Growth Of Mammary Cells Research Articles

Abstract 1991: Mutant GATA3 actively promotes the growth of normal and malignant mammary cells

Abstract GATA3 is a transcription factor expressed in luminal breast epithelial cells and is required for mammary gland development. Analysis of TCGA data reveals that somatic heterozygous mutations in GATA3 occur in up to 15% of estrogen receptor positive breast tumors, and that these tumors are diagnosed a median of eight years earlier than other estrogen receptor positive tumors, suggesting a more aggressive phenotype. These mutants have been proposed to be null alleles resulting in haploinsufficiency, however the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. Based on this disparity, we propose that there is a selective pressure to mutate and retain a portion of the GATA3 in breast cancer. Here we focus on the GATA3 mutants which lack the second zinc finger which is responsible for GATA motif binding. Expression of these mutants accelerated xenograft tumor growth by ZR751 cells, and transgenic expression in mouse mammary glands promoted precocious lobuloalveolar development. We have used integrated gene expression and ChIP-Seq profiling to demonstrate that these zinc-finger deleted proteins retain the ability to associate with the genome by tethering to complexes associated with FOXA1 and AP-2gamma recognition motifs, where they modulate the expression of adjacent genes. These data support a model in which the GATA3 mutations recently observed in breast cancer encode for active transcription factors which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of estrogen receptor positive breast cancer cell lines. Citation Format: Natasha Chandiramani, Esther Peterson, Paraic A. Kenny. Mutant GATA3 actively promotes the growth of normal and malignant mammary cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1991.

Abstract P5-06-01: The role of thyroid hormones in breast tumorigenesis: A translational study utilizing mouse models, cell culture and patient data

Abstract Breast cancer and thyroid hormone signaling have been linked since the 1960s. Breast cancer patients have a higher incidence of thyroid cancer, and thyroid cancer patients have a higher incidence of breast cancer than would be predicted by chance alone, supporting a link between thyroid hormone signaling and breast malignancy. Despite many correlative studies, the role and mechanism of thyroid hormone signaling in mammary tumorigenesis has not been elucidated. Past studies have not comprehensively evaluated thyroid hormones (T3, T4) and thyroid stimulating hormone (TSH) levels with breast cancer status in the same individual. The results are further confounded by issues of temporality; studies have either assessed thyroid hormone levels before or after diagnosis, prohibiting conclusions regarding whether thyroid disruption is a cause or effect of breast cancer. In vitro models demonstrate that mammary cells have thyroid hormone receptors and respond to thyroid hormones; however these studies have not been extended to in vivo models. In the current study, we took a translational approach by combining data from cell culture, mouse models and breast cancer patients from the City of Hope Cancer Registry (CHCR). We used the murine MMTV-PyMT model of breast cancer and treated mice with the anti-thyroid drug PTU; lowering T4 levels and increasing TSH. These mice developed significantly larger mammary tumors than untreated animals or those treated with T4 (p= 0.0012 and p=0.0183, respectively). Next, we showed that MCF10a cells in vitro are sensitive to both T4 and TSH added to the culture medium. We hypothesize that even subtle perturbations in normal thyroid hormone levels can stimulate mammary cell growth, increasing risk of transformation. We extended these findings to a small pilot study of 879 invasive and 136 in situ female breast cancer patients with comprehensive thyroid hormone lab analyses in the CHCR (total=984). Pre-treatment results were available for 44 women. TSH was significantly elevated for invasive versus in situ patients (Pt-test =0.016), regardless of the timing of the test. Pre-treatment TSH levels were significantly increased as the severity of disease increased (Pt-test =0.026). Women diagnosed stage 1 disease with no recurrence had a mean TSH level of 1.68 ± 1.87 (Standard Deviation: SD), whereas women diagnosed with stage 1 disease who returned with metastasis had a mean of 2.64 ± 1.72 SD. In the same women, free T4 and total T4 levels were lower and T3 levels were higher (Pt-test< 0.05) in women with invasive versus in situ disease. These studies support the hypothesis that even minimally dysregulated thyroid hormone levels may increase the risk of breast cancer development and progression to aggressive disease. Many women over the age of 50 suffer from sub-clinical hypothyroidism, and our results suggest that sub-clinical hypothyroidism increases breast cancer risk and disease progression. Interestingly, our data indicate that as disease progresses, dependence on TSH lessens, and the most striking differences may be seen in early and low grade disease. Collectively, our data highlight a need to further investigate the role of sub-clinical hypothyroidism in breast cancer. Citation Format: Franco A, Jolly LA, Russell S, Goldstein L, DeHart J. The role of thyroid hormones in breast tumorigenesis: A translational study utilizing mouse models, cell culture and patient data. [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 P5-06-01.

