Spontaneous Mammary Tumor Model Research Articles

Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9high CSCs. In addition, SOX9high and ABCA12high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

429 Eribulin Synergizes with STING Agonists by Enhancing Type 1 Interferon Expression and Improves Antitumor Efficacy as Combination Treatment

OBJECTIVES/GOALS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks effective targeted treatment options. TNBC's greater degree of immunogenicity than other breast tumors makes immunotherapy a viable strategy. Strategies to improve the immunotherapy response includes targeting the cGAS-STING innate immune pathway with STING agonists. METHODS/STUDY POPULATION: We have previously shown in vitro that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Separately, eribulin also significantly enhances type I interferon expression induced by STING agonists measured by qRT-PCR through a second TBK1-dependent mechanism downstream of STING activation through detecting higher amounts of phosphorylated IRF-3 by western blot protein analysis. Mechanisms of eribulin-mediated interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizing agent paclitaxel. RESULTS/ANTICIPATED RESULTS: We determine that the enhancement of type I interferon expression by eribulin is pharmacologically synergistic with multiple STING agonists. The significant enhancement by eribulin led us to evaluate the antitumor efficacy of eribulin in combination the STING agonist ADU-S100 in a challenging spontaneous mammary tumor model MMTV-PyVT. We show that the combination treatment significantly decreased tumor growth which allowed for longer survival compared to other groups. This is particularly interesting because of our previous studies showing that eribulin alone, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleen and draining lymph nodes of BALB/c mice with 4T1 tumors through flow cytometric analysis. DISCUSSION/SIGNIFICANCE: These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin can act as an immune adjuvant in addition to its anti-mitotic effect.

Tumor-derived Jagged1 promotes cancer progression through immune evasion.

Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating Tcells to inhibit Tcell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased Tcell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

Flash Irradiation Inhibits Breast Onset and Spares Radiation-Induced Liver Injury

<h3>Purpose/Objective(s)</h3> To clarify the efficacy of FLASH radiotherapy (FLASH-RT) in inhibiting mammary tumorigenesis and normal tissue sparing. <h3>Materials/Methods</h3> We used a spontaneous mammary tumor model of MMTV-PyMT mice for the study. Prior to the formation of mammary tumor, MMTV-PyMT mice (3–6-week-old) were treated with a single fraction dose of 9-16 Gy FLASH-RT or conventional radiotherapy (CONV-RT) using a mice-applicator designed targeting five pair of mammary glands. Tumor growth was detected by thorough palpation of all 10 mammary glands twice a week. Liver function, H&E staining and TUNEL staining of liver tissues were performed to determine the irradiation-induced liver injury. Inflammatory cytokines of TNF-a, IFN-g, IL-6 and IL-10 in the liver tissue and in the serum on Days 6 (D6), 18 (D18) and 31 days (D31) post-irradiation (IR) were tested by ELISA. DHE staining in frozen sections of tumor tissue and liver tissue at 4 weeks post-IR were performed, respectively. <h3>Results</h3> Median days for first tumor occurrence were 65, 62, 58.5 and 52 days of age, while median days for the involvement of 10th mammary gland were 79, 76, 76 and 63 days of age for FLASH (14-16 Gy), FLASH (9-12 Gy), CONV (9-12 Gy) and Control (0 Gy) groups, respectively. Tumor weight at 12 weeks after birth in FLASH and CONV were significantly lower than controls. On 18 days after IR, serum ALT level was significantly increased in the CONV compared with the samples in FLASH. H&E staining of liver in 7 weeks after 9-12 Gy IR revealed that FLASH caused less histological abnormalities in comparison to the samples from CONV, which was similar to the tissue of control. The number of TUNEL-positive hepatocytes in the FLASH was less than that of the CONV but similar to that of the control. In liver tissue, TNF-a, IFN-g and IL-6 significantly increased in CONV compared with FLASH and control; however, IFN-g and IL-6 also significantly increased in FLASH compared with control, while IL-10 significantly decreased in CONV compared with control. Serum IL-6, IFN-g and TNF-a levels in FLASH did not markedly change on D6, D18 and D31 post-IR compared with control. Serum IL-6, IFN-g and TNF-a levels were elevated on D6 and continuously increased on D18 and D31 in CONV, but began to increase on D6, and then recovered to the control level on D18 or D31 in FLASH. Serum IL-10 levels decreased at D6, D18 and D31 in CONV, but began to decrease at D6, and then recovered to the control level at D18 or D31 in FLASH. FLASH induced reactive oxygen species (ROS) accumulation in the tumor tissue but not in normal tissue; while absence of ROS accumulation in either tumor or normal tissue under CONV. Notably, the ROS level of normal liver tissue did not differ under CONV or FLASH. <h3>Conclusion</h3> We found that mammary tumorigenesis was inhibited by FLASH without significant liver injury compared with CONV-RT. The alteration of serum and liver inflammatory cytokines induced by CONV-RT was mildly altered by FLASH. This unique benefit can by partially explained by the accumulatio of ROS in tumor tissue after FLASH.

