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The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor which in certain cancer types drives pro-survival processes that facilitate tumorigenesis, malignant cell migration, invasion, and metastasis. Much of AHR's pro-tumorigenic action is due to its activation by the oncometabolite, kynurenine. Because of this AHR antagonists are being actively investigated as new anti-tumor therapy. In this study we compared the effects of treatment with the AHR antagonists, BAY2416964 and GNF351, to that of AHR knockout in PyMT murine mammary cancer cells. BAY2416964 and GNF351 effectively inhibited kynurenine-dependent increases in Cyp1a1 and Cyp1b1 mRNA levels. CRISPR/Cas9-generated PyMT AhrKO cells exhibited reduced cell proliferation compared with controls, but treatment with 1 mM BAY2416964 for 96 h had no effect on the proliferation of wildtype cells. To further examine the differences between AHR knockout and short term BAY2416964, we generated long-term BAY2416964 (LT-BAY) cells by exposing wildtype cells to 1 mM BAY2416964 for at least 6 weeks. Similar to AhrKO cells, LT-BAY cells exhibited reduced cell proliferation and migration compared with wildtype cells. No differentially expressed genes (DEGs) were identified in wildtype cells exposed to 1 mM BAY2416964 for 24 h; however, 46.4% of DEGs overlapped between AhrKO and LT-BAY cells including gene regulated cell proliferation. Our data reveal long-term pharmacological inhibition of AHR by BAY2416964 closely resembles AHR loss in a mouse model of breast cancer. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor which in certain cancer types drives pro-survival processes that facilitate tumorigenesis, malignant cell migration, invasion, and metastasis. Much of AHR’s pro-tumorigenic action is due to its activation by the oncometabolite, kynurenine. Because of this AHR antagonists are being actively investigated as new anti-tumor therapy. In this study we compared the effects of treatment with the AHR antagonists, BAY2416964 and GNF351, to that of AHR knockout in PyMT murine mammary cancer cells. BAY2416964 and GNF351 effectively inhibited kynurenine-dependent increases in Cyp1a1 and Cyp1b1 mRNA levels. CRISPR/Cas9-generated PyMT AhrKO cells exhibited reduced cell proliferation compared with controls, but treatment with 1 μM BAY2416964 for 96 h had no effect on the proliferation of wildtype cells. To further examine the differences between AHR knockout and short term BAY2416964, we generated long-term BAY2416964 (LT-BAY) cells by exposing wildtype cells to 1 μM BAY2416964 for at least 6 weeks. Similar to AhrKO cells, LT-BAY cells exhibited reduced cell proliferation and migration compared with wildtype cells. No differentially expressed genes (DEGs) were identified in wildtype cells exposed to 1 μM BAY2416964 for 24 h; however, 46.4% of DEGs overlapped between AhrKO and LT-BAY cells including gene regulated cell proliferation. Our data reveal long-term pharmacological inhibition of AHR by BAY2416964 closely resembles AHR loss in a mouse model of breast cancer.

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation. [ABSTRACT FROM AUTHOR]

The multifunctional protein staphylococcal nuclease domain‐containing protein 1 (SND1) is conserved and has been implicated in several aspects of tumor development, such as proliferation, epithelial–mesenchymal transition, and immune evasion. Despite this, the precise role of SND1 in the initiation and metastasis of mammary gland tumors remains largely unexplored. In this study, we utilized a mouse model of breast tumors induced by polyomavirus middle T antigen (PyMT) to demonstrate that the knockout of SND1 significantly delayed the onset of primary mammary tumor formation induced by PyMT. Histological staining and cytometric analysis were conducted to confirm the reduction of tumor‐initiating cells and lung metastasis following depletion of SND1. Additionally, our findings demonstrate that enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a crucial epigenetic modifier implicated in PyMT‐induced breast tumors, serves as an essential mediator of SND1‐promoted primary mammary tumor formation. Mechanistic investigations revealed that SND1 functions as a transcriptional co‐activator of hypoxia‐inducible factor 1 subunit alpha (HIF1α), thereby regulating the downstream target gene EZH2 and promoting tumorigenesis. Overall, this study provides novel insights into the role of SND1 as a co‐activator of HIF1α in the acceleration of PyMT‐induced spontaneous breast tumor formation through the promotion of EZH2 transcription. The findings provide novel insights into the relationship between SND1 and the formation of tumor‐initiating cells. [ABSTRACT FROM AUTHOR]

