E0771 Cells Research Articles

Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.

Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression.

Abstract A15: SMC2 role in regulating tumor angiogenesis via FGF signaling

Abstract Fibroblast growth factors (FGFs) play a crucial role during embryonic development, tissue homeostasis, wound healing and angiogenesis. FGF-binding protein 1 (FGFBP1) is a secreted protein that binds FGFs stored in the extracellular matrix and transports them to their receptor, therefore they can modulate FGF signaling. FGFBP1 contributes to the progression of many cancers including colon, pancreas and squamous cell cancer. It acts as an angiogenic switch during tumor development. E0771 cells, a murine mammary carcinoma cell line, was subcutaneously injected into syngeneic C57BL/6 mice. While these cells form aggressive tumors within two weeks and metastasize to the lungs in wild type FGFBP1 (FGFBP1+/+) mice, there was no tumor growth or metastasis in FGFBP1 knock out (FGFBP1-/-) mice. A genome-wide shRNA library screen revealed a novel role for structural maintenance of the chromosome 2 (SMC2) in compensating for the loss of FGFBP1 in the stroma of FGFBP1-/- mice. E0771 cells with SMC2 knock down (KD) or CRISPR targeting SMC2 formed aggressive tumors in FGFBP1-/- mice and metastasized to the lungs. Surprisingly, FGFBP1 expression level was 32 folds lower in SMC2 KD E0771 cells yet FGF2 was 16 folds up in the tumors from SMC2 KD E0771 cells. Tumor histology shows impaired and leaky angiogenesis in FGFBP1-/- compared to FGFBP1+/+ mice. Furthermore, higher levels of GM-CSF and G-CSF were detected in the conditioned media (CM) of SMC2 KD E0771 cells compared to control cells suggesting differential immune cell recruitment. Also, an endothelial monolayer exposed to CM from SMC2 KD E0771 cells showed delayed wound closure and loose junctions suggesting a tendency for endothelium disintegration and initiation of blood vessel formation. Interestingly, the CM effect was blocked using PD173074, a pan FGFR inhibitor. In conclusion, SMC2 demonstrate a role in regulating FGF signaling which affects the course of angiogenesis during tumor development. Note: This abstract was not presented at the conference. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Weiner M. Louis, Anna T. Riegel, Anton Wellstein. SMC2 role in regulating tumor angiogenesis via FGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A15.

Abstract P2-05-11: Sphingosine-1-phosphate signaling promotes metastatic niches and lung metastasis in obesity-related breast cancer

Abstract INTRODUCTION: The link between obesity and elevated breast cancer mortality is well known, however, the underlying mechanisms are poorly understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Previously, we found that obesity increases levels of S1P not only in breast tumors, but also in the lung. "Metastatic niches" are specialized microenvironments in distant organs primed by factors from cancer cells. We hypothesized that S1P secreted from the primary tumor could promote formation of a "metastatic niche" in the lung, which assists circulating cancer cells to form metastatic lesions. Further, HFD-induced obesity increases S1P secretion from the primary tumor, which could promote the formation of "metastatic niches" in the lung and lung metastasis. The aim of this study is to test these hypotheses. METHODS: A mouse model utilizing tail vein injection of E0771 syngeneic breast cancer cells was used. Prior to tail vein injections of naive E0771 cells, mice were treated with conditioned media from E0771 breast cancer cells overexpressing SphK1 (K1-CM) or that from E0771 cells cultured with the vector control (CT-CM). Histological analysis, RT-qPCR, and western blot were used. RESULTS: The lungs after K1-CM treatment demonstrated much more infiltration of macrophages with greater IL-6 secretion than lungs from CT-CM mice in areas without metastasis. Furthermore, SphK1, S1P receptor 1 and IL-6 expression were all significantly higher in the lungs of mice treated with K1-CM than with CT-CM, suggesting that S1P secreted from the primary tumor promotes formation of a metastatic niche in the lung. Next, mice were fed with HFD or ND for 12 weeks before treatment with SphK1-CM, and lungs were examined 7 days after intravenous injection of E0771 cells. Histological analysis demonstrated that there were significantly more lung metastases in mice on HFD than in mice on ND. Importantly, treatment with FTY720, a functional antagonist of S1P receptor 1, significantly reduced the lung metastases in HFD fed animals. Immunofluorescent staining revealed higher expression of IL-6 and greater number of F4/80 positive macrophages in mice fed with HFD compared with mice fed with ND, whereas FTY720 dramatically suppressed both IL-6 and macrophage infiltration in the lung of HFD-fed mice. HFD-induced obesity also increased pERK, pAKT, pStat3, and pp65 in the lung, and FTY720 suppressed these signaling pathways. CONCLUSION: Our results suggest that S1P plays a role in the formation of "metastatic niches" in the lung and lung metastasis of breast cancer, and obesity promotes this process. S1P will be a promising target for treatment of breast cancer metastasis, especially in condition with obesity. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Nagahashi M, Tsuchida J, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Sphingosine-1-phosphate signaling promotes metastatic niches and lung metastasis in obesity-related breast cancer. [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 P2-05-11.