Abstract 138: TNBC induces immune suppressive tumor microenvironments through microRNAs

Abstract Metastatic breast cancer is the second leading cause of cancer-related deaths for US women. The triple negative breast cancer subtype (TNBC) is one of the major contributors to breast cancer mortality, among all types of breast cancer. There are no effective treatments available for metastatic tumors. Therefore, identifying and targeting novel tumorigenesis pathways will facilitate developing effective therapeutic approaches that could ultimately lead to effective treatment for breast cancer, particularly metastatic cancer and TNBC. Our results demonstrate that miR-146a promotes tumorigenesis and metastasis of breast cancer. We found that miR-146a was markedly overexpressed in TNBC cell lines. Notably, miR-146a deficiency in a spontaneous mammary tumor model leads to reduced tumor growth and metastasis. Notably, miR-146a shifts the polarization of macrophages into immunosuppressive phenotypes. Our studies suggest that miR-146a may contribute to tumor development through modulation of tumor microenvironments. Note: This abstract was not presented at the meeting. Citation Format: Beichu Guo, Melvin Kantono, Wei Sun. TNBC induces immune suppressive tumor microenvironments through microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 138.

A Versatile Tumor Gene Deletion System Reveals a Crucial Role for FGFR1 in Breast Cancer Metastasis

RCAS avian viruses have been used to deliver oncogene expression and induce tumors in transgenic mice expressing the virus receptor TVA. Here we report the generation and characterization of a novel RCAS-Cre-IRES-PyMT (RCI-PyMT) virus designed to specifically knockout genes of interest in tumors generated in appropriate mutant mouse hosts. FGF receptor 1 (FGFR1) is a gene that is amplified in human breast cancer, but there have been no definitive studies on its function in mammary tumorigenesis, progression, and metastasis in vivo in spontaneous tumors in mice. We used the retroviral tumor knockout, or TuKO, strategy to delete fgfr1 in PyMT-induced mammary tumors in K19-tva/fgfr1loxP/loxP mice. The similarly injected control K19-tva mice developed mammary tumors exhibiting high metastasis to lung, making this an ideal model for breast cancer metastasis. The fgfr1 TuKO tumors showed significantly decreased primary tumor growth and, most importantly, greatly reduced metastasis to lung. In contrast to previous reports, FGFR1 action in this spontaneous mammary tumor model does not significantly induce epithelial-to-mesenchymal transition. Loss of FGFR1 does generate a gene signature that is reverse correlated with FGFR1 gene amplification and/or upregulation in human breast cancer. Our results suggest that FGFR1 signaling is a key pathway driving breast cancer lung metastasis and that targeting FGFR1 in breast cancer is an exciting approach to inhibit metastasis.

Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer.