Determination of the mammary epithelial cell that serves as the cell of origin for breast cancer is key to understand tumor heterogeneity and clinical management. In this study, we aimed to decipher whether Rank expression in the presence of PyMT and Neu oncogenes might affect the cell of origin of mammary gland tumors. We observed that Rank expression in PyMT+/− and Neu+/− mammary glands alters the basal and luminal mammary cell populations already in preneoplasic tissue, which may interfere with the tumor cell of origin restricting their tumorigenesis ability upon transplantation assays. In spite of this, Rank expression eventually promotes tumor aggressiveness once tumorigenesis is established. [ABSTRACT FROM AUTHOR]

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Abstract Background Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled. Methods We developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression. Results Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling. Conclusions Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.

Background: Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled.Methods: We developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression.Results: Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling.Conclusions: Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Progenitor cells are considered an important cell of origin of human malignancies. However, there has not been any single gene that can define mammary bipotential progenitor cells, and as such it has not been possible to use genetic methods to introduce oncogenic alterations into these cells in vivo to study tumorigenesis from them. Keratin 6a is expressed in a subset of mammary luminal epithelial cells and body cells of terminal end buds. By generating transgenic mice using the Keratin 6a (K6a) gene promoter to express tumor virus A (tva), which encodes the receptor for avian leukosis virus subgroup A (ALV/A), we provide direct evidence that K6a(+) cells are bipotential progenitor cells, and the first demonstration of a non-basal location for some biopotential progenitor cells. These K6a(+) cells were readily induced to form mammary tumors by intraductal injection of RCAS (an ALV/A-derived vector) carrying the gene encoding the polyoma middle T antigen. Tumors in this K6a-tva line were papillary and resembled the normal breast-like subtype of human breast cancer. This is the first model of this subtype of human tumors and thus may be useful for preclinical testing of targeted therapy for patients with normal-like breast cancer. These observations also provide direct in vivo evidence for the hypothesis that the cell of origin affects mammary tumor phenotypes.

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7–10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus‐polyoma middle T‐antigen (MMTV‐PyMT) oncogene. The MMTV‐PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild‐type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin‐deficient breast cancers. What's new? Nischarin is ubiquitously expressed in the mammary gland and regulates cell adhesion and migration. A role for Nischarin was also described in breast cancer, in which it is frequently under‐expressed. Here, the authors show that disruption of the Nischarin gene delays mammary gland development. Nischarin‐disrupted mice bearing the PyMT oncogene showed enhanced tumor growth and metastasis. Nischarin deletion decreased the activity of AMPK and its downstream effectors. Metformin had a strong inhibitory effect on the growth of tumors with no functional Nischarin expression. The findings suggest a potential use for metformin as a cancer therapeutic, particularly in Nischarin‐deficient breast cancers. [ABSTRACT FROM AUTHOR]

The expression of cellular reporters to label cancer cells, such as green fluorescent protein (GFP) and luciferase, can stimulate immune responses and effect tumor growth. Recently, a mouse model that expresses GFP and luciferase in the anterior pituitary gland was generated to tolerize mice to these proteins; the "Glowing Head" mouse. Mice were obtained from a commercial vendor, bred, and then used for tumor growth and metastasis studies. The transgene expression of luciferase was assessed within tumor-naïve mice as well as mice with mammary tumors or metastases. Tumor-free mice with white fur, compared to black fur, allowed for stronger luciferase transgene expression to be observed in the pituitary, sternum, and femur. Growth of four different luciferase-expressing mouse cancer cell lines readily occurred in the mammary gland. Though sternum expression of the luciferase transgene occurred in cancer-free mice, growth or death of luciferase positive cancer cells in the lung could be observed. Liver metastases seeded by portal vein injections of luciferase positive cancer cell lines were completely distinct from luciferase transgene expression. Though lung and brain metastasis studies have limitations, the Glowing Head mouse can be useful to inhibit immune system rejection of luciferase or GFP expressing cancer cells. This mouse model is most beneficial for studies of mammary tumors and liver metastases. [ABSTRACT FROM AUTHOR]

