Induced Breast Tumors Research Articles

CD44 targeting hyaluronic acid coated lapatinib nanocrystals foster the efficacy against triple-negative breast cancer

Lapatinib (LPT) is an orally administered drug for the treatment of metastatic breast cancer. For expanding its therapeutic horizon, we have prepared its nanocrystals (LPT-NCs) that were subsequently coated with hyaluronic acid (HA) to produce LPT-HA-NCs. The detailed in-vitro and in-vivo investigation of LPT-HA-NCs showed the superior anticancer activity due to active targeting to CD44 receptors than the counterparts LPT-NCs and free LPT. In the triple negative 4T1 cells induced breast tumor bearing female Balb/C mice; LPT-HA-NCs treatment caused significant retardation of tumor growth and overall increase in animal survival probability because of their higher tumor localization, increased residence time. Our findings clearly suggest that HA coated LPT-NCs formulation enhances the activity of LPT against triple negative breast cancer. It exhibited magnificent therapeutic outcome at low dose thus presenting a strategy to reduce dose administrations and minimize dose related toxicity.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

Phenotypical Characteristics of Chemically Induced Mammary Tumor.

The histological and immunohistochemical type of chemically induced (injection of N-methyl-N-nitrosourea into the mammary gland) breast tumor was studied in Wistar females. The tumor induced by N-methyl-N-nitrosourea was moderately differentiated adenocarcinoma with infiltrative growth lacking estrogen-α and human epidermal growth factor receptors, and expressing progesterone receptors; tumor cells were characterized by high proliferative activity. This variant of chemically induced breast tumor corresponds to human breast cancer luminal type B.

Abstract 1331: TGFβ1 induces breast tumor kinase overexpression in triple negative breast cancer via p38 MAPK signaling to glucocorticoid receptors

Abstract Triple negative breast cancer (TNBC) is the deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. It has a heterogeneous pathology and pathogenicity, but it is defined by the lack of estrogen receptor, progesterone receptor, and Her2 epidermal growth factor receptor expression. Because targeted therapy through these receptors is not possible, treatment relies on chemotherapy and surgery, which are often inadequate. Thus, the identification of possible molecular targets is critically important in TNBC. Breast tumor kinase (Brk) is a soluble tyrosine kinase that is overexpressed in 85% of BCs and a driver of aggressive and metastatic phenotypes. Overexpression of Brk mRNA and protein occurs in TNBC by unknown mechanisms. The glucocorticoid receptor (GR), a very potent modulator of cytokine mediated actions of the immune system, is emerging as a mediator of chemoresistance and recurrence in TNBC. We previously demonstrated that GR signaling cooperates with physiologic stress signaling mediated by hypoxia inducible factors HIF-1a and HIF-2 to modulate the expression of Brk mRNA and protein in TNBC cells. Moreover, phosphorylation of GR at ser134 by p38 MAPK is essential for GR regulation of Brk expression. P38 is an essential Ser/Thr kinase that regulates cellular transduction of growth factors, such as Hepatocyte Growth Factor (HGF), and cytokines (e.g. TGFβ1) in TNBC cells, and was previously shown to be co-expressed with Brk in human breast tumors. Herein, we further probed mechanisms of crosstalk between key cytokines, GR, and p38 signaling in the regulation of Brk overexpression. We hypothesize that TGFβ1 signaling modulates EMT and metastasis in part by increasing the expression of Brk in TNBC. Treatment of MDA-MB-231 cells with TGFβ1 for 1, 2, 24 and 48 hours increased Brk protein expression relative to vehicle controls. Additionally, TGFβ1 increased both HIF1 and HIF2 protein levels (at 24 and 48 hours respectively). TGFβ1 regulated Brk expression at the level of mRNA, as measured using RT-PCR. Moreover, TGFβ1 synergized with activated GR to further increase Brk mRNA levels. In contrast, mRNA levels of HIF1 and HIF2 were not modulated by TGFβ1, suggesting that the observed protein increases are due to stabilization of HIFs. Finally, TGFβ1 robustly induced p38-dependent phosphorylation of GR at serine 134. This phosphorylation event promoted ligand-independent GR transcriptional activity at the Brk promoter. Human breast tumors significantly co-express active p38 MAPK and Brk. Our molecular model implicates TGFβ1 signaling (via p38 MAPKs) to phospho-GR in the aberrant overexpression of Brk in TNBC. We conclude that blocking of the TGFβ1 pathway may provide a strategy to inhibit Brk mediated TNBC tumor progression. This work was supported by NIH/NCI R01 CA192178 (to CAL) and T32 GM008244-24. Citation Format: Carlos J. Santos Perez, Tarah Regan Anderson, Carol A. Lange. TGFβ1 induces breast tumor kinase overexpression in triple negative breast cancer via p38 MAPK signaling to glucocorticoid receptors [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 1331. doi:10.1158/1538-7445.AM2017-1331

Co-Inflammatory Roles of TGFβ1 in the Presence of TNFα Drive a Pro-inflammatory Fate in Mesenchymal Stem Cells.

