Animal Models Of Breast Cancer Research Articles

Phenformin suppresses proliferation and induces apoptosis in endometrial cancer cell lines

Objective: Obesity and diabetes have been linked to worse outcomes in women with endometrial cancer. Metformin is a biguanide drug that is widely used for the treatment of type 2 diabetes and has been found to have antitumorigenic effects via AMPK activation and inhibition of the mTOR pathway. Phenformin is another biguanide with antidiabetic activity that was withdrawn from the market due to a small risk of lactic acidosis (64 cases per 100,000 patient-years), which was higher than that seen with metformin. Studies in breast cancer animal models suggest that phenformin may be more potent for inhibiting tumor growth than metformin. Thus, our goal was to assess the effect of phenformin on proliferation and apoptosis in endometrial cancer cell lines.

SMAD5 expression and inhibition of the mitotic kinase aurora-A on sensitivity of breast cancer cells to chemotherapy.

e13516 Background: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A will lead to increased sensitivity to chemotherapy. Methods: The hypothesis was tested in vitro. Cellular proliferation was tested using MTT assay and protein levels were determined on Western blots. Results: In ER+ cell line, MCF7, constitutive activation of Raf-1/MAPK signaling pathway leads to aberrant activation of Aurora-A kinase activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to parental MCF7 cells. This effect is even more pronounced with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by novel inhibitor, Alisertib. This was further explored in triple negative cells (MDA-MB-231) and cells expressing Her-2/neu (BT474) which express higher levels of Aurora-A. Combination of Alisertib and paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are investigating a role of Aurora-A in developing chemoresistance through activating SMAD5. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. Conclusions: Inhibition of Aurora-A by Alisertib resulted in decreased SMAD5 nuclear phosphorylation and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be extremely useful in developing more effective treatments for breast cancer patients both in neo-adjuvant, adjuvant and metastatic settings.

A Challenge for Medicinal Chemistry by the 17β-hydroxysteroid Dehydrogenase Superfamily: An Integrated Biological Function and Inhibition Study

Members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) superfamily perform distinct multiple catalyses by the same enzyme, apparently contradictory to the long-held beliefs regarding the high specificity of enzymes. Surprisingly, these multi-catalyses can combine synergistically in vitro and in vivo and their dysfunction may result in the stimulation of breast or prostate cancer. 17β-HSD1 possesses high estrogen activation activity, while its androgen inactivation is significant for decreasing the week concentration of dihydrotestosterone (DHT) in breast cancer cells, an important factor for cell proliferation. 17β-HSD5 can also carry out multiple catalyses in hormone-dependent cancer cells. In addition to 17β-HSDs 1 and 5 some other family members possess such dual-activity as well, and their inhibition decreases hormone- dependent cancer proliferation. The multi-specificity of 17β-HSD1 is structurally based on the pseudo-symmetric androgens that can accommodate the narrow enzyme substrate tunnel by both normal and alternative binding. The atypical family member 17β-HSD5 possesses a spacious binding site, which is accessible to several substrates. Expression of 17β- HSD1 can also control other estrogen-responsive elements such as pS2, and can regulate steroid-hormone receptors. The fundamental involvement of 17β-HSD1 in catalysis and gene regulation underlies its close relationship to breast cancer, attributable to its long evolutionary process. These observations stimulated detailed study of steroid-converting enzyme inhibition. The most significant efforts in designing 17β-HSD1 inhibitors in decades have progressed through structure activity relationship studies supported by the availability of both small and protein molecule structures, with the elimination of residual estrogenic activity in the inhibitors. The first non-estrogenic inhibitors of 17β-HSD1 to show activity in vivo (breast cancer animal model) are now reported.

