Neu Mice Research Articles

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

Metastasis is altered through multiple processes regulated by the E2F1 transcription factor

The E2F family of transcription factors is important for many cellular processes, from their canonical role in cell cycle regulation to other roles in angiogenesis and metastasis. Alteration of the Rb/E2F pathway occurs in various forms of cancer, including breast cancer. E2F1 ablation has been shown to decrease metastasis in MMTV-Neu and MMTV-PyMT transgenic mouse models of breast cancer. Here we take a bioinformatic approach to determine the E2F1 regulated genomic alterations involved in the metastatic cascade, in both Neu and PyMT models. Through gene expression analysis, we reveal few transcriptome changes in non-metastatic E2F1−/− tumors relative to transgenic tumor controls. However investigation of these models through whole genome sequencing found numerous differences between the models, including differences in the proposed tumor etiology between E2F1−/− and E2F1+/+ tumors induced by Neu or PyMT. For example, loss of E2F1 within the Neu model led to an increased contribution of the inefficient double stranded break repair signature to the proposed etiology of the tumors. While the SNV mutation burden was higher in PyMT mouse tumors than Neu mouse tumors, there was no statistically significant differences between E2F WT and E2F1 KO mice. Investigating mutated genes through gene set analysis also found a significant number of genes mutated in the cell adhesion pathway in E2F1−/− tumors, indicating this may be a route for disruption of metastasis in E2F1−/− tumors. Overall, these findings illustrate the complicated nature of uncovering drivers of the metastatic process.

Participation of FAS- and TNF-Dependent Pathways in Apoptosis Mechanisms in Hypothalamus in Physiological and Pathological Aging

Cellular resistance to apoptosis can be related to the activity of cytokine-dependent signaling. Thus, the goal of the work is to study the mechanisms of cytokine-dependent, FAS/TNF-mediated regulation of apoptosis of neurosecretory cells in physiological and pathological (overexpression of the oncogene HER-2/Neu) aging. Transgenic, accelerated-aged HER-2/Neu mice of different ages and wild-type FVB/N mice were examined. The apoptosis level of neurons in hypothalamic sections (supraoptic and paraventricular nuclei) (TUNEL) and the expression of caspase-8, CD178 (FASL), FAS, FADD, and TRADD (Western blotting) was determined. It is shown that a proinflammatory component participates in the aging process. The expression of FAS, adapter proteins associated with the death domain (FADD and TRADD), and caspase-8 is activated in the hypothalamus in FVB/N (wild-type) mice during aging, and it correlates with an increase in the apoptosis level. HER-2/Neu expression leads to suppression of the extrinsic apoptotic pathway. In this case, the reception of an apoptotic signal (FAS-receptor expression) and its further transmission (expression of FADD and TRADD) is suppressed. However, a sufficiently high TRADD expression in young transgenic mice and an increased expression of TNF-α in old mice can activate one of the survival pathways (NF-κB, ERK or PI3K-AKT). Thus, the HER-2/Neu tyrosine kinase receptor plays a role in the mechanism of cell resistance to age-dependent apoptosis, and the FAS/TNF-signaling pathway is one of the targets of HER-2/Neu.

Anti‐cancer Effects of Melatonin, Estrogen, and Progesterone Hormone Therapy in MCF‐7 and MDA‐MB‐231 Breast Cancer Cells