Melanoma differentiation associated gene-7/Interleukin-24 (MDA-7/IL-24) is a novel member of the IL-10 gene family that selectively induces apoptosis and toxic autophagy in a broad spectrum of human cancers, including breast cancer, without harming normal cells or tissues. The ability to investigate the critical events underlying cancer initiation and progression, as well as the capacity to test the efficacy of novel therapeutics, has been significantly advanced by the development of genetically engineered mice (GEMs) that accurately recapitulate specific human cancers. We utilized three transgenic mouse models to better comprehend the in vivo role of MDA-7/IL-24 in breast cancer. Using the MMTV-PyMT spontaneous mammary tumor model, we confirmed that exogenously introducing MDA-7/IL-24 using a Cancer Terminator Virus caused a reduction in tumor burden and also produced an antitumor "bystander" effect. Next we performed xenograft studies in a newly created MMTV-MDA-7 transgenic model that over-expresses MDA-7/IL-24 in the mammary glands during pregnancy and lactation, and found that MDA-7/IL-24 overexpression delayed tumor growth following orthotopic injection of a murine PDX tumor cell line (mPDX) derived from a tumor formed in an MMTV-PyMT mouse. We also crossed the MMTV-MDA-7 line to MMTV-Erbb2 transgenic mice and found that MDA-7/IL-24 overexpression delayed the onset of mammary tumor development in this model of spontaneous mammary tumorigenesis as well. Finally, we assessed the role of MDA-7/IL-24 in immune regulation, which can potentially contribute to tumor suppression in vivo. Our findings provide further direct in vivo evidence for the role of MDA-7/IL-24 in tumor suppression in breast cancer in immune-competent transgenic mice.

Tumor Endothelial Cells with Distinct Patterns of TGFβ-Driven Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFβ stimulation. Although some TECs strikingly upregulate α smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFβ treatment. Compared with NECs, SMA(+) TECs were 40% less motile in wound-healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreen(Cdh5-Cre) reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA(+) TECs, suggesting that not all endothelial cells (EC) respond identically to TGFβ in vivo. Indeed, examination of 84 TGFβ-regulated target genes revealed entirely different genetic signatures in TGFβ-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFβ-regulated genes but operates in tandem with TGFβ to upregulate others. ECs challenged with TGFβ secrete bFGF, which blocks SMA expression in secondary cultures, suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFβ-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature.

Natural history of tumor growth and immune modulation in common spontaneous murine mammary tumor models.

Recent studies in patients with breast cancer suggest the immune microenvironment influences response to therapy. We aimed to evaluate the relationship between growth rates of tumors in common spontaneous mammary tumor models and immune biomarkers evaluated in the tumor and blood. TgMMTV-neu and C3(1)-Tag transgenic mice were followed longitudinally from birth, and MPA-DMBA-treated mice from the time of carcinogen administration, for the development of mammary tumors. Tumor-infiltrating CD4(+) and CD8(+) T-cells, FOXP3(+) T-regulatory cells, and myeloid-derived suppressor cells were assessed by flow cytometry. Serum cytokines were evaluated in subsets of mice. Fine needle aspirates of tumors were collected and RNA was isolated to determine levels of immune and proliferation markers. Age of tumor onset and kinetics of tumor growth were significantly different among the models. Mammary tumors from TgMMTV-neu contained a lower CD8/CD4 ratio than that of other models (p < 0.05). MPA-DMBA-induced tumors contained a higher percentage of FOXP3(+) CD4(+) T-cells (p < 0.01) and MDSC (p < 0.001) compared with the other models. Individuals with significantly slower tumor growth demonstrated higher levels of Type I serum cytokines prior to the development of lesions compared to those with rapid tumor growth. Moreover, the tumors of animals with more rapid tumor growth demonstrated a significant increase in the expression of genes associated with Type II immunity than those with slower-progressing tumors. These data provide a foundation for the development of in vivo models to explore the relationship between endogenous immunity and response to standard therapies for breast cancer.

Abstract SY40-04: Immune control of spontaneous tumor development

Abstract Long recognized as an evolutionarily ancient cell type to control development, tissue homeostasis, and immune responses to pathogens, macrophages are being rediscovered as regulators of several chronic disorders including cancer. Using a spontaneous mammary tumor model, here we show that tumor growth induces the differentiation and expansion of a unique subset of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). Compared to MTMs, TAMs do not exhibit an “alternatively activated macrophage” phenotype, but express the adhesion molecule Vcam1, and have high proliferative capacity upon their differentiation from CCR2-expressing inflammatory monocytes. Importantly, TAM differentiation is dependent on the transcriptional regulator of the Notch signaling pathway, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses, and suppresses tumor growth. These findings demonstrate that TAMs represent a novel lineage of mononuclear phagocytes with critical regulatory functions in promoting T cell tolerance to tumors. Citation Format: Ming Li. Immune control of spontaneous tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY40-04. doi:10.1158/1538-7445.AM2014-SY40-04

A model of spontaneous mouse mammary tumor for human estrogen receptor- and progesterone receptor-negative breast cancer.