Abstract B31: Pak1 and β-catenin inhibition blocks tumor progression in ErbB2-driven breast cancer models

Abstract p21-activated kinase-1 (Pak1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. However, its role in tumorigenesis in vivo, and the particular signaling pathways affected, are not defined. In this work, we examined the distinct roles of Pak1 and Pak2 in cellular and animal models of ErbB2-driven breast cancer. We found that inhibition of Pak1, but not Pak2, impedes transformation by ErbB2 in a 3D cell culture system, but that loss of either Pak1 or Pak2 causes loss of both Erk and Akt activation. A phospho-proteomic screen revealed that Pak1-deficient, but not Pak2-deficient, ErbB2 cells showed almost total loss of β-catenin expression that is accompanied by a significant reduction in almost all known β-catenin target genes. In MMTV-ErbB2 transgenic mice, loss of Pak1 prolonged survival, and mammary tissues of such mice showed loss of β-catenin. Expression of a β-catenin mutant bearing a phospho-mimetic mutation at Ser 675, a specific Pak1 phosphorylation site, restored the ability of ErbB2 to transform Pak1-deficient mammary epithelial cells. Lastly, we showed that small molecule inhibitors of Pak or β-catenin blocked transformation by ErbB2 in 3D culture and tumorigenesis by ErbB2 in mouse xenografts, and combined inhibition by both agents was synergistic. These findings establish Pak1 as a new target in ErbB2-driven breast cancer and define a new mechanism of action primarily through the β-catenin, but not the Erk or Akt, signaling pathways. Citation Format: Luis E. Arias-Romero, Olga Villamar-Cruz, Jonathan Chernoff. Pak1 and β-catenin inhibition blocks tumor progression in ErbB2-driven breast cancer models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B31.

Bazedoxifene Exhibits Antiestrogenic Activity in Animal Models of Tamoxifen-Resistant Breast Cancer: Implications for Treatment of Advanced Disease

There is compelling evidence to suggest that drugs that function as pure estrogen receptor (ER-α) antagonists, or that downregulate the expression of ER-α, would have clinical use in the treatment of advanced tamoxifen- and aromatase-resistant breast cancer. Although such compounds are currently in development, we reasoned, based on our understanding of ER-α pharmacology, that there may already exist among the most recently developed selective estrogen receptor modulators (SERM) compounds that would have usage as breast cancer therapeutics. Thus, our objective was to identify among available SERMs those with unique pharmacologic activities and to evaluate their potential clinical use with predictive models of advanced breast cancer. A validated molecular profiling technology was used to classify clinically relevant SERMs based on their impact on ER-α conformation. The functional consequences of these observed mechanistic differences on (i) gene expression, (ii) receptor stability, and (iii) activity in cellular and animal models of advanced endocrine-resistant breast cancer were assessed. The high-affinity SERM bazedoxifene was shown to function as a pure ER-α antagonist in cellular models of breast cancer and effectively inhibited the growth of both tamoxifen-sensitive and -resistant breast tumor xenografts. Interestingly, bazedoxifene induced a unique conformational change in ER-α that resulted in its proteasomal degradation, although the latter activity was dispensable for its antagonist efficacy. Bazedoxifene was recently approved for use in the European Union for the treatment of osteoporosis and thus may represent a near-term therapeutic option for patients with advanced breast cancer.

Abstract 4521: A novel targeted triggered release nanoparticle against cancer cells of diverse histological origin.

Abstract The paradigm of cancer treatment has changed substantially over the last decade. The discovery of new therapeutic targets within the tumor cell and the recognition of the interdependence with the tumor microenvironment have been the basis of such changes. However, no common ground was yet found for the treatment of cancer within its diverse histological origin. Conventional therapy against human canc¬er, relying on the use of drugs that do not differentiate between cancer and normal cells, is progressively giving room to novel therapeutic strategies. Specificity became mandatory for treatment effectiveness and target selection one of the main priorities. In this scenario, delivery systems rise as promising approaches, for the reason they can improve pharmacokinetic features of anticancer drugs and efficiently address a particular payload to a selected target, namely through exploitation of high specificity and affinity between cell-surface receptors and targeting ligands coupled at their surface. We evidence the overexpression of a target cell surface receptor (nucleolin) in several cancer cell lines of diverse histological origins: breast (hormone-dependent and triple negative subtypes), colon, prostate and small cell and non-small cell lung cancer. We demonstrate the ability of the nucleolin receptor to specifically internalize a F3-targeted sterically stabilized lipid-based formulation. Moreover, we show that the unique combination of targeting specificity and intracellular triggered release of a therapeutic agent, doxorubicin, results in enhanced cytotoxic activity in those specific cell lines, when compared to the non-targeted counterpart. Active accumulation of the targeted nanoparticle into human orthotopic tumors, implanted in the mammary fat pad of nude mice, was registered for a time-point as short as 4 h, reaching 48% of the injected dose/g of tissue. Twenty-four hours post-injection accumulation in the tumor tissue was 33-fold higher than the non-targeted counterpart. In mice treated with the targeted nanoparticle containing doxorubicin, significant decrease of the tumor viable rim area and microvascular density, as well as limited invasion to surrounding healthy tissues were observed (as opposed to other tested controls). Moreover, the clinical potential of such strategy was demonstrated by the successful specific cellular association by breast cancer cells harvested from tumors of patients submitted to mastectomy. Overall, we provide evidence of a nanoparticle with specificity to cancer cells of diverse histological origins and proven therapeutic efficacy in an animal model of breast cancer, which may be a breakthrough in the development of anticancer therapies, especially when considering cancer cell heterogeneity. Citation Format: Vera Moura, Manuela Lacerda, Paulo Figueiredo, Maria L. Corvo, Maria M E Cruz, Raquel Soares, Sérgio Simões, João Moreira. A novel targeted triggered release nanoparticle against cancer cells of diverse histological origin. [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 4521. doi:10.1158/1538-7445.AM2013-4521

