysis did not show transcriptional enhancement of Wnt/β-catenin target genes but instead showed a translational gene signature that also correlated with elevated FGFR1 and FGFR2 in human breast cancer data sets. Additionally, iFGFR1 activation enhanced recruitment of RNA to polysomes, resulting in a marked increase in protein expression of several different Wnt/β-catenin target genes. FGF pathway activation stimulated extracellular signal-regulated kinase and the phosphorylation of key translation regulators both in vivo in the mouse model and in vitro in a human breast cancer cell line. Our results suggest that cooperation of the FGF and Wnt pathways in mammary tumorigenesis is based on the activation of protein translational pathways that result in, but are not limited to, increased expression of Wnt/β-catenin target genes (at the level of protein translation). Further, they reveal protein translation initiation factors as potential therapeutic targets for human breast cancers with alterations in FGF signaling. Cancer Res; 70(12); 4868–79. ©2010 AACR.