Abstract IA13: Clonal dynamics of normal and malignant human mammary cell growth in xenografts

Abstract Most human breast cancers have diversified genomically and biologically by the time they become clinically evident and little is known about their origin from normal human mammary cells, or the cellular and molecular mechanisms that lead to their genesis and evolution. We have developed methods to quantify, purify and characterize different subsets of normal human mammary cells and have used these to identify properties that may influence their propensity for transformation. We have also developed methods for inducing the rapid transformation in vivo of these purified subsets following their transplantation into immunodeficient mice. The results demonstrate the ability of a single oncogene (KRASG12D) to induce the formation of serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. Both primary and secondary tumors are phenotypically heterogeneous and transcriptome analyses of primary tumors assign them to a “normal-like” category. DNA barcoding of the cells at the time of their initial transduction with KRASG12D has revealed a dramatic change in the numbers and sizes of clones they generate after 2 weeks in vivo. DNA barcoding also showed the unexpected appearance of many “new” clones in tumors generated upon passage into secondary recipients, thus recapitulating some features of in vivo passaged human breast cancer cell lines and patients’ tumor xenografts. This system challenges previous concepts about the process of human mammary oncogenesis and provides a new system for analyzing factors that can influence its speed, efficiency and heterogeneity of outcomes. Citation Format: Connie J. Eaves, Long Nguyen, Davide Pellacani, Nagarajan Kannan, Sylvan Lefort, Sneha Balani, Claire Cox, Tomo Osako, Samuel Aparicio, Martin Hirst. Clonal dynamics of normal and malignant human mammary cell growth in xenografts. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA13.

Abstract A74: Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions

Abstract Breast cancer is one of the frequently diagnosed and life-threatening cancer diseases in women worldwide, especially, the triple negative breast cancer (ER-/PR-/HER2- TNBC) is a clinically challenging breast cancer disease due to lack of efficient targeted therapeutics. Recently, the development of phytocompounds derived from traditional medicinal herbs into potential chemotherapeutic or chemopreventive agent for human cancer management has aroused a great interest. In our previous study, we identified deoxyelephantopin (DET), a major germacranolide sesquiterpene lactone from a traditional medicinal herb Elephantopus scaber L., which could significantly suppress TS/A (ER+) mammary cancer cell growth, motility and metastasis in vitro andin vivo; however, a relatively less suppressive effect was observed in human TNBC cell line, MDA-MB-231. We thus designed and created a DET derivative (designated DETD) by semi-organic synthesis, which exhibited a 4-fold less in IC50 value than DET in inhibiting MDA-MB-231 cell proliferation. To address the modes of action of both authentic DET and novel DETD compounds against TNBC, we established and used mass spectrometry (MS)-based quantitative proteomics, direct binding assay of DET-near infrared (NIR) fluorescent compound conjugate, and mammary tumor model to gain the mechanistic insight. Our data showed that DET and DETD can markedly induce the reactive oxygen species (ROS) production in early stage of treatment, that may further promote nonautophagic cell death through cytoplasmic vacuoles production. Furthermore, we observed the release of exosome vesicles from MDA-MB-231 cells was elevated by DET or DETD treatment compared to the vehicle control. Quantitative investigation of exosomal proteome showed that DET and DETD responsive exosomal proteins were involved in the regulation of EIF2 signaling, mTOR signaling, regulation of eIF4 and p70S6K signaling, CDK5 signaling, and inhibition of angiogenesis. Of note, the immunofluorescence cell staining showed that DET-NIR was co-localized with exosomes of MDA-MB-231 cells, on the other hand, several nuclear exosomal proteins of mouse TNBC 4T1 cells could interact directly with DET. Together results of this study suggest that DET and DETD compounds may serve as a good candidate in treatment of TNBC disease, through modulation of oxidative stress and specific exosomal proteins in cancer cells. Citation Format: Jeng-Yuan Shiau, Kyoko Nakagawa-Goto, Kuo-Hsiung Lee, Wai-Leng Lee, Jacquelyn Gervay-Hague, Lie-Fen Shyur. Modulation of oxidative stress and exosome activity by phytoagent deoxyelephantopin (DET) and its derivative treatment in suppressing triple negative breast cancer cell functions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A74.