Abstract P2-05-28: The impact of the plant lignin secoisolariciresinol diglycoside on preclinical models of estrogen receptor positive breast cancer

Abstract Background: Several preclinical studies indicate that secoisolariciresinol diglycoside (SDG), a polyphenolic plant lignin found most abundantly in flaxseeds, inhibits the progression of both estrogen receptor (ER) positive and negative mammary tumors. SDG is metabolized by gut bacteria into the biologically active metabolites enterolactone (ENL) and enterodiol (END), which are known to have anti-estrogenic activity. However, the mechanisms mediating SDG's anti-tumor effects remain poorly understood. Methods: In a dose-determination pilot study linked to an ongoing clinical trial of SDG in women at high risk for breast cancer, 18 week old C57BL/6 mice were randomized to a control diet or SDG-supplemented diets (25 or 74 mg/kg of food) for 8 weeks prior to euthanization, and the levels of serum and tissue SDG metabolites (particularly ENL and END), metabolic hormones and inflammatory markers were measured. In an ongoing tumor study, 12-week old C57BL/6 and foxn1 nu/nu mice were randomized to the control or control plus SDG (100 mg/kg of food, a dose projected to match ENL and END metabolite levels achieved in the clinical trial) diet regimen. After 8 weeks on diet, they will receive orthotopic injections of E0771 mouse mammary tumor cells or BT-483 human breast cancer cells (both ER positive), continuing on the same diets until euthanization. Cell culture studies examining the impact of biologically relevant concentrations of ENL and END on E0771 and BT-483 cells are also in progress. Results: In comparison to those maintained on the control diet, the higher dose SDG diet reduced estrogen and pro-inflammatory signaling in the pilot study mice, as evidenced by higher interleukin 10 and lower C-reactive protein mammary fat pad expression as well as lower circulating levels of the adipokines leptin and resistin, which have been linked to chronic inflammation. High dose SDG also decreased serum insulin and glucose levels, indicating improved metabolic function. Because serum ENL and END levels in the pilot study did not reach those achieved in the SDG clinical trial, a 100 mg/kg SDG dose was chosen for the tumor study. Cell culture studies indicate that ENL (150 nM) inhibits E0771 and BT-483 cell proliferation and ER alpha:beta expression ratio. Conclusions: Preliminary data suggests that the anti-tumor effects of SDG's metabolites may be mediated through multiple mechanisms, including improvements in metabolic function and inflammatory signaling as well as modulation of breast cancer cell gene expression. The results of the ongoing tumor study will inform the design of additional cell culture studies aimed at further defining these mechanisms. Citation Format: Bowers LW, Ford NA, Rossi EL, Shamsunder MG, Kimler BF, Fabian CJ, Hursting SD. The impact of the plant lignin secoisolariciresinol diglycoside on preclinical models of estrogen receptor positive breast cancer. [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 P2-05-28.

Abstract P1-03-03: Obesity increases the lipid mediator, sphingosine-1-phosphate in the tumor and tumor microenvironment, and promotes tumor progression

Abstract INTRODUCTION: While obesity is an established independent prognostic factor for breast cancer patients, the underlying mechanisms how obesity promotes breast cancer progression are not well understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Our hypothesis is that obesity increases levels of S1P in both tumor and its microenvironment, which promotes obesity-induced inflammation and breast cancer progression. The aim of this study is to test the hypothesis. METHODS: A high fat diet (HFD) was fed C57Bl/6 mice, which is the most commonly used murine diet-induced obesity model. E0771 cells derived from C57Bl/6 mice were implanted into mammary fat pads of mice that were fed with HFD or normal diet (ND) for 12 weeks prior to the implantation. Western blot, immunofluorescent staining, RT-qPCR and LC-ESI-MS/MS assays were used. RESULTS: Mice fed with HFD for 20 weeks developed severe obesity with an almost 2 fold increase of body weight compared with ND fed mice. E0771 mouse breast cancer cells were implanted into mammary fat pad of C57Bl/6 mice, and the mice fed with HFD developed significantly larger tumors within 30 days than those fed with ND. Expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the tumors of HFD fed mice, compared to those fed with ND. Furthermore, immunofluorescent analysis with anti-F4/80 antibody showed that tumors from HFD fed animals recruited significantly more tumor associated macrophages than those from ND fed animals. Expression of SphK1 and S1PR1, but not SphK2, was increased in the tumors from mice fed HFD. Mass spectrometry analysis revealed that while S1P levels in the normal breast mammary fat pad were increased with HFD feeding, S1P levels were even higher in breast tumors. Consistent with increased SphK1 and S1P in tumors, S1P was also significantly increased in the tumor interstitial fluid, which is a component of the tumor microenvironment and bathes cancer cells in the tumor. S1P levels were also increased in the serum of the tumor-implanted animals fed with HFD compared with those fed with ND, while minimal changes in S1P were evident in the serum of non-tumor bearing mice fed with HFD. Furthermore, S1P levels in the lungs of tumor-implanted animals fed with HFD were significantly higher than those fed with ND. CONCLUSION: S1P is increased not only in tumor, but also in tumor microenvironment such as tumor interstitial fluid by obesity. Our results suggest that S1P has a role in obesity-induced inflammation and the cancer progression. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Tsuchida J, Nagahashi M, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Obesity increases the lipid mediator, sphingosine-1-phosphate in the tumor and tumor microenvironment, and promotes tumor progression. [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 P1-03-03.

Meroxest improves the prognosis of immunocompetent C57BL/6 mice with allografts of E0771 mouse breast tumor cells.