A novel melatonin, estrogen (E2), and progesterone (P4) hormone therapy (MEPT) was developed as a safe bio‐identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer (BC) risk and mortality of all types of BC in postmenopausal women. MEPT delayed tumor onset and reduced tumor incidence in Neu female mice (Dodda et al., 2019; Front Oncol). For other BCs, its actions are currently unknown. In this study, MEPT’s anti‐cancer actions (cell viability, migration) were tested in MCF‐7 (human ER+/PR+) and MDA‐MB‐231 (human TNBC with constitutively active ras mutation) using equivalent concentrations of melatonin (1nM), E2 (50pM), and P4 (10nM) observed in serum after an oral dose that produced anti‐tumor actions in neu mice (Dodda et al 2019; Front Oncol). Potential mechanisms underlying MEPT’s anti‐cancer actions were assessed by western blot (pERK1/2, pERK5, NF‐kB, RUNX2, β1‐INTEGRIN, RANKL, and ELF‐5) using MEK1/2 (PD98059) and MEK5 (Bix02189) inhibitors and radioligand binding using 2‐[125I]‐iodomelatonin or [3H]estradiol binding for melatonin and estrogen receptor expression, respectively. For MCF‐7 cells, BIX02189 combined with MEPT blocked MEPT‐mediated inhibition of migration and MEPT‐mediated ELF5 induction in MCF‐7 cells (vs. vehicle) and decreased pERK5 (vs. Bix02189) suggesting that MEK5, through pERK1/2 and ELF5, were involved in the anti‐cancer actions of MEPT in MCF‐7 cells. For MDA‐MB‐231 cells, although PD98059 and Bix02189 alone inhibited viability and migration, when added in combination with MEPT, no further blockade or enhancement occurred suggesting that MEK1/2 and MEK5 were not involved in MEPT’s anti‐cancer actions in MDA‐MB‐231 cells. MEPT’s differential actions on MCF‐7 and MDA‐MB‐231 cells were most likely attributed to unique melatonin/melatonin receptor actions within each cell. Both MCF‐7 and MDA‐MB‐231 cells express high‐affinity melatonin receptors (MCF‐7: Kd = 13 pM; Bmax = 20.55 fmol/mg protein vs. MDA‐231: Kd = 1.7 nM; Bmax = 60.09 fmol/mg protein) but only in MCF‐7 cells were [3H]‐estradiol binding sites detected (MCF‐7: Kd = 2.2 nM; Bmax = 8.6 pmol/mg protein) and stimulatory actions of E2 or P4 on MCF‐7 cell proliferation observed. These findings demonstrate novel anti‐cancer actions of MEPT in ER+ BC and TNBC cells through distinct mechanisms. Future studies assessing MEPT’s actions against ER+ BC and TNBC in vivo are warranted.Support or Funding InformationThis study was funded by the Marie‐Clement Rodier, C.S.Sp. Endowed Chair.

Everolimus Inhibits the Progression of Ductal Carcinoma In Situ to Invasive Breast Cancer Via Downregulation of MMP9 Expression.

We evaluated the role of everolimus in the prevention of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) progression. The effects of everolimus on breast cancer cell invasion, DCIS formation, and DCIS progression to IDC were investigated in a 3D cell culturing model, intraductal DCIS xenograft model, and spontaneous MMTV-Her2/neu mouse model. The effect of everolimus on matrix metalloproteinase 9 (MMP9) expression was determined with Western blotting and IHC in these models and in patients with DCIS before and after a window trial with rapamycin. Whether MMP9 mediates the inhibition of DCIS progression to IDC by everolimus was investigated with knockdown or overexpression of MMP9 in breast cancer cells. Everolimus significantly inhibited the invasion of human breast cancer cells in vitro. Daily intragastric treatment with everolimus for 7 days significantly reduced the number of invasive lesions from intraductal DCIS foci and inhibited DCIS progression to IDC in the MMTV-Her2/neu mouse mammary tumor model. Mechanistically, everolimus treatment decreased the expression of MMP9 in the in vitro and in vivo models, and in breast tissues from patients with DCIS treated with rapamycin for 1 week. Moreover, overexpression of MMP9 stimulated the invasion, whereas knockdown of MMP9 inhibited the invasion of breast cancer cell-formed spheroids in vitro and DCIS in vivo. Knockdown of MMP9 also nullified the invasion inhibition by everolimus in vitro and in vivo. Targeting mTORC1 can inhibit DCIS progression to IDC via MMP9 and may be a potential strategy for DCIS or early-stage IDC therapy.

Analysis of HER-2/neu Receptor Expression and the Level of Neuronal Apoptosis in HER-2/neu Transgenic Mice during Aging.

We studied neuronal death in the sensorimotor cortex, hippocampus, and supraoptic and paraventricular nuclei of the hypothalamus and dynamics of HER-2/neu expression in late ontogenesis in young and old transgenic HER-2/neu mice. Wild-type FVB/N mice served as the control. The intensity of apoptosis (TUNEL) and HER-2/neu expression (Western blotting) in the same brain regions were measured. HER-2/neu was detected in the cortex, hippocampus, and hypothalamus of transgenic and wild-type mice, and its expression increased with age. The effect of HER-2/neu on the intensity of cell death in various brain regions depended on the stage of ontogenesis and animal genotype. Enhanced expression of HER-2/neu determines low rate of cell death in the studied brain regions during pathological ageing.