Abstract LB-254: XBP1-NFkappaB signaling promotes antiestrogen resistance in breast cancer animal model.

Abstract There are nearly 3 million breast cancer survivors in the United States, with more than 230,000 women being newly diagnosed with invasive breast cancer each year. Of total breast cancer patients, around 70 percent are estrogen receptor (ER) positive and often receive endocrine therapy. Endocrine resistance, either de novo or acquired, is a major hurdle for treating ER positive breast cancer patients. Unfolded protein response (UPR) is known to be activated and promote endocrine resistance in breast cancer cells. As an essential component of URP signaling, X-box binding protein 1 (XBP1) was shown up-regulated in endocrine resistant breast cancer. XBP1 was demonstrated as an important mediator for endocrine resistance in vitro cell culture model in our previous findings. In this study, the effect of XBP1 in endocrine resistance in animal model was examined. XBP1 contains two alternative splice variants: XBP1(S) and XBP1(U). In order to differentiate the role of each variant, we generated XBP1 mutant constructs that overexpress specifically only either the XBP1(S) or XBP1(U). The XBP1 over-expressing human breast cancer cells have been injected into the mammary fat pad of female nude mice. The preliminary data shows that the over-expression of both XBP1 splice variants promotes tumor growth in vivo. More importantly, tumors with over-expressed XBP1 show less sensitivity to tamoxifen treatment. Our previous finding revealed that NFkappaB signaling is regulated by XBP1 and required for antiestrogen resistance in cultured cells. In the current study, we have treated XBP1-overexpressed breast tumor bearing animals with NFkappaB inhibitor Parthenolide, and find that it enhances tamoxifen sensitivity. In conclusion, our data demonstrate that XBP1-NFkappaB signaling plays an important role in antiestrogen resistance in vivo. Citation Format: Rong Hu, Alan Zwart, Anni Warri, Robert Clarke. XBP1-NFkappaB signaling promotes antiestrogen resistance in breast cancer animal model. [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 LB-254. doi:10.1158/1538-7445.AM2013-LB-254

Bexarotene Induces Cellular Senescence in MMTV-Neu Mouse Model of Mammary Carcinogenesis

Previous studies have shown that retinoids and rexinoids can prevent breast cancer in animal models and in women with increased risk of developing the disease. The cellular effects of these vitamin A analogues have been primarily associated with induction of differentiation and inhibition of proliferation. In this study, we tested the hypothesis that bexarotene (LGD1069, Targretin), a rexinoid, can not only inhibit cell proliferation but also induce cellular senescence in mammary epithelial cells, premalignant lesions, and tumors of the MMTV-Neu model of mammary carcinogenesis, which develops estrogen receptor-negative tumors. Mice with palpable mammary tumors were treated for 4 weeks with bexarotene at 80 or 40 mg/kg body weight, and senescent cells were determined by SA-β-Gal assay. Bexarotene decreased in a dose-dependent manner the multiplicity of premalignant lesions and tumors, and this was associated with inhibition of cell proliferation and induction of cellular senescence and apoptosis. By double labeling of senescent cells, first by SA-β-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARβ, but not p53, were upregulated by bexarotene in mammary tumors and in breast cancer cell lines, suggesting involvement of multiple signaling pathways in mediating the senescence program of rexinoids. These findings indicate that, in addition to cell proliferation and apoptosis, cellular senescence could be used as a potential biomarker of response in breast cancer prevention and therapy studies with rexinoids and possibly with other antitumor agents.