IntroductionRecently, we have reported the antitumor properties of a new family of synthetic merosesquiterpenes, among which meroxest is highlighted, since it has high activity and specificity for ER+ breast cancer cells. In this paper, we characterize allografts of ER+ E0771 mouse breast tumor cells in immunocompetent C57BL/6 mice, and also analyze the effect of meroxest on the prognosis of the disease.Material and methodsTwenty female C57BL/6 mice were injected with 106 E0771 cells. Once the tumors reached the appropriate size, the mice were divided into two groups, one control and another treated orally with 15 mg/kg of meroxest. After 20 days, tumor samples were taken for histopathological study and for determination of the expression of the prognostic markers Ki67 and vascular endothelial growth factor (VEGF) by immunofluorescence.ResultsIn sections stained with hematoxylin-eosin, we observed that tumors have a well-defined capsule enclosing E0771 tumor cells. The central area of tumors contains necrotic regions with leukocyte infiltration. Meroxest treatment significantly reduces tumor size (68%, p < 0.05), induces changes in its structure, decreases the degree of leukocyte infiltration, and significantly reduces the expression of Ki67 (33%, p < 0.05) and VEGF (82%, p < 0.05).ConclusionsMeroxest improves the prognosis of mice since it reduces leukocyte infiltration, and decreases the expression of Ki67 and VEGF markers. Consequently, the merosesquiterpene could become a useful antiangiogenic drug in the treatment of breast cancer. These results encourage us to deepen the study of meroxest, in order to find more evidence that supports the convenience of its evaluation in a clinical study or trial.

Abstract 3909: Iron oxide nanoparticle-leptin receptor antagonist: A novel targeted adjuvant therapy for triple negative breast cancer

Abstract Background: Leptin is a cytokine secreted by adipose tissue that binds its receptor, OB-R, leading to increased growth and survival of cancer cells. High levels of leptin have been correlated with poorer outcomes in breast cancer patients, particularly those afflicted with triple negative breast cancer (TNBC). We have created a leptin peptide receptor antagonist, LPrA2, which has been shown to block leptin signaling in several cancer types. To increase its effectiveness, LPrA2 was coupled to iron oxide nanoparticles (IONPs). We aim to determine if IONP-LPrA2 will serve as an adjuvant treatment, when combined with chemotherapeutics, for human TNBC and mouse TNBC-like (Tamoxifen-resistant) breast cancer cells E0771-TAM. Methods: Conjugation of LPrA2 to IONPs was confirmed by Western Blot and inhibition assays. To evaluate the effects of IONP-LPrA2 in mouse TNBC-like cells, the E0771 cell line (progesterone receptor and HER2 negative) was made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Human TNBC and E0771 cells, both responsive and unresponsive to estrogen (E0771-TAM), were treated with recombinant leptin and culture medium containing fetal bovine serum plus chemotherapeutics and IONP-LPrA2. Cell cycle progression, cell viability and apoptosis were analyzed using the Cellometer Vision Image Cytometer ®. Results: IONP-LPrA2 was found to cause significant decrease in DNA synthesis during the S phase of the cell cycle in TNBC and TNBC-like cells when compared to E0771 cells. Additionally, IONP-LPrA2 showed additive effects on reduction of cell viability and apoptosis when combined with chemotherapeutics. Conclusion: These findings indicate that IONP-LPrA2 may be useful in the treatment and prevention of TNBC. Further, IONP-LPrA2 treatment may increase the effectiveness and allow for reduction of the dosage of chemotherapeutics. This will in turn attenuate undesired side effects of chemotherapy. These results may be beneficial for breast cancer patients, particularly those with TNBC who are overweight or obese lacking targeted therapy thus suffering the poorest outcomes. Citation Format: Tia L. Harmon, Adriana Harbuzariu, Lily Yang, Ruben R. Gonzalez-Perez. Iron oxide nanoparticle-leptin receptor antagonist: A novel targeted adjuvant therapy for triple negative breast cancer. [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 3909. doi:10.1158/1538-7445.AM2015-3909