Toxic acrolein production due to Ca2+ influx by the NMDA receptor during stroke

Background and purposeN-Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca2+, contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. MethodsA unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. ResultsWhen the size of brain infarction was decreased by N1, N4, N8-tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca2+, the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca2+ increase. ConclusionThe results indicate that toxicity of Ca2+ during brain infarction is correlated with the increase of acrolein.

Ovariectomy is associated with metabolic impairments and enhanced mammary tumor growth in MKR mice.

Obesity and type 2 diabetes (T2D) are associated with an increased risk of breast cancer incidence and mortality. Common features of obesity and T2D are insulin resistance and hyperinsulinemia. A mammary tumor promoting effect of insulin resistance and hyperinsulinemia was demonstrated in the transgenic female MKR mouse model of pre-diabetes inoculated with mammary cancer cells. Interestingly, in MKR mice, as well as in other diabetic mouse models, males exhibit severe hyperglycemia, while females display insulin resistance and hyperinsulinemia with only a mild increase in blood glucose levels. This gender-specific protection from hyperglycemia may be attributed to estradiol, a key player in the regulation of the metabolic state, including obesity, glucose homeostasis, insulin resistance, and lipid profile. The aim of this study was to investigate the effects of ovariectomy (including the removal of endogenous estradiol) on the metabolic state of MKR female mice and subsequently on the growth of Mvt-1 mammary cancer cells, inoculated into the mammary fat pad of ovariectomized mice, compared with sham-operated mice. The results showed an increase in body weight, accompanied by increased fat mass, elevated blood glucose levels, and hypercholesterolemia, in ovariectomized MKR mice. In addition, mammary tumor growth was significantly higher in these mice. The results suggest that ovarian hormone deficiency may promote impaired metabolic homeostasis in the hyperinsulinemic MKR female mice, which in turn is associated with an increased growth of mammary tumors.

Abstract 2877: Exercise, alone and in combination with a whole tumor cell vaccine reduces mammary tumor cell growth and enhances anti-tumor immunity