Breast cancer (BC) is the most frequently malignancy in women. Therefore, establishment of an animal model for the development of preventative measures and effective treatment for tumors is required. A novel heterogeneous spontaneous mammary tumor animal model of Kunming mice was generated. The purpose of this study was to characterize the spontaneous mammary tumor model. Histopathologically, invasive nodular masses of pleomorphic tubular neoplastic epithelial cells invaded fibro-vascular stroma, adjacent dermis and muscle tissue. Metastatic spread through blood vessel into liver and lungs was observed by hematoxylin eosin staining. No estrogen receptor (ER) or progesterone receptor (PR) immunoreactivity was detected in their associated malignant tumors, human epidermal growth factor receptor-2 (HER-2) protein weak expression was found by immunohistochemistry. High expression of vascular endothelial growth factor (VEGF), moderate or high expression of c-Myc and cyclin D1 were observed in tumor sections at different stages (2, 4, 6 and 8 weeks after cancer being found) when compared with that of the normal mammary glands. The result showed that the model is of an invasive ductal carcinoma. Remarkably in the mouse model, ER and PR-negative and HER2 weak positivity are observed. The high or moderate expressions of breast cancer markers (VEGF, c-Myc and cyclin D1) in mammary cancer tissue change at different stages. To our knowledge, this is the first report of a spontaneous mammary model displaying colony-strain, outbred mice. This model will be an attractive tool to understand the biology of anti-hormonal breast cancer in women.

The Effect of Antineoplastic Drugs in a Male Spontaneous Mammary Tumor Model

Male breast cancer is a rare disease. The limited number of clinical cases has led to the primary treatments for men being derived from female breast cancer studies. Here the transgenic strain FVB/N-Tg(MMTV-PyVT)634Mul/J (also known as PyVT) was used as a model system for measuring tumor burden and drug sensitivity of the antineoplastic drugs tamoxifen, cisplatin, and paclitaxel on tumorigenesis at an early stage of mammary carcinoma development in a male mouse model. Cisplatin treatment significantly reduced tumor volume, while paclitaxel and tamoxifen did not attenuate tumor growth. Cisplatin treatment was shown to induce apoptosis, grossly observed by reduced tumor formation, through reduced Bcl-2 and survivin protein expression levels with an increase in caspase 3 expression compared to control tumors. Tamoxifen treatment significantly altered the hormone receptor expression levels of the tumor, while additionally upregulating Bcl-2 and Cyclin D1. This suggests an importance in hormonal signaling in male breast cancer pathogenesis. The results of this study provide valuable information toward the better understanding of male breast cancer and may help guide treatment decisions.

Abstract 3268: Histological and biological effects of substituted quinolines (gap junction enhancers) in a spontaneous mammary tumor model

Abstract Effective use of antineoplastic drugs depends on the ability to balance the killing of tumor cells against the inherent toxicity to the host. Antineoplastic agents that act primarily on rapidly dividing and growing cells produce multiple side effects and are dose limiting. In recent years, there has been the development of biologic response modifiers aimed at enhancing innate anti-tumor defense mechanisms. Previous publications showed that substituted quinolines possess potent inhibitory activities against breast cancer cells through the enhancement of gap junction intercellular communication. These drugs decrease 71% of xenograft breast tumors in nude mice. The objective of this project is to evaluate the effect of substituted quinolines in a spontaneous mouse mammary tumor model (PyVT). PyVT mice were used at three different stages (pre, early and late) to characterize histological and biological changes in the treated animals compared to controls. Substituted quinolines cause a decrease of tumor growth rate by 112%, 77%, and 57% in the pre, early, and late stages of tumor development, respectively. Treatment during the pre and early stages of tumor formation also resulted in a decrease in the total number of tumors. Western blot analysis of the pre stage tumors show that the gap junction protein connexin 43 is more expressed in treated animals compared to control and the contrary for connexin 46. In the early and late stages, connexin 43 is expressed less in treated mice than controls, and vice versa for connexin 46. The expression of these connexins in control tissue suggests that this is due to an overall increase in connexin 43 and a decrease in connexin 46 during tumor development and metastasis. Histology of vital organs showed no significant alteration in treated animals compared to controls. These findings provide evidence that substituted quinolines cause a significant tumor reduction in a spontaneous mammary tumor model with no obvious adverse effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3268. doi:1538-7445.AM2012-3268