Background: The gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC, PVT1 and FAM84B, is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus.Methods: The MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT, MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website.Results: Mice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC, FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes.Conclusion: Deletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development. [ABSTRACT FROM AUTHOR]

Metastatic breast cancer is currently incurable. It has recently emerged that different metabolic pathways support metastatic breast cancer. To further uncover metabolic pathways enabling breast cancer metastasis, we investigated metabolic differences in mouse tumors of differing metastatic propensities using mass spectrometry-based metabolomics. We found that sialic acid metabolism is upregulated in highly metastatic breast tumors. Knocking out a key gene in sialic acid metabolism, Cmas, inhibits synthesis of the activated form of sialic acid, cytidine monophosphate-sialic acid and decreases the formation of lung metastases in vivo. Thus, the sialic acid pathway may be a new target against metastatic breast cancer.

Growing cancers are known to modify immune responses through suppressive mechanisms manifested within the local tumor microenvironment. Accumulating evidence indicates that secreted tumor products can also influence on distant immunological compartments, including myelopoiesis in the bone marrow. However, it is unknown if a similar effect can occur to regulate B-cell lymphopoiesis in breast cancer. Examining the MMTV-PyMT murine model of breast cancer, we show a complete block in bone marrow B-cell lymphopoiesis, which is dependent on tumor burden. We also observed an increase in the total number of splenic B cells and an elevated frequency of marginal zone B cells. By using in vitro assays of B-cell lymphopoiesis, we show that tumor-secreted molecules directly inhibit B-cell progenitor proliferation and favor maturation. These data demonstrate a profound sensitivity of B-cell lymphopoiesis to the accumulation of ectopically produced molecules during tumor growth in PyMT. [ABSTRACT FROM AUTHOR]

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (~7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency. [ABSTRACT FROM AUTHOR]

Background: Clinical studies implying the sunitinib multi-kinase inhibitor have led to disappointing results for breast cancer care but mostly focused on HER2-negative subtypes. Preclinical researches involving this drug mostly concern Triple Negative Breast Cancer (TNBC) murine models. Here, we explored the therapeutic efficacy of sunitinib on a PyMT-derived transplanted model classified as luminal B (HER2-positive) and monitored the response to treatment using both in vivo and ex vivo approaches. Methods: Tumour-induced animals were treated for 9 (n = 7) or 14 (n = 8) days with sunitinib at 40 mg/kg or with vehicle only. Response to therapy was assessed in vivo by monitoring glucose tumour metabolism and hypoxia using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and [18F]fluoromisonidazole ([18F]FMISO) Positron Emission Tomography (PET). After primary tumour excision, ex vivo digital microscopy was performed on treated and control samples to estimate vascular density (CD31), apoptosis (Tunel), proliferation (Ki-67), Tumour-Associated Macrophage (TAM) infiltration (F4/80), metabolism (GLUT1) and cellular response to hypoxia (HIF1 alpha). The drug impact on the metastasis rate was evaluated by monitoring the PyMT gene expression in the lungs of the treated and control groups. Results: Concomitant with sunitinib-induced tumour size regression, [18F]FDG PET imaging showed a stable glycolysis-related metabolism inside tumours undergoing treatment compared to an increased metabolism in untreated tumours, resulting at treatment end in 1.5 less [18F]FDG uptake in treated (n = 4) vs control (n = 3) tumours (p < 0.05). With this small sample, [18F]FMISO PET showed a non-significant decrease of hypoxia in treated vs control tumours. The drug triggered a 4.9 fold vascular volume regression (p < 0.05), as well as a 17.7 fold induction of tumour cell apoptosis (p < 0.001). The hypoxia induced factor 1 alpha (HIF1 alpha) expression was twice lower in the treated group than in the control group (p < 0.05). Moreover, the occurrence of lung metastases was not reduced by the drug. Conclusions: [18F]FDG and [18F]FMISO PET were relevant approaches to study the response to sunitinib in this luminal B (HER2-positive) model. The sunitinib-induced vascular network shrinkage did not significantly increase tumour hypoxia, suggesting that tumour regression was mainly due to the pro-apoptotic properties of the drug. Sunitinib did not inhibit the metastatic process in this PyMT transplanted model. [ABSTRACT FROM AUTHOR]