Abstract Regular, moderate exercise (EX) can reduce both the incidence and recurrence of breast cancer (BC), and improve survival. Numerous biological mechanism(s) have been proposed to explain these beneficial clinical effects of EX. However, little work has been done to examine the effect of EX on immunomodulation, i.e. the balance between anti-tumor immunity and the emergence of immunosuppressive cells, in particular myeloid derived suppressor cells (MDSC). We have previously demonstrated that moderate EX significantly enhances antigen-specific CD4+ and CD8+ T cell responses and reduces regulatory T cell function in tumor free-mice. Thus, the goal of the current study was to determine if EX could enhance effector function and reduce immunosuppression in tumor bearing mice, and determine if there were any synergistic effects of EX and the administration of a whole tumor cell vaccine on the aforementioned outcomes. Female Balb/c mice were randomized into EX or sedentary control (SED) groups (n = 14-16/group) and had access to running wheel or standard cages, respectively, for 12 weeks prior to the injection of 5×10^4 lucerifase-transfected 4T1.2 tumor cells into the 4th mammary fat pad. Mice were further randomized into vaccination (n = 6-8/group) or vehicle control (n = 4/group) and administered 1×10^6 irradiated 4T1.2 cells (VAC) or HBSS (VEH) at day 7, 14, 21, and 28 post tumor injection. All mice were fed the AIN-76A diet; however, the EX mice (n = 14) were fed 90% of caloric intake of SED animals to remain in energy balance (prevent weight gain) over the course of the study. Primary tumor growth was quantified, and at sacrifice (day 35) organs were collected to assess the following endpoints: splenic antigen-specific CD8+ effector function and myeloid derived suppressor cell (MDSC) accumulation, and metastatic burden in femurs. EX mice, with or without vaccine, weighed significantly less than SED mice (p<0.001). There was a significant effect of both vaccination and EX on primary tumor growth (F(24,200) = 7.386, p<0.001), splenic IFN-γ production (KW = 11.43, p = 0.010); and the accumulation of MDSC (F(3,28) = 6.486, p = 0.021). However, there was only an EX effect on metastatic burden (p = 0.027). There was a synergistic effect of the combination of vaccine+EX on tumor growth, but no other endpoint. These results demonstrate that exercise alone (i.e. in a prevention model, 12 weeks prior to tumor implantation) is highly effective in reducing primary tumor growth and metastases in an aggressive tumor, and significantly shifted the balance of effector and immunosuppressive factors in the direction of anti-tumor immunity. Furthermore, these results demonstrate that exercise is a viable intervention that may yield significant clinical benefit when used in combination with therapeutic cancer vaccines. This work is supported by internal PSU pilot funds for CJR; T32AI074551-03 for WJT. Citation Format: William J. Turbitt, Donna Sosnoski, Andrea Mastro, Connie Rogers. Exercise, alone and in combination with a whole tumor cell vaccine reduces mammary tumor cell growth and enhances anti-tumor immunity. [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 2877. doi:10.1158/1538-7445.AM2015-2877

Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells

Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.

Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse.

The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5-3.5days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice.

A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment.

BackgroundRadiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance.MethodsWe have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation.ResultsOf ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds.ConclusionsFor the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1481-9) contains supplementary material, which is available to authorized users.

Effect of Thyroid Function on MNU-Induced Mammary Carcinogenesis

Mammary cancer is a disease that affects many women. Extensive research has been conducted to elucidate which variables are involved in the development of this cancer. Studies have highlighted thyroid function as a modulator of tumor growth and development. Thyroxine and 3,3',5-triiodothyronine are responsible for regulating the development, differentiation, homeostasis, and metabolism of cells in the body including mammary tissue. Thyroid hormones also have estrogen-like effects on mammary cancer cell growth by regulating the estrogen receptor. The present study was designed to determine whether medically induced hyperthyroidism increases the multiplicity, prevalence, and mammary tumor burden in rats; and to elucidate whether surgically induced hypothyroidism conversely attenuates the rate of mammary cancer cell growth. Female Sprague-Dawley rats were randomly divided into three groups (euthyroid-control, hyperthyroid, and hypothyroid). Hyperthyroidism was induced via oral administration of levothyroxine; whereas, hypothyroidism was induced by thyroidectomy. Mammary carcinogenesis was induced with a single intraperitoneal injection of N-methyl-N-nitrosurea (MNU). Rats were sacrificed at 38 weeks, and the mammary tumors were excised, fixed for histology and analyzed. Analysis included evaluation of malignancy and immunohistochemistry for ER. MNU-induced mammary carcinogenesis among the groups resulted in a significant difference in tumor burden. The hyperthyroid group had a statistically higher tumor burden than did the euthyroid group, and the hypothyroid group had no tumors of mammary tissue origin at 38 weeks. All excised mammary tumors were ER alpha negative. These data support the hypothesis that thyroid function is one of potentially many factors that contribute to modulation of MNU-induced mammary tumor growth.