Impact of obesity on breast cancer recurrence and minimal residual disease

BackgroundObesity is associated with an increased risk of breast cancer recurrence and cancer death. Recurrent cancers arise from the pool of residual tumor cells, or minimal residual disease (MRD), that survives primary treatment and persists in the host. Whether the association of obesity with recurrence risk is causal is unknown, and the impact of obesity on MRD and breast cancer recurrence has not been reported in humans or in animal models.MethodsDoxycycline-inducible primary mammary tumors were generated in intact MMTV-rtTA;TetO-HER2/neu (MTB/TAN) mice or orthotopic recipients fed a high-fat diet (HFD; 60% kcal from fat) or a control low-fat diet (LFD; 10% kcal from fat). Following oncogene downregulation and tumor regression, mice were followed for clinical recurrence. Body weight was measured twice weekly and used to segregate HFD mice into obese (i.e., responders) and lean (i.e., nonresponders) study arms, and obesity was correlated with body fat percentage, glucose tolerance (measured using intraperitoneal glucose tolerance tests), serum biomarkers (measured by enzyme-linked immunosorbent assay), and tissue transcriptomics (assessed by RNA sequencing). MRD was quantified by droplet digital PCR.ResultsHFD-Obese mice weighed significantly more than HFD-Lean and LFD control mice (p < 0.001) and had increased body fat percentage (p < 0.001). Obese mice exhibited fasting hyperglycemia, hyperinsulinemia, and impaired glucose tolerance, as well as decreased serum levels of adiponectin and increased levels of leptin, resistin, and insulin-like growth factor 1. Tumor recurrence was accelerated in HFD-Obese mice compared with HFD-Lean and LFD control mice (median relapse-free survival 53.0 days vs. 87.0 days vs. 80.0 days, log-rank p < 0.001; HFD-Obese compared with HFD-Lean HR 2.52, 95% CI 1.52–4.16; HFD-Obese compared with LFD HR 2.27, 95% CI 1.42–3.63). HFD-Obese mice harbored a significantly greater number of residual tumor cells than HFD-Lean and LFD mice (12,550 ± 991 vs. 7339 ± 2182 vs. 4793 ± 1618 cells, p < 0.001).ConclusionThese studies provide a genetically engineered mouse model for study of the association of diet-induced obesity with breast cancer recurrence. They demonstrate that this model recapitulates physiological changes characteristic of obese patients, establish that the association between obesity and recurrence risk is causal in nature, and suggest that obesity is associated with the increased survival and persistence of residual tumor cells.

Dynamic role of the codon 72 p53 single-nucleotide polymorphism in mammary tumorigenesis in a humanized mouse model

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBA-induced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Effects of cytoflavin on neuronal apoptotic processes in the murine cerebral cortex on a model of physiologicaland pathological aging

To investigate the role of cytoflavin in mechanisms for the apoptotic regulation of cerebral cortical cells during physiological and pathological aging (in the presence of HER-2/neu overexpression). HER-2/neu transgenic mice were used; wild-type FVB/N mice served as controls. The levels of apoptosis (TUNEL) and the expression of its associated proteins (p53, CD95, Mcl-1, p-AKT, and p-ERK) (Western blotting) were estimated in the sensorimotor cortex. Activation of fundamental AKT and ERK survival pathways promotes a low level of cell death in young FVB/N mice; the extrinsic receptor mechanism of apoptosis is observed to be initiated by aging. The high p-AKT levels in the cortical cells provide suppressed cell death in transgenic mice regardless of their age. After cytoflavin administration, the old wild-type mice show a lower level of apoptosis in the cortical neurons apparently due to the increased expression of the anti-apoptotic protein Mcl-1, while the old transgenic mice exhibited suppression of the AKT and ERK survival pathways and, accordingly, activation of the extrinsic receptor and p53-dependent apoptosis pathways. Thus, cytoflavin exerts a pronounced neuroprotective effect during physiological and accelerated aging, while its effect on the level of neuronal apoptosis is ambiguous and depends on the genetic line of animals. So, this is a moderate stimulation of apoptosis when its level is low in HER-2/neu mice with a high level of carcinogenesis, as well as a decrease in the high level of apoptosis in old wild-type animals, which prevents neurodegeneration.