Abstract 903: Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice

Abstract Metformin has been shown to decrease breast cancer incidence in diabetic women, suggesting that improvement of metabolic status may be a useful approach to decrease breast cancer risk. As the putative cellular target of metformin, AMP-activated protein kinase (AMPK) not only regulates energy metabolism in liver, muscle, and adipose tissue but also inhibits protein synthesis and induces cell cycle arrest and apoptosis. Naringenin is a flavonoid abundant in citrus fruits that may have beneficial effects on metabolic health and tumorigenesis. Our in vitro data showed that naringenin inhibited proliferation of E0771 murine breast cancer cells, increased AMPK activity, and reduced expression of cell cycle regulatory proteins Cyclin D1 and anti-apoptotic Bcl-2 protein. In this study, we evaluated whether dietary naringenin acts like metformin to suppress mammary tumor growth in a mouse model of postmenopausal obesity. Ovariectomized female C57BL/6J mice were fed a high-fat diet (60% energy as fat) for 3 weeks to induce obesity. Then mice were randomized to experimental diets of HF (continued the high-fat diet), LN (high fat + 1% wt/wt naringenin in diet), HN (high fat + 3% wt/wt naringenin in diet), and Met (high fat + 180 mg/kg metformin in water) for 5 weeks. After 2 weeks of treatment, 5 × 10(5) E0771 cells were injected into one thoracic mammary fat pad. Mice were maintained on study diets for another 3 weeks. For all mice tumor weight correlated with weight gain in the first 5 weeks prior to cell inoculation (r = 0.51, p &amp;lt; 0.01). There was no difference in fasting glucose levels among the groups on week 8. No difference was observed between the HF and LN groups regarding final body weight, food intake, tissue weights, and tumor mass. HN significantly decreased final body weight, food intake, and adipose depot mass. mRNA expression of two inflammatory cytokines, interleukin 6 and monocyte chemoattractant protein-1 were decreased in perigonadal fat of the HN group. Although tumor growth (cm2) decreased over time in HN mice, there was no significant difference in tumor mass between HF and HN groups at necropsy (p = 0.4). HN did not affect AMPK activity in tumor tissue, but decreased Bcl-2 protein expression. Additionally, dietary naringenin resulted in a dose ­dependent accumulation in plasma, tumor, and mammary fat. In comparison to naringenin enriched diets, metformin significantly reduced tumor growth and final tumor weight, without affecting body weight, food intake, and tissue weights. Metformin decreased protein expression of Cyclin D1 in mammary tumors. However, we did not observe AMPK activation and changes in Bcl-2 protein levels in tumors. Taken together, these data suggest that metformin may inhibit mammary tumor growth without affecting metabolic status as assessed by body weight, adiposity, and fasting glucose. There may be different mechanisms of action for naringenin and metformin to alter mammary tumorigenesis. Citation Format: Jia-Yu Ke, Yung-Hsuan Hsiao, Shana R. Straka, Lisa D. Yee, Martha A. Belury. Comparison of the citrus flavonoid naringenin and metformin for effects on breast cancer in obese ovariectomized mice. [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 903. doi:10.1158/1538-7445.AM2015-903

Abstract 3173: The NLRC4 inflammasome promotes breast cancer progression in diet-induced obese mice

Abstract Obesity is not only a known risk factor for the development of ER+ breast cancer in postmenopausal woman, but is also associated with a poor clinical outcome across all subtypes of breast cancer. Obesity is also one of the only known risk factors for the development of invasive ductal carcinoma (IDC) versus ductal carcinoma in-situ (DCIS; non-invasive). A number of hypotheses have been proposed to explain the association between obesity and breast cancer progression. One such hypothesis is that the chronic inflammation in adipose tissue associated with obesity, especially adipose tissue in the mammary glands, creates a microenvironment that promotes breast cancer progression by acting as a reservoir of pro-inflammatory cytokines (including interleukin IL-1β and IL-6). One source of pro-inflammatory cytokines in adipose tissue is activated inflammasomes in infiltrating macrophages. Inflammasomes are multiprotein complexes whose main function is the activation of IL-1β and IL-18 during inflammation via Caspase 1-mediated proteolytic cleavage. Chronic inflammation, NLRP3 inflammasome activation and IL-1β are thought to be a major cause of obesity-associated insulin resistance and are involved in many human diseases including type-2 diabetes and cancer. However, the mechanism linking obesity and breast cancer progression remains unknown. We hypothesize that obesity-induced inflammasome activation in mammary adipose tissue results in active IL-1β which in turn promotes breast cancer progression. Using a syngeneic orthotopic transplant model of breast cancer in C57Bl/6 mice (Py8119 cells and E0771 cells) we have shown that diet-induced obese (DIO) mice (mice fed a high fat diet) have increased tumor growth and metastasis compared to control mice (mice fed normal chow). Tumors from DIO mice have increased macrophage infiltration and caspase-1 activation compared to tumors from control mice. Additional studies have shown that treating DIO mice with an IL1R1 (the receptor for IL-1) blocking antibody reduces tumor growth to that observed in control mice. Furthermore, NLRC4 deficient DIO mice had reduced tumor growth (compared to wild-type DIO) mice, while there was no decrease in tumor growth in NLRP3 deficient DIO mice, indicating that NLRC4 inflammasome activation promotes breast cancer growth in obese mice. Finally, tumors from DIO mice had increased vascularization (as evidenced by an increase in CD31+ cells) compared to control mice, which was decreased in NLRC4 deficient mice or mice treated with the anti-IL1R1 antibody. This data indicates that obesity induced NLRC4 inflammasome activation in infiltrating macrophages in the mammary gland promotes breast cancer progression possibly through increased angiogenesis in the tumor microenvironment. Thus, targeting inflammasome activation or the IL-1/IL1R1 axis represents a rational target for the treatment of breast cancer in obese patients. Citation Format: Ryan Kolb, Nicholas Borcherding, Yinghong Liu, Fang Yuan, Qing Xie, Fayyaz Sutterwala, Weizhou Zhang. The NLRC4 inflammasome promotes breast cancer progression in diet-induced obese mice. [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 3173. doi:10.1158/1538-7445.AM2015-3173

Abstract 898: Exercise inhibits the growth of breast cancer and reduces fat mass in postmenopausal obese mice associated with a circulating angiostatic phenotype and the inhibition of angiogenesis in those tissues