The anti-cancer effects of carotenoids and other phytonutrients resides in their combined activity

Epidemiological studies have consistently shown that regular consumption of fruits and vegetables is strongly associated with reduced risk of developing chronic diseases, such as cancer. It is now accepted that the actions of any specific phytonutrient alone do not explain the observed health benefits of diets rich in fruits and vegetables as nutrients that were taken alone in clinical trials did not show consistent preventive effects. The considerable cost and complexity of such clinical trials requires prudent selection of combinations of ingredients rather than single compounds. Indeed, synergistic inhibition of prostate and mammary cancer cell growth was evident when using combinations of low concentrations of various carotenoids or carotenoids with retinoic acid and the active metabolite of vitamin-D. In this study we aimed to develop simple and sensitive in vitro methods which provide information on potent combinations suitable for inclusion in clinical studies for cancer prevention. We, thus, used reporter gene assays of the transcriptional activity of the androgen receptor in hormone-dependent prostate cancer cells and of the electrophile/antioxidant response element (EpRE/ARE) transcription system. We found that combinations of several carotenoids (e.g., lycopene, phytoene and phytofluene), or carotenoids and polyphenols (e.g., carnosic acid and curcumin) and/or other compounds (e.g., vitamin E) synergistically inhibit the androgen receptor activity and activate the EpRE/ARE system. The activation of EpRE/ARE was up to four fold higher than the sum of the activities of the single ingredients, a robust hallmark of synergy. Such combinations can further be tested in the more complex in vivo models and human studies.

Anticancer Effects of γ-Tocotrienol Are Associated with a Suppression in Aerobic Glycolysis.

Aerobic glycolysis is an established hallmark of cancer. Neoplastic cells display increased glucose consumption and a corresponding increase in lactate production compared to the normal cells. Aerobic glycolysis is regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling pathway, as well as by oncogenic transcription factors such as c-Myc and hypoxia inducible factor 1α (HIF-1α). γ-Tocotrienol is a natural isoform within the vitamin E family of compounds that displays potent antiproliferative and apoptotic activity against a wide range of cancer cell types at treatment doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in mouse +SA and human MCF-7 breast cancer cells. Treatment with γ-tocotrienol resulted in a dose-responsive inhibition of both +SA and MCF-7 mammary tumor cell growth, and induced a relatively large reduction in glucose utilization, intracellular ATP production and extracellular lactate excretion. These effects were also associated with a large decrease in enzyme expression levels involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and lactate dehydrogenase A. γ-Tocotrienol treatment was also associated with a corresponding reduction in the levels of phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but not HIF-1α or glucose transporter 1 (GLUT-1). In summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol are mediated, at least in the part, by the concurrent inhibition of Akt/mTOR signaling, c-Myc expression and aerobic glycolysis.

309 RNA sequencing and in silico analysis identifies an unannotated antisense long non-coding RNA involved in cancer progression

metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. Also, the orthotopic growth of syngeneic pancreatic and mammary tumor cells was significantly reduced after injection in Tie2hPTX3 mice and led to increased survival compared to control mice. Finally, double transgenic TRAMP/Tie2-hPTX3 mice showed a significant delay of multistage prostate tumor onset and progression in respect to TRAMP mice. Our findings demonstrate for the first time that in vivo delivery of PTX3 exerts a dramatic impact on tumor growth, vascularization and metastasis. These results have set the basis for the identification of a low molecular weight PTX3-derived molecule that recapitulates the FGF-trap activities of PTX3 and exhibits promising therapeutic potential for FGF-dependent tumors.