Abstract 379: Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer

Abstract Purpose: Approximately 20% of breast cancers (BC) among US women overexpress HER2, an oncoprotein that stimulates increased cellular proliferation and survival. HER2 acts through two major signaling pathways, PI3K/Akt and Raf/MAPK, and its overexpression is associated with aggressive disease, resistance to therapy, and poor prognosis. Our laboratory has previously demonstrated in preclinical models that calorie restriction (CR) modulates HER2 BC pathogenesis, decreasing incidence and increasing latency in transgenic MMTV-HER2/neu mice. Our current study aims to determine the impact of CR on HER2 signaling and cancer cell phenotypes, including proliferation, cell cycle progression, and apoptosis. Methods: In vitro experiments were performed using a BC cell line derived from the MMTV-HER2/neu mouse model. To mimic CR, cells were treated with nutrient restricted media containing either reduced glucose (1mM), reduced serum (1%) or both (1mM/1%). The impact of CR on proliferation and survival was measured using growth curve experiments, and flow cytometric analysis of cell cycle and apoptosis. HER2 signaling proteins were assessed by western blotting. Results: Relative to cells grown in control media (25mM glucose/10% BCS): a) cells grown in glucose restricted (1mM) + serum restricted (1%) media grew 87% slower (p&amp;lt;0.01); b) cells grown in serum-restricted media (1%) with control glucose levels (25mm) grew 86% slower (p&amp;lt;0.01); and c) cells grown in glucose restricted media (1mM) with control serum levels (10%) grew 67% slower (p&amp;lt;0.05). Serum restricted groups, regardless of glucose levels, also exhibited increased G1 cell cycle arrest following 24 hours of treatment and increased percentage of apoptotic cells following 72 hours of treatment compared to control. In contrast, glucose restriction alone did not significantly affect apoptosis compared to control; however, it induced a G2/M arrest, possibly explaining reduction in growth. Western blotting analysis revealed that serum restricted cells had reduced levels of pAkt (Ser473) but no differences in Akt, pERK, or ERK, compared to the control and 1mM groups following 24 hours of CR treatment. Conclusions: In vitro models of CR, specifically reduced serum proteins alone or in combination with glucose, reduced activation of PI3K/Akt in HER2 BC cells. This altered signaling was associated with reduced tumorigenic potential, as evidenced by decreased growth and increased apoptosis. These findings suggest that combining a nutrient restriction regimen or pharmacologic mimetic of CR with HER2-targeted therapy, which also inhibits PI3K/Akt signaling, may act synergistically. Future experiments will test the hypothesis that CR modulates the efficacy of HER2 targeted therapies through in vitro and in vivo models of HER2 BC. Citation Format: Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Ciara H. O'Flanagan, Laura W. Bowers, Stephen D. Hursting. Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 379.

Stromal Stiffness Increases YAP-Dependent Mediated Cell Survival of Breast Cancer Cells

The Extracellular Matrix (ECM) provides biochemical and biophysical cues that activate key molecular pathways that direct cell fate in cancer cell development. These cues are primarily transduced through the integrin family of receptor ECM stiffness, which are known to enhance integrin activity and enhance the malignant behavior of tumors by activating the YAP-TAZ transcription factors. The stroma of cancerous tissues is a potent and active mitigator of malignant transformation. The mechanism by which stromal tension acts to promote resistance has yet to be identified. In the present study, we investigate whether ECM stiffness mediates therapy resistance in premalignant invasive breast cancer tumors by regulating integrin-dependent YAP activity. We used 3D organotypic cultures of invasive breast cancer cells (T4-2 WT, and T4-2 YAP-KO cells) cultured on polyacrylamide gels of varying stiffness (Low [400 Pa], and High [4K Pa]). To determine whether stromal stiffness increases YAP-mediated cell survival, we monitored proliferation (Ki-67) and cell survival (TUNEL) post-irradiation treatment (IR, 8 Gy). Additionally, we looked at the direct effect of YAP activation on tumor grow by injecting T4-2 WT or T4-2 YAP-KO breast cancer cells in Neu mice treated either with a YAP-inhibitor verteporfin (100mg/kg) or a control (IPTG, 10mM/M) for two weeks (5 treatments). Our study shows that irradiated breast cancer cells grown on stiff ECM in vitro lead to a significant increase in YAP nuclear localization. Furthermore, using ANOVA analysis, we observed a significant elevated cell proliferation (P < 0.05) and enhanced cell survival (P < 0.05) leading to the activation of the YAP-TAZ signaling pathways. Our results also showed that inhibition of YAP signaling in stiff ECM cultures treated with IR resulted in a significant reduction of proliferation of breast cancer cells cultured in vitro as well as a reduction of tumor growth in mice (P < 0.05). Overall, these results provide evidence that stiff micro-environmental conditions of breast cancer cells are a potent mitigator of therapy resistance whose effects are mediated through YAP activity.