Evolution of the tissue selective estrogen complex (TSEC)

Estrogens mediate gene transcription and signaling of numerous cellular processes in a variety of tissues, including the bone, breast, and endometrium, through binding and activation of estrogen receptors (ERs). Estrogen-mediated ER agonist activity has shown benefit in conditions related to estrogen deficiency in women; however, these effects have been associated with stimulation of breast and uterine tissues. Due to the complexity of ER signaling, selective estrogen receptor modulators (SERMs) can exhibit ER agonist or antagonist activity depending on the target tissue. A newer approach to menopausal therapy, the tissue selective estrogen complex (TSEC), pairs a SERM with one or more estrogens with the goal of maintaining the benefits of estrogens without the stimulatory effects on the breast and uterus. Preclinically, different TSECs have been associated with distinct gene expression profiles compared with each other and with their individual SERM/estrogen components. Studies in cultured breast cancer cells and animal models have demonstrated a lack of estrogen-induced stimulation with TSECs in the mammary gland and endometrium. In the breast, biochemical analyses indicate that degradation of the ER is an important mechanism by which TSECs exert their antagonistic effects. TSECs have also shown positive effects similar to estrogens in other tissue types, including bone and the central nervous system, although mechanisms underlying these activities are less clear. Overall, preclinical studies have shown that estrogens, SERMs, and TSECs each exert distinct and tissue specific molecular and pharmacologic effects.

The MMTV-PyVT Transgenic Mouse as a Multistage Model for Mammary Carcinoma and the Efficacy of Antineoplastic Treatment

Animal models are commonly used to analyze the mechanism of carcinogenesis as well as the development and screening of potent drugs. Although numerous animal models of breast cancer have been used in research, few display multiple stages of tumorigenesis. The transgenic strain FVB/N-Tg(MMTV-PyVT) 634 Mul/J (also known as PyVT) was established to determine the effect of mammary gland-specific expression of the polyomavirus middle T antigen. Here the PyVT model with three distinct stages of tumor development (Pre, Early, and Late stages) was used as a model system for measuring tumor growth, tumor burden, and metastasis of mammary carcinomas. Additionally the expression profile of the molecular markers, survivin and Ki-67, was determined. Three antineoplastic compounds were tested over a 14-day period to determine their efficacy at attenuating tumor growth at each stage of development. Interestingly cisplatin and paclitaxel were determined to be ineffective anticancer drugs, while tamoxifen significantly reduced tumor growth in the Pre and Early stages of tumor formation.

Abstract P6-01-03: Decreasing the metastatic potential in Triple Negative Breast Cancer through the miR-17 cluster

Abstract Background: In animal models of breast cancer, caloric restriction (CR), or a 25–30% reduction of calories, has decreased tumor initiation and progression. We sought to determine if CR could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (RT) and alter the molecular profile of triple negative breast cancer (TNBC), which displays a poor prognosis. Methods/Results: In two murine models of TNBC (4T1 which is highly metastatic and 67NR which is locally aggressive), mice with palpable tumors were treated with radiation, caloric restriction (30% intake reduction) or both. 3 times per week measurements revealed significant tumor regression with RT or diet modification, and an even greater regression is observed combining diet modification with RT. Concurrently, many reports have shown that TNBC is most distinct from other breast tumor subtypes due to up-regulation of the miR-17-92 cluster which we have shown by RT-PCR is downregulated with both CR and radiation (RT) and even further decreased with the combination. cDNA arrays done on all conditions revealed that the top 5 statistically significant GO terms with high fold changes were all associated with extracellular matrix and metastases. In silico analysis demonstrated 5 ECM related genes that were targets of the miR-17 cluster: collagen 4 alpha 1 (COL4A1), collagen 4 alpha 3 (COL4A3), laminin alpha 3 (LAMA3), metallopeptidase inhibitor 2 and 3 (TIMP2 and TIMP3) and these were inversely correlated with the miRs. Conclusions: CR, or altering the metabolism of breast cancer, can be combined with RT to decrease progression of TNBC tumors and that the miR-17-92 cluster is also decreased with these conditions. cDNA arrays suggest that extracellular matrix (ECM) proteins play a large role in the mechanism of action of CR and RT combined. Combination therapy with CR and RT can decrease the metastatic potential in TNBC and in part does this through the miR-17-92 cluster by way of controlling ECM. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-03.