High plasticity is a hallmark of mesenchymal stem cells (MSCs), and as such, their differentiation and activities may be shaped by factors of their microenvironment. Bones, tumors, and cardiomyopathy are examples of niches and conditions that contain MSCs and are enriched with tumor necrosis factor α (TNFα) and transforming growth factor β1 (TGFβ1). These two cytokines are generally considered as having opposing roles in regulating immunity and inflammation (pro- and anti-inflammatory, respectively). Here, we performed global gene expression analysis of human bone marrow-derived MSCs and identified overlap in half of the transcriptional programs that were modified by TNFα and TGFβ1. The two cytokines elevated the mRNA expression of soluble factors, including mRNAs of pro-inflammatory mediators. Accordingly, the typical pro-inflammatory factor TNFα prominently induced the protein expression levels of the pro-inflammatory mediators CCL2, CXCL8 (IL-8), and cyclooxygenase-2 (Cox-2) in MSCs, through the NF-κB/p65 pathway. In parallel, TGFβ1 did not elevate CXCL8 protein levels and induced the protein expression of CCL2 at much lower levels than TNFα; yet, TGFβ1 readily induced Cox-2 and acted predominantly via the Smad3 pathway. Interestingly, combined stimulation of MSCs by TNFα + TGFβ1 led to a cooperative induction of all three inflammatory mediators, indicating that TGFβ1 functioned as a co-inflammatory cytokine in the presence of TNFα. The cooperative activities of TNFα + TGFβ1 that have led to CCL2 and CXCL8 induction were almost exclusively dependent on p65 activation and were not regulated by Smad3 or by the upstream regulator TGFβ-activated kinase 1 (TAK1). In contrast, the TNFα + TGFβ1-induced cooperative elevation in Cox-2 was mostly dependent on Smad3 (demonstrating cooperativity with activated NF-κB) and was partly regulated by TAK1. Studies with MSCs activated by TNFα + TGFβ1 revealed that they release factors that can affect other cells in their microenvironment and induce breast tumor cell elongation, migration, and scattering out of spheroid tumor masses. Thus, our findings demonstrate a TNFα + TGFβ1-driven pro-inflammatory fate in MSCs, identify specific molecular mechanisms involved, and propose that TNFα + TGFβ1-stimulated MSCs influence the tumor niche. These observations suggest key roles for the microenvironment in regulating MSC functions, which in turn may affect different health-related conditions.

Abstract A40: Class IIa HDAC inhibition promotes an anti-tumor macrophage phenotype that induces breast tumor regression and inhibits metastasis

Abstract While tumor-associated macrophages (TAMs) often have net pro-tumor effects, their embedded location and their untapped potential provide impetus to the discovery of strategies to turn them against tumors. We recently reported that a first in class selective class IIa HDAC inhibitor (TMP195) influenced human monocyte responses to colony stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumor microenvironment and reduces tumor burden and pulmonary metastases through macrophage modulation. TMP195 induces recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumors. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumor reduction. These data introduce class IIa HDAC inhibition as a novel means to harness the anti-tumor potential of macrophages to enhance cancer therapy. Citation Format: Jennifer L. Guerriero, Alaba Sotayo, Holly Ponichtera, Alexandra Pourzia, Sara Schad, Ruben Carrasco, Lazo Suzan, Roderick Bronson, Anthony Letai. Class IIa HDAC inhibition promotes an anti-tumor macrophage phenotype that induces breast tumor regression and inhibits metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A40.