Abstract 3616: Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model

Abstract Breast cancer is the most common cancer among women worldwide. Hormone-mediated therapy to treat estrogen receptor alpha (ERα) positive breast cancers include the use of ER antagonist, Tamoxifen, aromatase inhibitors and other compounds that degrade ERα. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. Recent studies have identified, synthesized, and tested the clinical safety of ERβ-selective agonists. Given the tumor-suppressive properties of ERβ, it may be possible to use these compounds to induce or activate the ERβ and test their role in the chemoprevention and blocking the progression of breast cancer. We have investigated the therapeutic utility of ERβ agonists in the prevention and progression of breast cancer using MMTV-HER2/neu mice transgenic mouse model. MMTV-HER2/neu mice develop premalignant lesions at 4-5 months, and tumors starting at month 7 due to overexpression of the HER2/neu proto-oncogene. MMTV-HER2/neu mice were treated with an ERβ agonist, LY500307 examined the prevention and progression of mammary cancers in these mice. When compared to controls, ERβ agonist-treated mice exhibited a significant decrease in the development of preneoplastic changes. Differential gene expression analysis revealed a significant change in the expression of a number of genes in response to LY500307 treatment. Pathway analysis identified an enrichment for chemokines signaling pathways, particularly TNF, in blocking the development of preneoplastic changes resulting from treatment with ERβ agonists. Our studies also show a decrease in the formation of mammary tumors in HER-2/neu mice with preexisting preneoplastic changes when treated with LY500307. This study suggests that ERβ agonist treatment may be a valuable therapeutic option for the prevention in women at increased risk of breast cancer and in blocking the progression of hormone receptor positive breast tumors. Citation Format: Kumaraguruparan Ramasamy, Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2017-3616

Abstract LB-131: Targeting expression of EZH2 with increased H3K27 trimethylation activity drives and synergizes with oncogene to promote mammary tumorigenesis in genetically engineered mouse models

Abstract EZH2, a core component of the PRC2 complexes, functions as a transcription repressor by catalyzing histone H3K27 trimethylation. EZH2 mutation frequently occurs in hematologic cancers, and overexpression is found in several solid tumor types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression of EZH2 is associated with highly proliferative and aggressive types of breast and prostate tumors. Particularly, EZH2 was more frequently overexpressed in the triple-negative breast cancers (TNBCs), which do not express the genes for estrogen receptor, progesterone receptor or HER2, indicating that EZH2 may be a tumor driver and contribute to basal-like lineage choice of tumor cells. However, transgenic expression of wild type of EZH2 (EZH2WT) in mammary and prostate gland epithelial cells failed to develop tumors. These results indicate that overexpression of EZH2 in mammary and prostate tissues alone is not sufficient to drive tumorigensis, and activation of EZH2 may be required for initiating tumor development. To test this hypothesis, we generated a new transgenic mouse expressing an EZH2 mutant (EZH2T487A) which has increased enzymatic activity (Wei YK et al, 2011) in mammary glands. Our data showed that expression of the activating mutant EZH2T487A but not EZH2WT in mammary glands developed heterogeneous tumors with basal or/and basal-luminal bilineage phenotypes in more than 50% of offsprings. In addition, crossing the EZH2T487A transgenic mice with the well-established luminal mammary tumor mouse model Tg-MMTV-HER2/Neu mice developed luminal type of mammary tumors with shorter latency as compared to each single transgenic mouse, suggesting that EZH2T487A expression, leading to global increase of H3K27Me3 is insufficient to reprogram the HER2/Neu-driven tumor cell lineage commitment. TCGA database analysis uncovered that EZH2 overexpression in TNBCs often coincides with p53 mutation. Accordingly, we examined whether p53 inactivation synergizes EZH2 to drive tumorigenesis with basal-like tumors. By crossing MMTV-EZH2T487A mice with MMTV-Cre/Trp53flox/flox mice we generated mice with EZH2T487A;p53+/- genotype. These mice exclusively developed multiple tumors in all offsprings with early onset and poor survival as compared to their parental mice and the resultant tumors displayed TNBC characteristics. Our findings indicate that expression of the activating EZH2 alone could drive tumorigenesis, and in cooperation with oncogenic HER2 and p53, EZH2 activation accelerated tumor development but barely affected lineage choice of tumors induced by the predominant drivers. Importantly, the developed tumors in these mouse models exhibited the phenotypes of some subtypes of human breast cancers, in particular, the TNBCs. Therefore, such spontaneous breast tumors with both EZH2 overexpression and p53 mutation could be more faithfully to recapitulate human TNBC development, natural tumor microenvironment and immune response, which can be used for assessing breast tumor immunity and evaluating targeted and/or immunotherapies for TNBCs. Citation Format: Lei Nie, Yongkun Wei, Hirohito Yamaguchi, Longfei Huo, Weiya Xia, Rong Deng, Jun Tang, Cihui Zhu, Baozhen Ke, Mien-Chie Hung. Targeting expression of EZH2 with increased H3K27 trimethylation activity drives and synergizes with oncogene to promote mammary tumorigenesis in genetically engineered mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2017-LB-131