Understanding vascular growth and maturation in developing tumors has important implications for tumor progression, spread, and ultimately host survival. Modulating the signaling of endothelial G proteincoupled receptors (GPCRs) in blood and lymphatic vessels can enhance or limit tumor progression. Sphingosine 1-phosphate receptor 1 (S1PR1) is a GPCR for circulating lysophospholipid S1P that is highly expressed in blood and lymphatic vessels. Using the S1PR1- enhanced green fluorescent protein (eGFP) mouse model in combination with intravital imaging and pharmacologic modulation of S1PR1 signaling, we show that boundary conditions of high and low S1PR1 signaling retard tumor progression by enhancing or destabilizing neovasculature integrity, respectively. In contrast, midrange S1PR1 signaling, achieved by receptor antagonist titration, promotes abundant growth of small, organized vessels and thereby enhances tumor progression. Furthermore, in vivo S1PR1 antagonism supports lung colonization by circulating tumor cells. Regulation of endothelial S1PR1 dynamically controls vascular integrity and maturation and thus modulates angiogenesis, tumor growth, and hematogenous metastasis. [ABSTRACT FROM AUTHOR]

Metastasis is the primary cause of death in breast cancer patients, yet there are challenges to modeling this process in vivo. The goal of this study was to analyze the effects of injection site on tumor growth and metastasis and gene expression of breast cancer cells in vivo using the MMTV-PymT breast cancer model (Met-1 cells). Met-1 cells were injected into 5 sites (subcutaneous, mammary fat pad, tail vein, intracardiac, and intratibial), and tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Met-1 tumors were analyzed based on morphology and changes in gene expression in each tissue microenvironment. There were 6 permissible sites of Met-1 tumor growth (mammary gland, subcutis, lung, adrenal gland, ovary, bone). Met-1 cells grew faster in the subcutis compared to mammary fat pad tumors (highest Ki-67 index). Morphologic differences were evident in each tumor microenvironment. Finally, 7 genes were differentially expressed in the Met-1 tumors in the 6 sites of growth or metastasis. This investigation demonstrates that breast cancer progression and metastasis are regulated by not only the tumor cells but also the experimental model and unique molecular signals from the tumor microenvironment. [ABSTRACT FROM PUBLISHER]

The genetic basis of quantitative traits such as body weight and obesity is complex, with several hundred quantitative trait loci (QTLs) known to affect these and related traits in humans and mice. It also has become increasingly evident that the direct effects of these QTLs vary considerably depending on factors such as the sex of the individuals and their dietary environment, and we were interested to know whether this context-dependency also applies to indirect epistatic effects of QTLs as well. We therefore conducted a genome scan to search for epistatic effects on 13 different weight and adiposity traits in an F2 population of mice (created from an original intercross of the FVB strain with M16i, a polygenic obesity model) that were fed either a control or a high-fat diet and half of which harbored a transgene (PyMT) that caused the development of metastatic mammary cancer. We used a conventional interval mapping approach with SNPs to scan all 19 autosomes, and found extensive epistasis affecting all of these traits. More importantly, we also discovered that the majority of these epistatic effects exhibited significant interactions with sex, diet, and/or presence of PyMT. Analysis of these interactions showed that many of them appeared to involve QTLs previously identified as affecting these traits, but whose direct effects were variously modified by indirect epistatic effects of other QTLs depending on the sex, diet, or PyMT environment. It was concluded that this context dependency of epistatic effects is an important component of the genetic architecture of complex traits such as those contributing to weight and obesity.