Abstract LB-90: Molecular mechanism of TAZ-induced lung tumorigenesis

Abstract The transcriptional co-activator with PDZ-binding domain (TAZ) is a transcriptional co-activator and major component of an emerging signaling pathway called the Hippo pathway that plays critical roles in tumorigenesis, organ size control, stem cell renewal, drug resistance, etc. Recently, we have provided the first biological evidence that TAZ is over-expressed in non-small cell lung cancer (NSCLC) cells and over-expression of TAZ can transform HBE135 immortalized non-tumorigenic human lung epithelial cells (Zhou et al., 2011). However, the cellular genes mediating TAZ-induced transformation are unknown. In addition, there is no in vivo mouse model established to study the roles of TAZ in lung tumorigenesis in vivo. To address these issues, we have carried out the following experiments: First, a Dox-inducible lentiviral expression system was used to overexpress constitutively active TAZ (TAZ-S89A) in a mouse immortalized lung epithelial cell line (E10). Overexpression of TAZ-S89A in E10 cells caused increased cell proliferation, transformation, and tumor formation in vivo in nude mice. Next, tumorigenic stable cell lines (TAZS89A-TM) were established after isolating TAZS89A-induced tumors from mice. Second, to identify the downstream genes mediating TAZ-induced tumor formation, the technology of Next-generation sequencing (RNA-seq) was performed on the new tumorigenic cell line E10-TAZ-TM to identify downstream genes transcriptionally regulated by TAZ after induction of TAZ by Dox. By analyzing the data and confirming them using qRT-PCR, multiple significant oncogenes were found activated by TAZ overexpression. Third, these genes were knocked down by shRNAs in the tumorigenic E10-TAZS89A-TM cell line and tested whether they are important in TAZ-induced cell proliferation, transformation and tumor formation in mice. Finally, since previous studies indicate that TAZ is involved in mammary cell growth by interacting with transcription factor TEAD or through its WW domain, we also tested whether these genes are activated through TEAD-binding domain or WW-domain of TAZ. E10 stable cell lines overexpressing inducible TAZ mutant with amino acid mutations in TEAD-binding domain (TAZ-F52A/F53A) or WW domain (TAZ-W152A/P155A) were established, followed by qRT-PCR analysis. The TEAD-binding domain of TAZ was found necessary for activation of the identified genes and maintaining TAZ-induced oncogenic effects in lung cells. In conclusion, this project established the first in vivo xenograft mouse model system using TAZ-overexpressing lung epithelial cells, and identified the downstream target genes mediating TAZ-induced lung tumorigenesis. Citation Format: Adel B. Alharbi, Hongchao Shan, Yawei Hao, Xiaolong Yang. Molecular mechanism of TAZ-induced lung tumorigenesis. [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 LB-90. doi:10.1158/1538-7445.AM2014-LB-90

Abstract 2072: Chitinase-3-like-1 protein expression associated with pulmonary inflammation accelerates metastasis to the lung

Abstract Disseminated metastasis accounts for majority of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3-like-1 protein (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. We found that there are elevated levels of CHI3L1 in plasma of mammary tumor-bearing mice. However, the biological and physiological functions of CHI3L1 are still unclear. We previously found that CHI3L1 has an inhibitory role on the expression of interferon-gamma, and in up-regulation of pro-inflammatory mediators such MCP-1, IL-8 and MMP-9 all of which contribute towards tumor growth and metastasis. In determining the cause of high levels of CHI3L1 in plasma, we found that in addition to tumor cells, splenic macrophages and more interestingly pulmonary macrophages from mammary tumor bearers secrete high levels of CHI3L1. Additionally, allergic mice also expressed higher levels of CHI3L1 in circulation and lung tissue. More specifically, CD11b+Ly6C+ cells from the lungs of allergic mice produced higher amounts of CHI3L1 compared to control mice. We have previously established that pulmonary myeloid cells through their production of CHI3L1 set up the proper microenvironment for growth of incoming mammary tumor cells. Thus, we hyphothesize that CHI3L1 levels in the lungs of mice with allergic pulmonary inflammation could accelerate metatstasis. To understand the role of CHI3L1 in accelerating pulmonary metastasis, pulmonary inflammation was induced by sensitization with ragweed allergen. Allergic mice implanted with either 4T1 or 67NR mammary tumors had a 5-fold increase in tumor volume, and formation of metastatic foci in their lungs compared to control mammary tumor bearers. Further, allergic mice showed accelerated tumor growth and shorter survival. In contrast, CHI3L1 null mice showed decreased tumor volume and metastasis, and increased survival compared to control mice. In vivo administration of chitin (β-(1-4)-poly-N-acetyl D-glucosamine) microparticles, the ligand for CHI3L1, to control and allergic mammary tumor bearers resulted in a significant reduction in pulmonary metastasis and tumor growth. These studies suggest that CHI3L1 plays a role in tumor progression and that chitin microparticles can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful target for treatment of breast cancer. Citation Format: Stephania Libreros, Ramon Garcia-Areas, Vijaya Iragavarapu-Charyulu. Chitinase-3-like-1 protein expression associated with pulmonary inflammation accelerates metastasis to the lung. [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 2072. doi:10.1158/1538-7445.AM2014-2072