Abstract LB-456: The intraductal injection of 5-fluorouracil induces immune response to eradicate and prevent breast cancer

Abstract Chemotherapy has been shown to act as an immune response modifier. Many groups, incuding our own, are using chemotherapy to induce immune responses such as by using low doses of cyclophosphamide. Our previous work has demonstrated that anti-cancer agents can eliminate new/existing tumors with minimal toxicity in rat and mouse models of breast cancer. Here we demonstrate that intraductal (i.duc), but not the intravenous (IV) route of injection, of chemotherapy to mammary glands alters the tumor environment to effectively induce immune effector cells. We used the mouse HER2/neu (neu/N) transgenic, spontaneous mammary tumor model. 5-fluorouracil (5FU) was administered intraductally to the mammary glands on the left side of parous mice whereas the mammary glands on right side received no treatment (NT). The mammary tumor incidence in the 5FU treated side was significantly lower compared to mice that received NT, and IV treated group. Interestingly the incidence of mammary tumors in the untreated side of 5FU-treated mice was also significantly lower compared to the NT and IV group. We hypothesized that the protection afforded to the contralateral chain of mammary glands by ipsilateral i.duc administration of 5FU, may be mediated through an immunological mechanism. Twenty week parous mice were administered 5FU either through i.duc injection, only to left side teats, IV injection, or NT. The mice received 5FU 2 times in a 4 week interval. A week after the second treatment, the mice were sacrificed and the regional lymphnodes (RLN) of the 3rd and 4th mammary glands and spleen were removed. We isolated lymphocyte from RLN and spleen to analyze by flowcytometry. The number of CD8 T cell showed no change among the groups in the RLN but was significantly lower in the i.duc group in the spleen. The number of CD62LLOW+ T cell in 5FU treated side of RLN was significantly higher compared to the IV and NT group. On the other hand the number of CD62LLOW+ was low in the spleen. To study the population of systemic memory T cell in the peripheral blood, blood was harvested into Trucount tubes and analyzed with memory T cells surface marker. Twenty week parous mice was administered 5FU i.duc injection only to the left side, IV injection, or NT every 4weeks for a total of 3 times. Cancer cells were injected into the left side of 4th mammary gland fat pad of the mice at 16 weeks after initial procedure. One hundred μl blood was collected 5 days before and 5 days after cancer cell injection. The mice treated by i.duc 5FU showed increased numbers of CD95+/CD62L low effector memory T (TEM) cells, whereas the mice treated with IV 5FU did not recruit TEM cells significantly. There was no significant change in CD95+/CD62L high central memory T cells before and after treatment and among the groups. In summary, i.duc administration of 5FU into mammary glands effectively induced immune effector cells and prevented mammary tumor growth in neu/N transgenic mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-456. doi:10.1158/1538-7445.AM2011-LB-456

Elevated Expression of DecR1 Impairs ErbB2/Neu-Induced Mammary Tumor Development

Tumor cells utilize glucose as a primary energy source and require ongoing lipid biosynthesis for growth. Expression of DecR1, an auxiliary enzyme in the fatty acid beta-oxidation pathway, is significantly diminished in numerous spontaneous mammary tumor models and in primary human breast cancer. Moreover, ectopic expression of DecR1 in ErbB2/Neu-induced mammary tumor cells is sufficient to reduce levels of ErbB2/Neu expression and impair mammary tumor outgrowth. This correlates with a decreased proliferative index and reduced rates of de novo fatty acid synthesis in DecR1-expressing breast cancer cells. Although DecR1 expression does not affect glucose uptake in ErbB2/Neu-transformed cells, sustained expression of DecR1 protects mammary tumor cells from apoptotic cell death following glucose withdrawal. Moreover, expression of catalytically impaired DecR1 mutants in Neu-transformed breast cancer cells restored Neu expression levels and increased mammary tumorigenesis in vivo. These results argue that DecR1 is sufficient to limit breast cancer cell proliferation through its ability to limit the extent of oncogene expression and reduce steady-state levels of de novo fatty acid synthesis. Furthermore, DecR1-mediated suppression of tumorigenesis can be uncoupled from its effects on Neu expression. Thus, while downregulation of Neu expression may contribute to DecR1-mediated tumor suppression in certain cell types, this is not an obligate event in all Neu-transformed breast cancer cells.