Chemopreventive and anti‐angiogenic effects of dietary phenethyl isothiocyanate in an N‐methyl nitrosourea‐induced breast cancer animal model

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.

The influence of glucocorticoid signaling on tumor progression

The diagnosis of cancer elicits a broad range of well-characterized stress-related biobehavioral responses. Recent studies also suggest that an individual's neuroendocrine stress response can influence tumor biology. One of the major physiological pathways altered by the response to unrelenting social stressors is the hypothalamic-pituitary-adrenal or HPA axis. Initially following acute stress exposure, an increased glucocorticoid response is observed; eventually, chronic stress exposure can lead to a blunting of the normal diurnal cortisol pattern. Interestingly, recent evidence also links high primary tumor glucocorticoid receptor expression (and associated increased glucocorticoid-mediated gene expression) to more rapid estrogen-independent breast cancer progression. Furthermore, animal models of human breast cancer suggest that glucocorticoids inhibit tumor cell apoptosis. These findings provide a conceptual basis for understanding the molecular mechanisms underlying the influence of the individual's stress response, and specifically glucocorticoid action, on breast cancer and other solid tumor biology. How this increased glucocorticoid signaling might contribute to cancer progression is the subject of this review.

Abstract A36: Resveratrol decreases DNMTs in a human relevant animal model of breast cancer

Abstract Background: We previously reported that trans-resveratrol (resveratrol) downregulates the expression DNA methyltransferases (DNMTs) 1 and 3b in vitro and demethylates the tumor suppressor gene RASSF-1a in the breasts of women who are at increased breast cancer risk. Effects increased with increasing dose/circulating level of the agent, respectively. In the current report, we evaluated the effect of increasing doses of resveratrol on mammary tumor formation in the ACI rat, which develops estrogen dependent mammary carcinoma. Methods: 91 female rats were treated with a slow release estradiol (E2) pellet plus one of the following: 5 mg/kg/day (low dose) resveratrol (n=23), 25 mg/kg/day (high dose) resveratrol (n=22), 0.1mg/kg/week 5-azacytadine (aza) (n=22), an inhibitor of DNMTs which is used clinically, or control (n=24). The animals were examined weekly to assess for the presence of tumor formation. After 5.5 months of treatment, the animals were sacrificed and the mammary glands harvested, with half of each gland snap frozen and the other half formalin fixed. Specimens were evaluated by a board certified pathologist to confirm histology as benign or malignant. DNMT1 and 3b were measured by qRT-PCR. Results: Tumor formation was lowest (18% of rats) in the high dose resveratrol and 5-aza groups, intermediate in the low dose resveratrol group (26%) and highest in control (33%). DMNT1 expression was significantly (p&amp;lt;0.0001) influenced by treatment, while DNMT3b was not. On the other hand, DNMT3b expression was significantly (p&amp;lt;0.0001) influenced by histology (benign vs. malignant), whereas DNMT1 was not. Conclusion: Treatment with resveratrol and 5-aza significantly influence DNMT expression, which may be important in the prevention/delay of mammary tumor formation. This mechanism of action of resveratrol warrants further investigation. Citation Format: Wenyi Qin, Weizhu Zhu, Ke Zhang, Kaitlin Clarke, Edward R. Sauter. Resveratrol decreases DNMTs in a human relevant animal model of breast cancer. [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 A36.

Abstract A58: High Throughput Search for a Combination Cancer Preventive Treatment