Abstract 2151: Evaluation of a citrus flavonoid as a chemopreventive agent against breast cancer

Abstract Naringenin is a flavonone found in citrus fruits and tomatoes that may have anti-tumor properties. Previously, our lab discovered that naringenin induced the activity of AMP-activated protein kinase (AMPK) and reduced phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP) in MCF-7 human breast cancer cells. These properties have been linked with anti-tumor actions of drugs such as metformin and mTOR inhibitors. Based on these findings, we tested the ability of naringenin (0 uM - 200 uM) to modulate growth of MCF-7 human breast cancer cells and E0771 murine mammary tumor cells. In both models for cancer cell growth, naringenin dose-dependently decreased live cell number in a time-dependent manner. Furthermore, results of flow cytometry and propidium iodide staining assays showed accumulation of apoptotic cells after naringenin treatment. Naringenin also decreased expression of cyclin D1 and elevated expression of pro-apoptotic protein Bax suggesting that naringenin may inhibit the proliferation of mammary tumor cells by inducing cell cycle arrest and promoting apoptosis. To test for dose-dependent accumulation of naringenin in mammary gland tissue, female mice were fed varying doses of naringenin (control, 0%; low dose,1.0 wt%; or high dose, 3.0 wt% naringenin) in a high saturated fat diet for 11 weeks. Body weights and cumulative food intake were not different between diet groups. Dietary naringenin resulted in dose-responsive accumulation in mammary gland. Relative accumulation of naringenin in tissues was greatest in liver (low dose, 7.61 ± 1.14 umol/kg tissue and high dose, 37.04 ± 8.33 umol/kg tissue) followed by mammary gland and muscle which had a similar magnitude of naringenin accumulation (low dose ∼ 0.4 umol/kg tissue and high dose naringenin, 2.5 umol/kg tissue) of accumulation. Naringenin and monoglucuronide naringenin were two of the main forms of accumulated flavonoid in mammary gland raising the possibility that naringenin and/or metabolites are the active form of flavonoid responsible for anti-tumor activity against mammary cancer. Evaluating the species of naringenin metabolites in mammary gland that exert pro-apoptotic actions will be important to evaluate in order to understand the potential of naringenin-rich foods to contribute to prevention of breast cancer in women. Citation Format: Jia-Yu Ke, Min Tian, Kara L. Kliewer, Steve J. Schwartz, Ken M. Reidl, Shin-yu Tsai, Lisa D. Yee, Martha A. Belury. Evaluation of a citrus flavonoid as a chemopreventive agent against breast cancer. [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 2151. doi:10.1158/1538-7445.AM2014-2151

Abstract 3391: Alteration of mammary tumor cell behavior by FLIZ1

Abstract Fetal liver zinc finger protein (FLIZ1, ZC3H8) was previously described as a negative regulator of GATA3 expression in T cells. GATA3 also is an important determinant of mammary cell fate and breast tumor cell behavior. We hypothesized that FLIZ1 also regulates GATA3 in the mammary gland, thereby influencing normal and tumor cell processes controlled by GATA3. We identified tumor cell lines with high levels of FLIZ1 expression, and transfected them with vectors driving expression of FLIZ1-interfering RNAs. Stable transfectants (Flizout cells) were evaluated for ability to migrate in a wound healing assay, and were found to have decreased mobility. Flizout cells also grew more slowly and with more pronounced epithelial morphology in media containing charcoal-stripped serum, suggesting increased dependence on steroid hormones. Flizout cells were compared to control cells in proliferation assays, and were found to grow at equivalent rates in standard media, but Flizout cells grew at decreased rates in the presence of tamoxifen. We implanted Flizout cells and controls into the mammary glands of syngeneic female mice, which were then treated with tamoxifen or peanut oil vehicle alone. Flizout cells grew more slowly in tamoxifen-treated animals, and tissue weights were significantly less than controls. In order to determine if FLIZ1 plays a role in the normal mammary gland, we assessed its expression during stages of mouse mammary gland development by immunoblot, and found FLIZ1 expression increased during mammary involution. These data are consistent with a role for FLIZ1 in regulation of mammary cell growth and tumor cell behavior. Citation Format: Torri Anderson, Christopher Cali, Katherine Bell, Keith G. Danielson, Ashley Klein, Christina Martin, Sara Radecki, Adam Santoro, John A. Schmidt, Janice E. Knepper. Alteration of mammary tumor cell behavior by FLIZ1. [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 3391. doi:10.1158/1538-7445.AM2014-3391