Electro-Acupuncture Therapy Increases Serum Interferon-γ Levels in Rats with 7, 12 Dimethylbenz(α)anthracene (DMBA)-Induced Breast Tumors

Objective:To determine the effect of electro-acupuncture (EA) treatment on serum levels of interferon-γ (IFN-γ) in rats with 7,12-dimethylbenz(α)anthracene (DMBA)-induced breast tumors.Methods:Twenty five female Wistar rats were divided randomly into 5 groups: normal group (N; neither DMBA-induced nor treated with EA); control group (C; DMBA-induced only); EA 3 days : (DMBA-induced + EA for 3 days); EA 5 days: (DMBA-induced + EA for 5 days); EA 10 days: (DMBA-induced + EA for 10 days) group. Animals were acclimatized from day 1 to day 7. Subcutaneus injections of DMBA 10mg/kg BW was administered every second day, from days 7 to 35. Acupuncture was performed every second day from day 42. Rats were sacrificed on the second day after the last acupuncture, breast tumors excised and stained histological sections were analysed by light microscopy. At sacrifice, blood was extracted from the heart for measurement of serum IFN-γ by ELISA.Results:All of the DMBA-induced rats developed tumors. Electro-acupuncture significantly increased IFN-γ levels in DMBA induced rats, when compared to control group.Conclusions:Our findings suggest that EA significantly increases IFN-γ levels in DMBA-induced breast tumors.

Abstract B038: Class IIa HDAC inhibition promotes an antitumor macrophage phenotype that induces breast tumor regression and inhibits metastasis

Abstract While tumor-associated macrophages (TAMs) often have net pro-tumor effects, their embedded location and their untapped potential provide impetus to the discovery of strategies to turn them against tumors. We recently reported that a first in class selective class IIa HDAC inhibitor (TMP195) influenced human monocyte responses to colony stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a macrophage-dependent autochthonous mouse model of breast cancer to demonstrate that in vivo TMP195 treatment alters the tumor microenvironment and reduces tumor burden and pulmonary metastases through macrophage modulation. TMP195 induces recruitment and differentiation of highly phagocytic and stimulatory macrophages within tumors. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade in this model significantly enhances the durability of tumor reduction. These data introduce class IIa HDAC inhibition as a novel means to harness the antitumor potential of macrophages to enhance cancer therapy. Citation Format: Jennifer L. Guerriero, Alaba Sotayo, Holly Ponichtera, Alexandra Pourzia, Sara Schad, Ruben Carrasco, Suzan Lazo, Roderick Bronson, Anthony Letai. Class IIa HDAC inhibition promotes an antitumor macrophage phenotype that induces breast tumor regression and inhibits metastasis [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B038.

Abstract P3-03-03: A mouse model of sporadic breast tumor with a conditional P53 mutation

Abstract Missense mutations in the tumor suppressor gene p53 are present in more than 50% of human tumors. In particular, the arginine-to-histidine mutation at codon 175 (p53R175H) has been found in more than 4% of human breast cancers. p53 mutations fall into two general categories: germline mutations that are associated with hereditary tumors, and somatic mutations that cause sporadic tumors. Mouse models for cancers induced by p53 germline mutations have been established and characterized. However, hitherto there are no accurate animal models for sporadic tumors induced by p53 somatic missense mutations, although this mechanism of inactivating p53 occurs in a large fraction of human cancers. We have generated a mouse allele, p53WM, that carries coding sequences for both wild type (WT) and R172H mutant p53 (corresponding to R175H in humans) at the p53 endogenous locus. The coding sequence for WT p53 is flanked by loxP sites and therefore can be deleted by Cre recombinase. In the absence of Cre, following radiation, the p53WM/+ mice have the similar ability as the p53 WT mice to activate p53, as demonstrated by transcriptional activation of p53 targets and induction of apoptosis. In addition, like WT mouse embryonic fibroblasts (MEFs), the p53WM/+ MEFs also underwent cell cycle arrest following radiation. After crossing p53WM/+ mice with Zp3-Cre mice which express the Cre recombinase in oocytes, the coding sequence for WT p53 was removed, and the mutant p53R172H allele was regenerated as determined by sequencing, western blot analysis and real time reverse transcription PCR. To investigate whether a somatic p53R172H mutation in mammary epithelium can induce breast tumors, adenoviruses expressing Cre (Ad-Cre) were injected into the mammary duct of p53WM/+ mice. Limiting the adenovirus dose allowed us to induce the p53R172H mutation in 1 of 20, 100 or 1000 cells, generating a mutant p53 surrounded by normal cells which more accurately mimics the clinical situation of sporadic tumors. Meanwhile, p53WM/+; K14-Cre and p53WM/+; WAP-Cre mice have been generated, in which p53R172H mutation are induced through Cre transgene specifically in the mammary epithelial cells. Lastly, to examine how somatic p53 mutation cooperates with an oncogene in mammary tumor induction, MMTV-neu; p53WM/+ mice were also subjected to the mammary intraductal Ad-Cre injection. We are currently monitoring mice and will further investigate the molecular mechanisms of tumorigenesis by studying the loss of heterozygosity, and the co-evolution of tumor epithelial cells and their microenvironment. This mouse model is more likely to share the underlying molecular pathology with human sporadic tumors. Therefore, it will be more predictive of human responses to drugs, and thus a more valuable tool in preclinical testing. Citation Format: Zhang Y, Xiong S, Pant V, El-Naggar A, Lozano G. A mouse model of sporadic breast tumor with a conditional P53 mutation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-03-03.

Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

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 3171: IL-25/IL-17E does not induce breast tumor cell apoptosis and both IL-17A and IL-25/IL-17E contribute to breast cancer survival and proliferation associated with generation of pro-oncogenic low molecular forms of cyclin E

Abstract Pro-inflammatory IL-17 cytokines were initially described for their pathogenic role in chronic inflammatory diseases and subsequent accumulating evidence indicated their involvement in carcinogenesis. In the present study we focused our work on IL-17A and IL-17E which are found in the breast tumoral microenvironment. Cell proliferation and cytotoxic assays as well as Quantitative PCR and Western Blot analysis of oncogenic pathways were performed using several breast cancer cell lines and primary tumor cells. We report that IL-17A and IL-17E receptor subunits (IL17RA, RB and RC chains) are up-regulated in breast cancers versus normal samples. Moreover, IL-17E, which is undetectable in most normal breast tissues tested, is expressed at higher levels in tumors. We studied the molecular signaling following the stimulation of cell lines with IL-17A and IL-17E. We found that both cytokines induced cell proliferation and activation of pathways involved in cells survival. Accordingly, IL-17A and IL-17E promoted resistance to Docetaxel but failed to induce apoptosis as previously reported for IL-17E. Interestingly, we also revealed that both cytokines induced the generation of tumorigenic low molecular weight forms of cyclin E (LMW-E), high levels of which correlate strongly with a poor survival in breast cancer patients. These results show for the first time some of the molecular pathways activated by IL-17A and IL-17E that may participate to their pro-oncogenic activity in breast cancers. Citation Format: Jérôme Giustiniani, Hervé Curé, Christian Garbar, Yacine Merrouche, Nathalie Bonnefoy, Jérémy Bastid, Gilles Alberici, Jean-François Eliaou, Armand Bensussan. IL-25/IL-17E does not induce breast tumor cell apoptosis and both IL-17A and IL-25/IL-17E contribute to breast cancer survival and proliferation associated with generation of pro-oncogenic low molecular forms of cyclin E. [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 3171. doi:10.1158/1538-7445.AM2015-3171

Breast Cancer and Occupation: The Need for Action: APHA Policy Statement Number 20146, Issued November 18, 2014.

Breast cancer is the most prevalent cancer among women in the United States and other countries, making it a major public health concern. Despite significant scientific evidence about its known or suspected causes, research and prevention measures to identify and eliminate occupational and other environmental hazards and risk factors for breast cancer remain largely overlooked. As a result, hazards continue unabated for women generally, especially those who work outside the home. The science linking breast cancer and occupation in particular is growing. Researchers have identified commonly used chemicals that induce breast tumors in test animals. Animal studies link chemicals that mimic reproductive hormones to elevated breast cancer rates. Other animal and human studies link chemical exposures to increased breast cancer rates, including two recent investigations focused on occupational hazards. But the latter are the exception. Studies that attempt to identify and characterize workplace agents linked to breast cancer, as well as intervention studies focusing on the use of less toxic processes and substances, are limited. In what might be construed as a case of gender and social class bias, many research and funding agencies have ignored or downplayed the role of occupational studies despite their relevance to prevention efforts. Action required starts with making a national priority of promoting and supporting research on occupational and other environmental causes of breast cancer. Other public health actions include hazard surveillance and primary prevention activities such as reductions in the use of toxic materials, informed substitution, and green chemistry efforts. The original document is accessible at the APHA website, http://www.apha.org/policies-and-advocacy/public-health-policy-statements/policy-database/2015/01/07/14/55/breast-cancer-and-occupation.

PTEN/PIK3CA genes are frequently mutated in spontaneous and medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumours of tree shrews

Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3–4month-old female tree shrews were orally administrated 20mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.