Abstract Obesity and physical inactivity are important risk factors for cancers, especially for postmenopausal breast cancer. However, potential mechanisms of exercise in reducing obesity and preventing postmenopausal breast cancer are poorly understood. The animal models in this area are very limited. In the present study, we used a postmenopausal obese mouse model inoculated with mouse breast cancer (E0771) cells to examine the potential effects of long-term exercise in reducing fat mass and breast cancer progression. Ovariectomy (OVX) was performed in 12 sixty week-old female mice (C57BL/6J), then followed by a high-fat (60%) diet for 12 weeks. In the eighth week of the dietary program, the body weight (BW) was significantly increased from 32.1±1.8 to 42.5±2.3 g; P&amp;lt;0.01), then 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. The mice were randomized into two groups, exercise and sedentary (n = 6). The exercise was conducted in a voluntary running cage system, i.e., 19.7±2.8 m/min, 30 min in the morning and 30 min in the afternoon, 7days/week, for 4 weeks. The lean mass (LM) or fat mass (FM) composition was monitored by EchoMRI weekly. In the end of experiment, the mice were anesthetized by isoflurane 2 hours after the last exercise, and blood samples, visceral fat and tumors were collected for measuring the expression of endostatin, sFlt-1 and free-VEGF using ELISA and blood vessel density (BVD) using CD31 immunochemistry. There was a significant decrease in BW (40.5±1.9 vs. 45.7±2.3 g; P&amp;lt;0.01) and tumor weight (1.95±0.41 vs. 2.63±0.11 g; P&amp;lt;0.01) in exercised-group compared to sedentary group, respectively. Exercise significantly decreased FM (17.8±0.9 vs. 22.7±1.1 g; P&amp;lt;0.01), tumor VEGF levels (44.1±2.1 vs. 65.4±3.3 pg/mg protein; P&amp;lt;0.01), and BVD (147.2±6.3 vs. 168.3±5.1 BV#/mm^2; P&amp;lt;0.01) in comparison with the sedentary, respectively. Exercise significantly decreased a ratio of capillaries to adipocytes of visceral fat in comparison with sedentary group (0.63±0.02 vs. 0.89±0.05; P&amp;lt;0.01). Exercise significantly increased endostatin (33.3±1.6 vs. 24.1±1.2 ng/ml; P&amp;lt;0.01) and sFlt-1 (59.5±3.1 vs. 48.3±2.5 pg/ml; P&amp;lt;0.05), and decreased free-VEGF (42.5±2.1 vs. 61.3±3.1 pg/ml; P&amp;lt;0.01) in the circulation in comparison with sedentary group, respectively. In postmenopausal obese mice, a long term exercise significantly 1) inhibited breast tumor angiogenesis and growth, 2) limited fat mass accumulation by reducing a ratio of capillaries to adipocytes, and 3) induced a circulating angiostatic phenotype characterized by increased endostatin and sFlt-1 and decreased free VEGF. These findings support the hypothesis that exercise-induced circulating angiostatic phenotype may contribute to the prevention of breast cancer progression and obesity in postmenopausal women. Citation Format: Shelby A. Cole, Jian-Wei Gu. Exercise inhibits the growth of breast cancer and reduces fat mass in postmenopausal obese mice associated with a circulating angiostatic phenotype and the inhibition of angiogenesis in those tissues. [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 898. doi:10.1158/1538-7445.AM2015-898

Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study.

Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids. Occurrence of triglycerides and free fatty acids will be examined in bAT and similar lipidomic analyses will be carried out visceral adipose tissue, highly inflamed in obesity.

Alcohol promotes mammary tumor growth through activation of VEGF-dependent tumor angiogenesis.

Alcohol consumption has been recognized as a risk factor for breast cancer. Experimental studies demonstrate that alcohol exposure promotes the progression of existing mammary tumors. However, the mechanisms underlying this effect remain unclear. In the present study, the role of vascular endothelial growth factor (VEGF) in alcohol promotion of breast cancer development was investigated using a mouse xenograft model of mammary tumors and a three-dimensional (3D) tumor/endothelial cell co-culture system. For the mouse xenograft model, mouse E0771 breast cancer cells were implanted into the mammary fat pad of C57BL6 mice. These mice were exposed to alcohol in their drinking water. For the 3D co-culture system, E0771 cells and MDA-MB231 breast cancer cells were co-cultured with SVEC4-10EE2 and human umbilical vein endothelial cells, respectively. The results demonstrated that alcohol increased tumor angiogenesis and accelerated tumor growth. Furthermore, it appeared that alcohol induced VEGF expression in breast cancer cells in vitro and in vivo. Blocking VEGF signaling by SU5416 inhibited tumor angiogenesis in the 3D tumor/endothelial cell co-culture system. Furthermore, injection of SU5416 into mice inhibited alcohol-promoted mammary tumor growth in vivo. These results indicate that alcohol may promote mammary tumor growth by stimulating VEGF-dependent angiogenesis.

Abstract P2-05-06: Does the lipid mediator sphingosine-1-phosphate have a role in obesity-related cancer progression?