Abstract Identification of safer chemopreventive drugs is essential for the future of cancer preventive treatment. Although the feasibility for chemoprevention of estrogen receptor (ER) -positive breast cancers has been exemplified by the use of selective estrogen response modifiers (SERMs), we are still lacking preventive options for many other disease types, including breast cancers not dependent on ER status. Rexinoids are a class of agents that effectively reduce the incidence of both ER-positive and -negative tumors in animal models of breast cancer and thus may be better cancer preventive drugs than the current standard SERMs. However, significant side-effects at the effective clinical dose, including hypertriglyceridemia and weight gain, limit its generalized use. A phase II biomarker trial of the synthetic rexinoid, bexarotene (Targretin, LGD1069) demonstrated that bexarotene reduces expression of the proliferation markers Ki67 and Cyclin D1 in normal breast epithelium of postmenopausal women, suggesting that the cancer preventive effects of bexarotene are associated with its ability to elicit cell cycle blockade and inhibit proliferation of premalignant mammary epithelial cells. Here we hypothesize that the combination of repurposed drugs and some small molecule compounds with low dose rexinoid will minimize toxicity and result in effective anti-proliferative in premalignant cells. Therefore, we designed an image-based in vitro assay system to conduct high throughput screens to identify existing drugs and drug-like small molecules which enhance the growth suppressive effect of cancer preventive rexinoids. We used an automated 96 well platform to test the NIH clinical collection of drugs and a library of structurally diverse compounds (MayBridge Hitfinder) alone or in combination with 100nM bexarotene. Through multiparametric determination of cell counts, DNA content and cellular toxicity, we identified combination treatments of premalignant breast cells that fit the above criteria and report on dose-response studies and secondary screens for optimal dose and cell health. Validation of some of the candidate agents and plans for in vivo testing of their chemopreventive activity will be discussed. Citation Format: Ivan P. Uray, Powel H. Brown. High-throughput search for a combination cancer preventive treatment. [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 A58.

Dietary Administration of δ- and γ-Tocopherol Inhibits Tumorigenesis in the Animal Model of Estrogen Receptor–Positive, but not HER-2 Breast Cancer

Tocopherol, a member of the vitamin E family, consists of four forms designated as α, β, γ, and δ. Several large cancer prevention studies with α-tocopherol have reported no beneficial results, but recent laboratory studies have suggested that δ- and γ-tocopherol may be more effective. In two different animal models of breast cancer, the chemopreventive activities of individual tocopherols were assessed using diets containing 0.3% of tocopherol (α-, δ-, or γ-) or 0.3% of a γ-tocopherol rich mixture (γ-TmT). Although administration of tocopherols did not prevent human epidermal growth factor receptor 2 (HER2/neu)-driven tumorigenesis, δ- and γ-tocopherols inhibited hormone-dependent mammary tumorigenesis in N-methyl-N-nitrosourea (NMU)-treated female Sprague-Dawley rats. NMU-treated rats showed an average tumor burden of 10.6 ± 0.8 g in the control group at 11 weeks, whereas dietary administration of δ- and γ-tocopherols significantly decreased tumor burden to 7.2 ± 0.8 g (P < 0.01) and 7.1 ± 0.7 g (P < 0.01), respectively. Tumor multiplicity was also reduced in δ- and γ-tocopherol treatment groups by 42% (P < 0.001) and 32% (P < 0.01), respectively. In contrast, α-tocopherol did not decrease tumor burden or multiplicity. In mammary tumors, the protein levels of proapoptotic markers (BAX, cleaved caspase-9, cleaved caspase-3, cleaved PARP) were increased, whereas antiapoptotic markers (Bcl-2, XIAP) were inhibited by δ-tocopherol, γ-tocopherol, and γ-TmT. Furthermore, markers of cell proliferation (PCNA, PKCα), survival (PPAR-γ, PTEN, phospho-Akt), and cell cycle (p53, p21) were affected by δ- and γ-tocopherols. Both δ- and γ-tocopherols, but not α-tocopherol, seem to be promising agents for the prevention of hormone-dependent breast cancer.

Volumetric Contrast-Enhanced Ultrasound Imaging to Assess Early Response to Apoptosis-Inducing Anti-Death Receptor 5 Antibody Therapy in a Breast Cancer Animal Model