Abstract 240: Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC-bZip transcription factors in estrogen-induced breast carcinogenesis

Abstract Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C (VC) or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the role of CNC b-zip transcription factors in antioxidant-mediated prevention of breast cancer. Human non-neoplastic breast epithelial cell line MCF-10A was treated with E2, VC, VC + E2, Resveratrol (Res) and Res + E2 for up to 72 hrs. mRNA and protein expression levels of phase-II detoxifying enzymes SOD1, SOD2, SOD3, GPx, NQO1 and transcription factors from CNC b-zip family: Nrf1, Nrf2 and Nrf3 were quantified after treatment with E2 and compared with that of cells treated with antioxidants either alone or in combination with E2. The mRNA as well as protein expression levels of SOD3 and NQO1 were suppressed in MCF-10A cells treated with E2. There was no change in mRNA and protein expression levels of SOD1, SOD2 and GPX in MCF-10A cells treated with E2. Nrf2 mRNA and protein expression levels were also significantly suppressed in E2-treated MCF-10A cells. Vitamin C or Res treatment prevented E2-mediated decrease in SOD3, NQO1 and Nrf2 mRNA and protein expression levels. A significant increase in Nrf1 mRNA and protein expression levels in MCF-10A cells treated with E2 was noted. Further, treatment with antioxidants Res or VC also increased mRNA and protein expression levels of Nrf1. Nrf3 mRNA and protein expression levels were significantly increased in MCF-10A cells treated with E2. This induction was overcome by co-treatment with E2 and VC or Res. Our studies demonstrate that antioxidant-mediated protection against estrogen-induced breast cancer could be through up regulation of phase-II detoxifying enzymes via an Nrf-dependent pathway involving a complex interplay among Nrfs 1, 2 and 3. Note: This abstract was not presented at the meeting. Citation Format: Anwesha Chatterjee, Amruta Ronghe, Fatma Abdalla, Hari K. Bhat. Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC-bZip transcription factors in estrogen-induced breast carcinogenesis. [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 240. doi:10.1158/1538-7445.AM2014-240

Treatment of breast cancer cells with triclosan and octylphenol altered the expressions of cyclin D1 and p21 and induced breast tumor masses via an estrogen receptor-dependent signaling pathway in cellular and mouse xenograft models

Treatment of breast cancer cells with triclosan and octylphenol altered the expressions of cyclin D1 and p21 and induced breast tumor masses via an estrogen receptor-dependent signaling pathway in cellular and mouse xenograft models

Novel self-nanoemulsifying formulation of quercetin: Implications of pro-oxidant activity on the anticancer efficacy

The present work focuses on the anticancer potential of quercetin (QT) loaded self-nanoemulsifying drug delivery system (QT-SNEDDS) composed of Capmul MCM, Tween 20 and ethanol. In vitro cell culture studies revealed potential cell cytotoxicity of developed formulation mediated by its ability to induce DNA damage and apoptosis in MCF-7 cells. QT-SNEDDS at a dose of 50mg/kg demonstrated the antioxidant activity measured as function of prophylactic antitumor efficacy against DMBA induced breast tumors which revealed higher latency to the tumor growth as compared to free QT. This appreciation was further supported by normalized levels of tumor angiogenesis markers (MMP-2, MMP-9, TNF-α and IL-6). At higher doses (100mg/kg) the pro-oxidant activity was noted and exhibited significantly higher therapeutic anticancer efficacy (~65% tumor suppression) in the same model as compared to that of free QT (~20%). Finally, safety profile of developed formulation was established assessing various hepatotoxicity markers. From the Clinical EditorThis basic science study focuses on the anticancer potential of a specific quercetin loaded self-nanoemulsifying drug delivery system. At higher doses significantly higher therapeutic anticancer efficacy (~65% tumor suppression) was noted in the same model as compared to that of free quercetin (~20%).

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice

BackgroundThe major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice.MethodsWe used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey’s post hoc procedure.ResultsAnalysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of “PAM50” genes in the SK-BR-3 cell line from an ER-/Her-2+ to that resembling a “normal-like” phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin.ConclusionsThe SK-BR-3 cells and DMBA-induced tumors, both with an ER- and Her-2+ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer

Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidative stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female August Copenhagen Irish (ACI) rats were treated with E2, vitamin C, vitamin C + E2, BHA and BHA + E2 for up to 8 months. Superoxide dismutase 3 (SOD3) was suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased DNA damage, colony and mammosphere formation, and migration in SOD3 knocked down MCF-10A cells, and nuclear translocation of SOD3 in vitamin C-treated mammary tissues and in MCF-10A cells suggest protective role of SOD3 against DNA damage and mammary carcinogenesis. Our studies further demonstrate that SOD3, but not SOD2 and SOD1, is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidative stress and in the prevention of estrogen-mediated breast cancer.

Adoptive Transfer of Mammaglobin-A Epitope Specific CD8 T Cells Combined with a Single Low Dose of Total Body Irradiation Eradicates Breast Tumors

Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.