Abstract INTRODUCTION: Obesity, which is the number one health risk in US ranked by the CDC, is an established independent prognostic factor for breast cancer patients. While the link between obesity and elevated breast cancer mortality is well known, the underlying mechanisms are poorly understood. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression and inflammation. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and exerts its functions by binding to specific G protein-coupled receptors (S1PR1-5). We have recently shown that SphK1, but not SphK2, produces S1P that is exported from breast cancer cells, mediated by the ATP-binding cassette transporter, ABCC1. Furthermore, we discovered that another S1P transporter, Spns2, is important for the lymphatic network formation in the LNs. FTY720, which after phosphorylation is a S1PR1 functional antagonist, was recently approved by FDA for multiple sclerosis. FTY720 also has been suggested to have some anti-cancer actions. Our hypothesis is that obesity up-regulates SphK1, which produces more S1P; the elevated levels of S1P in both tumor and its microenvironment stimulate breast cancer progression. FTY720 is expected to disrupt the SphK1/S1P/S1PR1 axis, which is strengthened by obesity, and to reduce cancer metastasis and prolong survival. We tested our hypothesis by utilizing animal models of breast cancer with obesity treated with FTY720. METHODS: We utilized two different syngeneic breast cancer mouse models: 4T1-luc2 cells in BALB/c mice and E0771 cells in C57Bl/6 mice, both inoculated into mammary fat pads of mice fed with normal or high fat diet. FTY720 was given orally. Western blot, QPCR and LC-ESI-MS/MS assays were used. RESULTS: We observed that breast tumors in obese animals expressed higher levels of SphK1 and S1P transporters, such as ABCC1 and Spns2, as compared to animals on a normal diet. The levels of S1P in the plasma of obese mice were also elevated, which appears to be a consequence of the higher production of S1P by SphK1 in the tumor and its microenvironment. Importantly, tumor progression in both models was suppressed significantly by administration of FTY720. Interestingly, the cancer progression was suppressed more efficiently in the obese mice than the lean mice. Further, LC-ESI-MS/MS analysis showed that FTY720 suppressed S1P levels not only in the blood, but also in the tumor interstitial fluid. CONCLUSIONS: These results suggest that the S1P axis is strengthened by obesity, and has a role in cancer progression. Targeting the S1P axis with FTY720 may be useful for treating breast cancer in individuals with obesity. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and R01CA160688 (to K.T.). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-06.

Obesity induced a leptin‐Notch signaling axis in breast cancer

To investigate whether obesity induces a leptin-Notch signaling axis in breast cancer (BC), leptin-induced Notch was determined in human MCF-7 and MDA-MB231 and mouse E0771 cells and in E0771-BC hosted by syngeneic lean and diet-induced obesity (DIO) C57BL/6J female mice. Lean and DIO mice were treated for 3 weeks with leptin inhibitor (PEG-LPrA2) 1 week after the inoculation of E0771 cells. Leptin induced Notch1, 3 and 4 in BC cells, but Notch2 expression showed opposite pattern in MCF-7 compared to MDA-MB231 cells. Notch loss-of-function (DAPT and dominant negative [R218H] RBP-Jk [CSL/CBF1]) showed that a functional leptin-Notch signaling axis was involved in the proliferation and migration of E0771 cells. E0771-BC onset was affected by obesity (lean mice7/10 [70%] vs. DIO mice: 11/12 [92%]; Pearson χ(2) : p = 0.06]). PEG-LPrA2 significantly reduced BC growth (untreated: 19/42; [45%] vs. treated: 8/42 [19%]; Pearson χ(2) : p = 0.008). PEG-LPrA2 did not influence the caloric intake of mice but increased carcass and/or body weights of lean and DIO mice inoculated with E0771 cells, which could be related to the improvement of health conditions (less aggressive disease). Importantly, BC from obese mice had higher levels of Notch3, JAG1 and survivin than lean mice. Inhibition of leptin signaling reduced protein levels of Notch (NICD1, NICD4, Notch3, JAG1 and survivin) and significantly decreased mRNA expression of Notch receptors, ligands and targets. PEG-LPrA's effects were more prominent in DIO mice. Present data suggest that leptin induces Notch, which could be involved in the reported higher incidence and aggressiveness and, poor prognosis of BC in obese patients.

Abstract 1400: Obesity induced leptin-notch signaling axis in breast cancer.

Abstract Obesity leads to increased leptin levels and signaling which has been linked to the growth of breast cancer (BC). We early identified leptin as an inducer of Notch in BC cells. However, whether leptin-induction of Notch is instrumental to the development of BC is unknown. Objective: Therefore, we examined the impact of obesity and inhibition of leptin signaling (via LPrA2 antagonist) on the growth of syngeneic E0771-derived BC and tumor expression of Notch in lean and obese mice and in E0771 cell cultures. Methods: C57BL/6J female mice were fed either high-fat diet (HFD, 60% Kcal from fat) or normal diet (10% Kcal from fat). Obesity was defined as BW ≥ 20% BW Lean-control at week 5. Lean and diet-induced-obesity (DIO) mice were orthotopically inoculated with mouse E0771 cells in the lower left region of the mammary pad. One week later the mice were treated with either (1) pegylated leptin peptide receptor antagonist 2, PEG-LPrA2 or (2) PEG-Scrambled peptide (as control; 50μl i.v. tail vein/ 0.1 mM) following the two-arm design: (a) one and (b) two doses/weekly. Treatment lasted for 4 weeks. Tumor onset and growth was determined overtime and expression of Notch, its ligands, and crosstalk factors were assessed using western blot, immunohistochemistry, and ELISA. Leptin's effects on Notch and crosstalk factors were also investigated in E0771 cell cultures. Results: Obesity induced E0771-BC onset and growth and, increased the expression of Notch in vivo. Inhibition of leptin signaling had a higher impact on DIO mice than in lean mice by delaying onset and reducing tumor growth and the expression of Notch. Therefore, high levels of leptin in DIO-mice played a positive role in tumorigenesis. Similarly, leptin induced and PEG-LPrA2 decreased Notch expression in E0771 cells in vitro. Present data further validate the idea that the consumption of a high-fat diet leads to early BC onset and growth, which was linked to the leptin-Notch axis. Our results reinforce the potential use of inhibition of leptin signaling as a novel approach to fight breast cancer, especially in obese patients showing higher levels of leptin and incidence of BC. Funding: This work was supported by NIH/NCI 5SC1CA138658-03 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP), NIH/2G12RR003034-26; U54 MSM/TU/UAB Cancer Center Partnership; and 1R21CA153172-01A1 (to MTK). This research was also supported, in part, facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386). Citation Format: Monica Battle, Corey Gillespie, Tanisha McGlothen, Marta Torroella-Kouri, Ruben R. Gonzalez-Perez. Obesity induced leptin-notch signaling axis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1400. doi:10.1158/1538-7445.AM2013-1400

EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

The role of EGCG, a major green tea catechin in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Sixteen eight-wk-old female mice (C57BL/6 J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. Eight mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. EGCG treatment significantly reduced tumor weight over the control (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01), tumor CD (109 ± 20 vs. 156 ± 12 capillary #/mm^2; P < 0.01), tumor VEGF expression (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01), respectively. But, it has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. EGCG at 50 μg/ml significantly inhibited the activation of HIF-1α and NFκB as well as VEGF expression in cultured E0771 cells, compared to the control, respectively. These findings support the hypothesis that EGCG, a major green tea catechin, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression.

Abstract B103: Expression of n-3 fatty acid desaturase suppresses tamoxifen-resistant breast cancer in vitro and in fat-1 transgenic mice

Abstract Tamoxifen-resistance remains a significant clinical problem in breast cancer treatment. Currently, hormonal therapies can achieve 50-60% breast cancer remission rate in ERα+ patients, who will mostly develop resistance. We have hypothesized that fat-1 gene therapy, which introduce an n-3 fatty acid desaturase to convert n-6 to n-3 polyunsaturated fatty acids (PUFA), as an adjuvant treatment will enhance hormonal therapy of breast cancer. We have previously reported that C57BL/6 fat-1 transgenic mice suppress breast cancer growth and the inflammation/chemically induced lung metastasis of E0771 murine mammary tumors. This corroborates the experimental reports about the tumor suppression effect of n-3 PUFA, a main component of fish or fish oil. In this report, we will present experimental evidence that fat-1 gene expression suppresses tamoxifen-resistant human breast cancer in vitro; in addition, expression of n-3 fatty acid desaturase encoded by fat-1 gene in transgenic mice significantly suppresses murine tamoxifen-resistant E0771TamR mammary tumor growth in vivo. Methods: The tamoxifen-resistant human breast cancer cells MCF-7TamR and MDA-MB-231 (ER-), as well as the tamoxifen-sensitive human breast cancer T47D cells, were transfected with pST180 plasmid carrying fat-1 gene and the control empty pST128 plasmid. These cells were characterized in proliferation and caspase-3 apoptosis assays. The non-tumorigenic MCF-10A cells were also transfected with fat-1 gene to investigate the n-3 fatty acid desaturase effect on non-malignant human epithelial mammary cells. Two experimental groups of the fat-1 transgenic female C57BL/6 mice and wild type C57BL/6 control mice, were injected with tamoxifen-resistant E0771TamR tumor cells, or the parental E0771 cells, into breast fat pad of the 4th nipple and flank subcutaneously. Tumor sizes were measured daily until tumors reach 2 cm, when mice were sacrificed and tumor tissues and internal organs were harvested for fatty acid analysis and histology staining. Results: In cell culture studies, introduction of fat-1 gene significantly reduced the proliferation rate of the tamoxifen-resistant MCF-7TamR cells (37.3%), MDA-MB-231 cells (27.5%), and T47D cells (67.7%). The MCF-10A cells transfected with fat-1 gene did not show any change of cell proliferation rate compared to the control cells. The caspase-3 activities in MCF-7TamR cells, MDA-MB-231 cells, and T47D cells were respectively increased by 1.3-, 2.1- and 5.6-fold when expressing fat-1 gene, as compared to the control cells. In vivo, n-3 fatty acid desaturase expressed in fat-1 mice significantly suppressed growth of tamoxifen-resistant murine E0771TamR mammary tumors grown in flank and breast fat pad, as compared to the control mice. The fat-1 growth inhibition effect is more dramatic for E0771TamR tumors than the parental E0771 tumors in fat-1 mice. Fatty acid analysis has shown a significant decrease of long chain n-6/n-3 PUFA ratio in breast tumor tissues (33.5%), flank tumor tissues (33.9%), kidney (32.7%), muscle (43.8%), and adipose tissues (69.1%) in fat-1 mice compared to wild type mice. Conclusions: Introducing an n-3 fatty acid desaturase in tumor cells and transgenic animals has reduced long chain n-6/n-3 PUFA ratio and imposed an anti-tumor effect against tamoxifen-resistant breast cancer growth. Tamoxifen-sensitive breast cancer were also inhibited in Fat-1 gene therapy. Citation Format: Raghunatha Reddy, Ian Bayles, Qiuhong He. Expression of n-3 fatty acid desaturase suppresses tamoxifen-resistant breast cancer in vitro and in fat-1 transgenic mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B103.

P4-03-02: High Fat Diet-Induced Postmenopausal Obesity Promotes Tumor Angiogenesis and Breast Cancer Progression in Age-Relevant Ovariectomized Mice.