Booster immunizations with DNA plasmids encoding HER-2/neu prevent spontaneous mammary cancer in HER-2/neu transgenic mice over life span

Cancer vaccines are less effective at old than at young age because of immunosenescence. Besides, in preliminary observations we showed that the immunization with HER-2/neu DNA plasmid in transgenic young mice (standard immunization, SI) delays but not abrogate spontaneous mammary tumours progressively appearing during aging. In this study we evaluated whether booster immunizations (BI) of HER-2/neu transgenic mice with HER-2/neu DNA plasmids every 6 (ECD6), 3 (ECD3), or 1.5 (ECD1.5) months after SI induce a protective immunity that could be maintained over life span. The long term BI significantly improved the effect of SI increasing the number of tumour free mice at 110 weeks of age from 13% (SI) to 58% (BI). Both the number and the volume of tumour masses were reduced in BI than in SI groups. The protective effect of BI was associated with increased antibody production with isotype switching to IgG2a, augmented CD4 T cells, and increased in vivo cytotoxicity of HER-2/neu specific cytotoxic T lymphocytes, mainly in ECD1.5 and ECD3 groups. The transfer of sera from ECD1.5 mice to untreated HER-2/neu mice highly protected against tumour development than sera from SI mice. We conclude that BI induce a protective immunity effective over life span.

Abstract P4-15-02: Investigating the use estrogen receptor β agonists in the prevention of breast cancer using a transgenic mouse model

Abstract Breast cancer is the most common cancer among women worldwide. In the United States, 1 in 8 women will develop breast cancer during her lifetime, and in 2016 over 240,000 new cases will be diagnosed. Incidence rates differ by geographic location, with women living in Asia having the lowest rates. However, after migrating to the U.S and adopting a western diet their rates increase. This highlights the importance of lifestyle, including diet, in modulating breast cancer risk. Soy has been previously implicated as the dietary component contributing to the reduced breast cancer rates in Asian women. Compounds that can selectively activate Estrogen Receptor β have been identified in plants, including soy. Given the tumor-suppressive properties of ERβ, it may be possible to use these agonists in the chemoprevention of breast cancer. The objective of this study was to investigate the utility of using ERβ agonists in the prevention of breast cancer using a transgenic mouse model. MMTV-HER2/neu mice develop premalignant lesions at 4-5 months, and tumors starting at month 7 due to overexpression of the Her2/neu proto-oncogene. MMTV-HER2/neu mice were treated with 2 different ERβ agonists, S-equol or LY500307, for 3 months and evaluated for branching, hyperplasia and differential gene expression. When compared to controls, ERβ agonist-treated mice exhibited a significant decrease in branching and ductal hyperplasia, with no change in body weight. Differential gene expression analysis revealed 218 modified genes in response to S-equol treatment, and 258 genes modified by LY500307 treatment, with an overlap of 36 genes. Pathway analysis identified an enrichment for chemokines signaling pathways, particularly TNF, in the reversal of hyperbranching resulting from treatment with ERβ agonists. Although previous studies have demonstrated crosstalk between ER's non-genomic signaling and growth factor signal transductions pathway, this is the first study to demonstrate the impact of ERβ activation on HER2/neu mediated pre-neoplastic changes. Our study suggest that ER β agonist treatment may be a valuable therapeutic option for the chemoprevention of breast cancer in women at increased risk. Citation Format: Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Investigating the use estrogen receptor β agonists in the prevention of breast cancer using a transgenic mouse model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-15-02.