Curcumin restores sensitivity to retinoic acid in triple negative breast cancer cells.

BackgroundA major obstacle in the use of retinoid therapy in cancer is the resistance to this agent in tumors. Retinoic acid facilitates the growth of mammary carcinoma cells which express high levels of fatty acid-binding protein 5 (FABP5). This protein delivers retinoic acid to peroxisome proliferator-activated receptor β/δ (PPARβ/δ) that targets genes involved in cell proliferation and survival. One approach to overcome resistance of mammary carcinoma cells to retinoic acid is to target and suppress the FABP5/ PPARβ/δ pathway. The objective of this research was to investigate the effect of curcumin, a polyphenol extract from the plant Curcuma longa, on the FABP5/ PPARβ/δ pathway in retinoic acid resistant triple negative breast cancer cells.MethodsCell viability and proliferation of triple negative breast cancer cell lines (MDA-MB-231 and MD-MB-468) treated with curcumin and/or retinoic was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2’-deoxyuridine (BrdU). Expression level of FABP5 and PPARβ/δ in these cells treated with curcumin was examined by Western Blotting analysis and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Effect of curcumin and retinoic acid on PPARβ/δ target genes, PDK1and VEGF-A were also examined using qRT-PCR. Western Blotting was utilized to examine the protein expression level of the p65 subunit of NF-κB.ResultsTreatment of retinoic acid resistant triple negative breast cancer cells with curcumin sensitized these cells to retinoic acid mediated growth suppression, as well as suppressed incorporation of BrdU. Further studies demonstrated that curcumin showed a marked reduction in the expression level of FABP5 and PPARβ/δ. We provide evidence that curcumin suppresses p65, a transcription factor known to regulate FABP5. The combination of curcumin with retinoic acid suppressed PPARβ/δ target genes, VEGF-A and PDK1.ConclusionsCurcumin suppresses the expression level of FABP5 and PPARβ/δ in triple negative mammary carcinoma cells. By targeting the FABP5/PPARβ/δ pathway, curcumin prevents the delivery of retinoic acid to PPARβ/δ and suppresses retinoic acid-induced PPARβ/δ target gene, VEGF-A. Our data demonstrates that suppression of the FABP5/ PPARβ/δ pathway by curcumin sensitizes retinoic acid resistant triple negative breast cancer cells to retinoic acid mediated growth suppression.

Conjugates of 1'-aminoferrocene-1-carboxylic acid and proline: synthesis, conformational analysis and biological evaluation.

Our previous studies showed that alteration of dipeptides Y-Fca-Ala-OMe (III) into Y-Ala-Fca-OMe (IV) (Y = Ac, Boc; Fca = 1'-aminoferrocene-1-carboxylic acid) significantly influenced their conformational space. The novel bioconjugates Y-Fca-Pro-OMe (1, Y = Ac; 2, Y = Boc) and Y-Pro-Fca-OMe (3, Y = Boc; 4, Y = Ac) have been prepared in order to investigate the influence of proline, a well-known turn-inducer, on the conformational properties of small organometallic peptides with an exchanged constituent amino acid sequences. For this purpose, peptides 1–4 were subjected to detailed spectroscopic analysis (IR, NMR, CD spectroscopy) in solution. The conformation of peptide 3 in the solid state was determined. Furthermore, the ability of the prepared conjugates to inhibit the growth of estrogen receptor-responsive MCF-7 mammary carcinoma cells and HeLa cervical carcinoma cells was tested.