Morbidity and mortality of 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, an increased molecular understanding to identify biomarkers and therapeutic targets is needed. Breast cancer metastasis suppressor 1 (BRMS1) suppresses development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Little is known of Brms1 function throughout the initiation and progression of mammary carcinoma. The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using polyoma middle T oncogene-induced models. Brms1 expression did not significantly alter growth of the primary tumors. When expressed ubiquitously using a β-actin promoter, Brms1 suppressed pulmonary metastasis and promoted apoptosis of tumor cells located in the lungs but not in the mammary glands. Surprisingly, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in the mammary glands or lung, despite MMTV-induced expression within the lungs. These results strongly suggest that cell type-specific over-expression of Brms1 is important for Brms1-mediated metastasis suppression. [ABSTRACT FROM AUTHOR]

Metastasis virulence, a significant contributor to breast cancer prognosis, is influenced by environmental factors like diet. We previously demonstrated in an F2 mouse population generated from a cross between the M16i polygenic obese and MMTV-PyMT mammary cancer models that high fat diet (HFD) decreases mammary cancer latency and increases pulmonary metastases compared to a matched control diet (MCD). Genetic analysis detected eight modifier loci for pulmonary metastasis, and diet significantly interacted with all eight loci. Here, gene expression microarray analysis was performed on mammary cancers from these mice. Despite the substantial dietary impact on metastasis and its interaction with metastasis modifiers, HFD significantly altered the expression of only five genes in mammary tumors; four of which, including serum amyloid A ( Saa), are downstream of the tumor suppressor PTEN. Conversely, HFD altered the expression of 211 hepatic genes in a set of tumor free F2 control mice. Independent of diet, pulmonary metastasis virulence correlates with mammary tumor expression of genes involved in endocrine cancers, inflammation, angiogenesis, and invasion. The most significant virulence-associated network harbored genes also found in human adipose or mammary tissue, and contained upregulated Vegfa as a central node. Additionally, expression of Btn1a1, a gene physically located near a putative cis-acting eQTL on chromosome 13 and one of the metastasis modifiers, correlates with metastasis virulence. These data support the existence of diet-dependent and independent cancer modifier networks underlying differential susceptibility to mammary cancer metastasis and suggest that diet influences cancer metastasis virulence through tumor autonomous and non-autonomous mechanisms. [ABSTRACT FROM AUTHOR]

Mice transgenic for MUC1 (mucin 1) and polyomavirus middle T (PyMT) develop mammary carcinomas within 15 weeks with 100% penetrance. PyMT-induced mammary tumorigenesis is closely correlated with robust telomerase expression and activity. To assess the role of telomerase activation and telomere maintenance in mammary carcinoma tumorigenesis, we generated mice expressing MUC1 and PyMT (MMT mice) but deficient in the telomerase RNA component, mTerc, on the C57BL/6 background. Successive generational intercrosses of mTerc−/−MMT mice produced cohorts with progressively shorter telomeres that were audited for mammary tumor formation. Relative to MMT (N=14) and G0 mTerc+/− female controls (G0=14), mTerc−/−MMT females (G1=11, G2=15, G3=15 and G4=5) showed decreased tumor volumes and increased tumor latency—MMT=95.6 days; G0 mTerc+/−MMT=98.6 days versus G1, G2, G3 and G4 mTerc−/−MMT mice with latencies of 122.6, 138.9, 140.7 and 220.9 days, respectively (controls versus G1–G4, P<0.005). The progressive impairment of lung metastasis was also observed with each successive mTerc−/−MMT generation. The impairment of tumorigenesis was associated with decreased proliferation of mammary epithelial and tumor cells and increased apoptosis of tumor cells. Together, these results indicate that, in the setting of viral oncoprotein mammary tumorigenesis, telomerase-dependent telomere maintenance facilitates the formation and metastatic progression of mammary tumors. [ABSTRACT FROM AUTHOR]