Soluble receptor for advanced glycation endproducts (sRAGE) effectively reduces tumor growth as a single agent and in combination with doxorubicin in a spontaneous mammary tumor model

Introduction: The receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin family interacts with distinct ligands that have been implicated in various pathophysiologic states such as diabetes, atherosclerosis, inflammation as well as tumor growth. We have previously demonstrated that blockade of RAGE in tumors raised in mice from rat C6 glioma cells or murine Lewis lung carcinoma effectively reduced tumor growth, invasion and metastases. Blockade of RAGE has also previously been shown to halt the progression of breast tumors in a murine model that spontaneously produces breast tumors. We hypothesize that the addition of RAGE antagonists to standard chemotherapeutic agents for breast cancer may enhance the effectiveness of monotherapy. Methods: Female MMTV transgenic mice (5–8 weeks) were divided into four groups: (1) Control group-no treatment, (2) doxorubicin (DOX) 2 mg/kg IP weekly x 4 weeks (3) sRAGE 20 μg IP daily x 4 weeks and (4) DOX 2 mg/kg IP weekly x 4 weeks and sRAGE 20 μg IP daily x 4 weeks. The mice were sacrificed and tumors harvested on treatment day 35 and were weighed and measured. Results: There was no difference in tumor weight between the control mice (n = 9) and DOX-treated mice (n = 4) (0.192 g v. 0.193). The administration of sRAGE (n = 7) decreased tumor weight to 0.12 g and the combination of sRAGE and DOX (n = 8) decreased tumor weight by a third (0.192 g v. 0.061 g, p = 0.11). While the mean tumor volume in the control mice was 316.7 mm3, sRAGE decreased tumor volume to 89.65 mm3 and the combination of sRAGE and DOX decreased tumor volume by 6.5 fold (316.7 mm3 vs. 48.08 mm3, p = 0.006). The mean tumor volume in the DOX treated group was 500.9 mm3. Conclusions: RAGE antagonists suppress local tumor growth in transgenic mice with spontaneously occurring cancer in an early intervention model. The addition of sRAGE, a low molecular weight RAGE antagonist, to doxorubicin enhanced the effectiveness of this standard chemotherapy agent.

DNA vaccination with full-length or truncated neu induces protective immunity against the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

Genetic immunization against tumor antigens is an effective way to induce an immune response able to oppose cancer progression. Overexpression of HER-2/neu can lead to neoplastic transformation and has been found in many human primary breast cancers. We constructed DNA expression vectors encoding the full-length neu oncogene of rat cDNA (pCMV-NeuNT), the neu extracellular domain (pCMV-ECD), or the neu extracellular and transmembrane domains (pCMV-ECD-TM). We evaluated whether i.m. injection of these plasmids induces protection against the development of mammary tumors occurring spontaneously in FVB/N neu-transgenic mice. We found that pCMV-ECD-TM induced the best protection, whereas both pCMV-ECD and pCMV-NeuNT were less effective. The coinjection with a bicistronic vector for murine IL-12 increased the efficacy of pCMV-ECD and pCMV-NeuNT plasmids, and led to the same protection obtained with pCMV-ECD-TM alone. Anti-neuECD antibodies were detected in pCMV-ECD-TM vaccinated mice and, after coinjection with pCMV-IL12 plasmids, they appeared also in animals immunized with pCMV-ECD. Our data demonstrate the effectiveness of DNA vaccination using truncated Neu plasmids in inducing antitumor protection in a spontaneous mammary tumor model.