Chemo-immunotherapy induces tumor regression in a mouse model of spontaneous mammary carcinogenesis.

Tumor-specific immune tolerance represents an obstacle for the development of effective anti-tumor immune responses through cancer vaccines. We here evaluated the efficacy of chemo-immunotherapy in breaking tumor-specific immune tolerance in an almost incurable mouse model of spontaneous carcinogenesis.Transgenic HER-2/neu mice bearing large mammary tumors received the adoptive transfer of splenocytes and serum isolated from immune donors, with or without pre-conditioning with cyclophosphamide. Treatment efficacy was assessed by monitoring tumor growth by manual inspection and by magnetic resonance imaging. The same chemo-immunotherapy protocol was tested on tumor-free HER-2/neu mice, to evaluate the effects on tumor emergence.Our data show that chemo-immunotherapy hampered carcinogenesis and caused the regression of large mammary tumor lesions in tumor-bearing HER-2/neu mice. The complete eradication of a significant number of tumor lesions occurred only in mice receiving cyclophosphamide shortly before immunotherapy, and was associated with increased serum anti HER-2/p185 antibodies and tumor leukocyte infiltration. The same protocol significantly delayed the appearance of mammary tumors when administered to tumor-free HER-2/neu mice, indicating that this chemo-immunotherapy approach acted through the elicitation of an effective anti-tumor immune response. Overall, our data support the immune-modulatory role of chemotherapy in overcoming cancer immune tolerance when administered at lymphodepleting non-myeloablative doses shortly before transfer of antigen-specific immune cells and immunoglobulins. These findings open new perspectives on combining immune-modulatory chemotherapy and immunotherapy to overcome immune tolerance in cancer patients.

PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer

In the lactating mammary gland, the plasma membrane calcium ATPase2 (PMCA2) transports milk calcium. Its expression is activated in breast cancers, where high tumor levels predict increased mortality. We find that PMCA2 expression correlates with HER2 levels in breast cancers and that PMCA2 interacts with HER2 in specific actin-rich membrane domains. Knocking down PMCA2 increases intracellular calcium, disrupts interactions between HER2 and HSP-90, inhibits HER2 signaling, and results in internalization and degradation of HER2. Manipulating PMCA2 levels regulates the growth of breast cancer cells, and knocking out PMCA2 inhibits the formation of tumors in mouse mammary tumor virus (MMTV)-Neu mice. These data reveal previously unappreciated molecular interactions regulating HER2 localization, membrane retention, and signaling, as well as the ability of HER2 to generate breast tumors, suggesting that interactions between PMCA2 and HER2 may represent therapeutic targets for breast cancer.

Biomarkers of aging, life span and spontaneous carcinogenesis in the wild type and HER-2 transgenic FVB/N female mice.

FVB/N wild type and transgenic HER-2/neu FVB/N female mice breed at N.N. Petrov Research Institute of Oncology were under observation until natural death without any special treatment. Age-related dynamics of body weight, food consumption and parameters of carbohydrate and lipid metabolism, level of nitric oxide, malonic dialdehyde, catalase, Cu, Zn-superoxide dismutase, vascular endothelial growth factor were studied in both mice strains. The parameters of life span and tumor pathology were studied as well. Cancer-prone transgenic HER-2/neu mice developed in 100% multiple mammary adenocarcinomas and died before the age of 1year. Forty tree percent of long-lived wild type mice survived the age of 2years and 19%-800days. The total tumor incidence in wild type mice was 34%. The age-associated changes in the level of serum IGF-1, glucose and insulin started much earlier in transgene HER-2/neu mice as compared with wild type FVB/N mice. It was suggested that transgenic HER-2/neu involves in initiation of malignization of mammary epithelial cells but also in acceleration of age-related hormonal and metabolic changes in turn promoting mammary carcinogenesis.