Membrane-type I matrix metalloproteinase (MT1-MMP) is associated with multiple forms of cancer including mammary cancer. To directly evaluate the significance of MT1-MMP expression in tumor progression and metastasis using a genetically induced cancer model, we crossed MT1-MMP-deficient mice to MMTV–polyoma virus middle T-antigen (PyMT) mice. Expression of PyMT in the MT1-MMP-deficient background consistently resulted in hyperplasia of the mammary gland as seen in wild-type PyMT littermates. Following orthotopic transplantation of PyMT+ glands into the cleared mammary fat pad of syngeneic recipient mice, MT1-MMP-deficient tumors were palpable earlier than wild-type tumors. Moreover, MT1-MMP-deficient tumors grew to the experimental end point size quicker than control tumors, but demonstrated markedly reduced ability to metastasize to the lungs of recipient mice. Accordingly, MT1-MMP-deficient mice displayed an overall reduction in metastasis count of 50%. MT1-MMP was expressed solely in the stroma of PyMT-induced tumors and those metastatic nodules that formed in the lungs were devoid of MT1-MMP expression. Stromal fibroblasts isolated from MT1-MMP-deficient tumors did not degrade type I collagen suggesting that efficient dissemination of tumor cells is dependent on stromal cell remodeling of the tumor environment. The data demonstrate directly that MT1-MMP-mediated proteolysis by stromal cells is important in the metastatic process.Oncogene (2008) 27, 3274–3281; doi:10.1038/sj.onc.1210982; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

Abstract: Genetic depletion of macrophages in Polyoma Middle T oncoprotein (PyMT)-induced mammary tumors in mice delayed the angiogenic switch and the progression to malignancy. To determine whether vascular endothelial growth factor A (VEGF-A) produced by tumor-associated macrophages regulated the onset of the angiogenic switch, a genetic approach was used to restore expression of VEGF-A into tumors at the benign stages. This stimulated formation of a high-density vessel network and in macrophage-depleted mice, was followed by accelerated tumor progression. The expression of VEGF-A led to a massive infiltration into the tumor of leukocytes that were mostly macrophages. This study suggests that macrophage-produced VEGF regulates malignant progression through stimulating tumor angiogenesis, leukocytic infiltration and tumor cell invasion. [Copyright &y& Elsevier]

The breast epithelium comprises cells at different stages of differentiation, including stem cells, progenitor cells, and more differentiated epithelial and myoepithelial cells. Wnt signaling plays a critical role in regulating stem/ progenitor cells in the mammary gland as well as other tissue compartments. Furthermore, there is strong evidence suggesting that aberrant activation of Wnt signaling induces mammary tumors from stem/progenitor cells, and that Wnt exerts its oncogenic effects through LRP5/6-mediated activation of β-catenin and mTOR pathways. Recent studies using avian retrovirus-mediated introduction of oncogenes into a small subset of somatic mammary cells suggest that polyoma middle T antigen (PyMT) may also preferentially transform stem/progenitor cells. These observations suggest that stem/progenitor cells in the mammary gland may be especially susceptible to oncogenic transformation. Whether more differentiated cells may also be transformed by particular oncogenes is actively debated; it is presently unclear whether stem cells or differentiated mammary cells are more susceptible to transformation by individual oncogenes. Better stem cell and progenitor cell markers as well as the ability to specifically target oncogenes into different mammary cell types will be needed to determine the spectrum of oncogene transformation for stem cells versus more differentiated cells. [ABSTRACT FROM AUTHOR]

We have generated a mouse model for pancreatic cancer through the somatic delivery of oncogene-bearing avian retroviruses to mice that express TVA, the receptor for avian leukosis sarcoma virus subgroup A (ALSV-A), under the control of the elastase promoter. Delivery of ALSV-A-based RCAS vectors encoding either mouse polyoma virus middle T antigen (PyMT) or c-Myc to elastase-tv-a transgenic, Ink4a/Arf null mice induced the formation of pancreatic tumors. RCAS-PyMT induced pancreatic tumors with the histologic features of acinar or ductal carcinomas. The induced pancreatic lesions express Pdx1, a marker for pancreas progenitor cells, and many tumors express markers for both exocrine and endocrine cell lineages, suggesting that the tumors may be derived from progenitor cells. In contrast, RCAS-c-myc induced endocrine tumors exclusively, as determined by histology and detection of differentiation markers. Thus, specific oncogenes can induce the formation of different pancreatic tumor types in a single transgenic line, most likely from one or more types of multipotential progenitor cells. Our model appears to be useful for elucidating the genetic alterations, target cells, and signaling pathways that are important in the genesis of different types of pancreatic cancer. [ABSTRACT FROM AUTHOR]