Abstract Background: Obese postmenopausal women have 50% higher risk of breast cancer than non-obese women. The mechanisms of postmenopausal obesity-induced breast cancer are poorly understood due to lack of the established animal model that mimics postmenopausal obesity related to breast cancer progression. Material and Methods: Using age-relevant C57BL/6 mice, this study determined whether postmenopausal obesity increases VEGF expression, tumor angiogenesis, and breast tumor growth. Ovariectomy (OVX) was performed in 12 sixty week-old female mice (life span is about 140 weeks), then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the 8th wk of the dietary program, 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored using dial calipers for 4 wk. Body weights were monitored weekly. At the end of the experiment, blood samples, visceral fat, and tumors were collected for measuring VEGF expression using ELISA and intratumoral microvessel density (IMD) using CD31 immunochemistry. Results: Body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3±1.7 vs. 41.5±1.5 g; P&amp;lt;0.01). There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062±0.005 vs. 0.032±0.003; P&amp;lt;0.01). Postmenopausal obesity significantly increased breast tumor weight over the control (4.62±0.63 vs. 1.98±0.27 g; P&amp;lt;0.01) and IMD (173±3.7 vs. 139±4.3 IM#/mm^2; P&amp;lt;0.01). Tumor VEGF levels were higher in OVX/HF mice, compared to OVX/LF group (73.3±3.8 vs. 49.5±4.3 pg/mg protein; P&amp;lt;0.01). Plasma VEGF levels (69±7.1 vs. 48±3.5 pg/ml) and visceral fat VEGF levels (424.4±39.5 vs. 208.5±22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n=6; P&amp;lt;0.01). Interestingly, adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77±1.14 vs. 0.94±0.16 pg/mg tissue; n=6; P&amp;lt;0.01). The abdominal subcutaneous fat expressed more VEGF proteins than visceral fat in OVX/HF mice (692±72 vs. 431±44 pg/mg protein; n=6; P&amp;lt;0.01). There was a strong positive linear correlation between increased breast tumor weight and visceral fat weight in OVX mice (R2=0.7379; N=12; P&amp;lt;0.01). However, there was no significant difference in heart, or kidney weight/body weight ratio between postmenopausal obesity (OVX/HF) mice and the control mice (OVX/LF). Discussion: Our observations indicate that ovariectomy plus a high fat diet with the inoculation of E0771 (mouse ER+ breast cancer) cells in female wild type &amp;gt;60 week old mice can mimic human obesity-induced postmenopausal breast cancer. The increased tumor angiogenesis in postmenopausal obese mice was correlated with increased breast tumor growth, adipose tissue mass, and adipokines such as VEGF. These findings support the hypothesis that postmenopausal obesity promotes tumor angiogenesis and breast cancer progression, possibly through increased adipose tissue mass and adipokines such as VEGF that could systemically and locally affect breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-02.

P3-17-07: EGCG, a Green Tea Antioxidant Suppresses Breast Tumor Angiogenesis and Growth Via Inhibiting the Activation of HIF-1a and NFκB, and VEGF Expression.

Abstract Background Cancer cells are under greater oxidative stress than normal cells. Limited studies have showed that epigallocatechin-3-gallate (EGCG), a green tea antioxidant can afford protection against a variety of cancer types. The role of EGCG in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG as an antioxidant can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Material and Methods: 16 eight-wk-old female mice (C57BL/6J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. 8 mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunochemistry. Cultured E0771 cells were used for determining the direct effects of EGCG on proliferation (3H-thymidine incorporation), migration (Matrigel assay), VEGF expression (ELISA), the activation of HIF-1α and NFκB (motif binding assays, Active Motif). MCF-7 and MDA-MB-231 cells were also used for 3H-thymidine incorporation. Results: Oral EGCG treatment significantly reduced tumor weight over the control (0.37±0.15 vs. 1.16±0.30 g; P&amp;lt;0.01), tumor CD (109±20 vs. 156±12 capillary #/mm^2; P&amp;lt;0.01), tumor VEGF expression (45.72±1.4 vs. 59.03±3.8 pg/mg; P&amp;lt;0.01), respectively. EGCG treatment reduced plasma VEGF levels over the control mice (26.48±3.76 vs. 40.79±3.5 pg/ml; P&amp;lt;0.01). However, there were no differences in the body weight and heart weight between EGCG and the control groups. EGCG did not inhibit angiogenesis and VEGF expression in the heart and skeletal muscle of mice, compared to the control. EGCG at 50 mmol/L significantly inhibited the activation of HIF-1α (0.11±0.02 vs. 0.24±0.02; P&amp;lt;0.01) and NFκB (1.15±0.21 vs. 1.61±0.32; P&amp;lt;0.01) as well as VEGF expression (1752±49 vs. 2254±91 pg/mg; P&amp;lt;0.01) in cultured E0771 cells, compared to the control, respectively. EGCG caused a dose-related inhibition on the proliferation and migration in cultured E0771 cells. EGCG also caused a dose-related inhibition on the proliferation in cultured MCF-7 and MDA-MB-231 cells. Discussion: These findings support the hypothesis that EGCG, a green tea antioxidant, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Interestingly, oral EGCG treatment has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. This work will have important implications for translating EGCG therapy to human breast cancer treatment and prevention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-07.

α-Synuclein Expression Selectively Affects Tumorigenesis in Mice Modeling Parkinson's Disease

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn- dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over- expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.