Abstract 3735: Dietary sugar induces tumorigenesis in mammary gland partially through 12 lipoxygenase pathway

Abstract Background: Compelling evidence from numerous epidemiological studies worldwide shows that high-sugar diets are associated with increased breast cancer risk, but the data is not wholly consistent and the molecular mechanisms remain unclear. One proposed mechanism for the role of sugar in cancer development involves inflammation. Bioactive lipids, especially cyclooxygenases and lipoxygenases (LOXs) metabolites of arachidonic acid, are known to be involved in inflammation and cancers. Here we examined the effect of sucrose enriched diet in the development of primary and metastatic breast tumor and relevant molecular mechanism primarily focusing on 12-LOX pathway. Methods: Mice bearing three different types of breast tumors were fed with sucrose-enriched diets, and the primary tumors and lung metastasis were measured. The levels of 12- LOX protein expression and its arachidonate metabolite 12-HETE were determined in both tumor tissues from the mice and breast cancer cells in vitro. Results: A diet with 125 g/kg sucrose, a concentration equivalent to the average sugar consumption of American population (70 lb/person/year), notably shortened the breast tumorigenesis in MMTV-ErbB2/neu mice with increase of tumor incidence by 77%. Sucrose enriched-diets (125 g/kg) promoted the development of primary breast tumors in mice bearing mouse breast cancer 4T1 cells with an average tumor weight of 389.4±64.2 mg in the control animals vs. 534.0±90.5 mg in sucrose fed animals. The tumor weight was increased by 2.2-fold in mice injected with human triple negative breast cancer MDA-MB-231 cells upon sucrose diet feeding. Further, sucrose diet (125 g/kg) potentiated lung metastasis of 4T1 mouse mammary tumor evidenced by increasing the average numbers of lung nodules per mouse from 6.6±2.2 in control group to 13.0±5.0 in 125 g/kg sucrose group. The increased tumorigenicity of sucrose appears to be consistently associated with up-regulation of 12-LOX protein expression and 12-HETE production within the tumor tissues across the three breast tumorigenesis models. Additionally, 12-LOX and 12-HETE were induced in the glucose (30 mM) treated MDA-MB-231 cells and 12-HETE promoted MDA-MB-231 cell growth, invasion and migration. Proliferation of MDA-MB-231 cells cultured in 3-D matrigel was stimulated by glucose treatment, which was abrogated with the presence of 12-LOX inhibitor, Baicalein. Conclusion: Our data provides a strong evidence that added sugar (sucrose) accelerates the development of breast cancer partially through up-regulating expression of 12-LOX and production of 12-HETE. Therefore, dietary sugar induced 12-LOX signaling may play an important role in increased incidence for breast cancer that is associated with a high-carbohydrate diet and could be a promising target for intervention and risk-reducing strategies for breast cancer. Citation Format: Yan Jiang, Yong Pan, Patrea R. Rhea, Lin Tan, Mihai Gagea-Iurascu, Lorenzo Cohen, Peiying Yang. Dietary sugar induces tumorigenesis in mammary gland partially through 12 lipoxygenase pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2015-3735

Fish Oil Enhances T Cell Function and Tumor Infiltration and Is Correlated With a Cancer Prevention Effect in HER-2/neu But Not PyMT Transgenic Mice

Few studies have explored the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on immune modulation in murine models of mammary carcinogenesis. HER-2/neu and PyMT mice were randomized to 2 dietary interventions: AIN-93G-based diet with 1) 11% of diet (per gram weight) as corn oil (CO) or 2) 10% of diet as menhaden fish oil plus 1% of diet as corn oil (FO). FO significantly reduced the incidence and multiplicity of tumors (P < 0.001) in HER-2/neu, but not PyMT mice. FO-fed mice had significantly larger splenocyte counts than CO-fed mice in both the HER-2/neu and PyMT models; and in both models this was comprised of an increase in most cell types, including Gr-1+/CD11b+ cells. T cells from FO-fed HER-2/neu mice produced significantly more interleukin-2 (P = 0.004) and interferon-γ (P = 0.012) in response to in vitro stimulation with anti-CD3 (0.5 µg/ml). Lastly, FO-fed HER-2/neu mice had significantly more tumor immune infiltrates than CO-fed mice, including NK1.1+, F4/80+, and Gr-1+/CD11b+ cells (P ≤ 0.05). Greater Th1 cytokine production and significantly more tumor immune infiltrates in FO-fed Her2/neu mice may account for the cancer prevention effect of fish oil in this model.