The objective of this study was to determine whether volumetric contrast-enhanced ultrasound (US) imaging could detect early tumor response to anti-death receptor 5 antibody (TRA-8) therapy alone or in combination with chemotherapy in a preclinical triple-negative breast cancer animal model. Animal experiments had Institutional Animal Care and Use Committee approval. Thirty breast tumor-bearing mice were administered Abraxane (paclitaxel; Celgene Corporation, Summit, NJ), TRA-8, TRA-8 + Abraxane, or saline as a controlon days 0, 3, 7, 10, 14, and 17. Volumetric contrast-enhanced US imaging was performedon days 0, 1, 3, and 7 before dosing. Changes in parametric maps of tumor perfusion were compared with the tumor volume and immunohistologic findings. Therapeutic efficacy was detected within 7 days after drug administration using parametric volumetric contrast-enhanced US imaging. Decreased tumor perfusion was observed in both the TRA-8-alone- and TRA-8 + Abraxane-dosed animals compared to control tumors (P = .17; P = .001, respectively). The reduction in perfusion observed in the TRA-8 + Abraxane group was matched with a corresponding regression in tumor size over the same period. Survival curves illustrate that the combination of TRA-8 + Abraxane improves drug efficacy compared to the same drugs administered alone. Immunohistologic analysis revealed increased levels of apoptotic activity in the TRA-8-dosed tumors, confirming enhanced antitumor effects. Preliminary results are encouraging, and volumetric contrast-enhanced US-based tumor perfusion imaging may prove clinically feasible for detecting and monitoring the early antitumor effects in response to combination TRA-8 + Abraxane therapy.

Bone-Derived IGF Mediates Crosstalk between Bone and Breast Cancer Cells in Bony Metastases

The continuous release of bone-stored growth factors after bone resorption promotes the colonization of circulating cancer cells. However, the precise role of each of the various growth factors remains unclear. In this study, we investigated the role of bone-derived insulin-like growth factor (IGF) in the development of bone metastases in an animal model of breast cancer. We found that local stimulation of calvarial bone resorption before cell inoculation stimulated subsequent bone metastases to that site in vivo, although inhibition of bone resorption inhibited bone metastases. Anchorage-independent growth of cancer cells was stimulated by the culture supernatants from resorbed bones, which contained elevated levels of IGF-I. This stimulation was blocked by IGF type I receptor (IGF-IR) neutralizing antibody, but not antibody targeting other bone-stored growth factors including TGF-β, fibroblast growth factors, and platelet-derived growth factors. Although recombinant human IGF-I caused IGF-IR tyrosine autophosphorylation, followed by activation of Akt and NF-κB in cancer cells, dominant-negative inhibition of IGF-IR, Akt, or NF-κB significantly reduced bone metastases with increased apoptosis and decreased mitosis in metastatic cells. Together, our findings suggest that bone-derived IGF-I bridges the crosstalk between bone and metastasized cancer cells via activation of the IGF-IR/Akt/NF-κB pathway. Disruption of this pathway therefore may represent a promising therapeutic intervention for bone metastasis.

Tamoxifen-resistant, ER-positive MAC 51 cell line with a high metastatic potential developed from a spontaneous breast cancer mouse model

We developed and characterized an estrogen-responsive and ER-positive murine breast cancer cell line (MAC51) from a spontaneous breast cancer animal model. These cells are overexpressed with K8, K18 and K19 proteins in an immunofluoresence assay. Upregulation of ER alpha was observed in the immunofluoresence assay, real-time PCR analysis and western blot assay. A colocalization experiment in MAC 51 showed cytoplasmic colocalization of K18 and K19 proteins with ER α. Real-time analysis of tumor samples from engrafted animals, MAC 51, metastatic liver and metastatic ovary revealed overexpression of K8 and K18 compared to the respective controls. A hormone responsive experiment in immunodeficient mice showed highly significant decreases in estrogen and tumor volume after 14 days ovariectomization. The tumorogenicity assay showed higher (3 × 10 (5)) and lower (3 × 10(4)) concentrations of MAC 51 cells that developed tumors within 2weeks post-transplantation. Tumor morphology and histology resembled a sarcoma pattern but our spontaneous model appeared in an adenocarcinoma pattern. Metastasis to different organs occurred through hematogenous and lymphatic routes. We assessed the potency of the anticancer effect in MAC 51 cells by treating various anticancer drugs with E2, followed by studying apoptotic gene expression profiles. E2 and E2+ tamoxifen-treated cells showed upregulation of apoptotic genes caspase 1, 3, 9, P53 and Bcl-xl but the tamoxifen- and paclitaxel-treated cells did not upregulate the apoptotic genes. Tamoxifen-resistant, ER-positive and high metastatic potential cell lines from murine origin are very rare. Also, estrogen greatly induced apoptosis in this cell line, hence MAC 51 has a greater application potential to evaluate low doses of estrogen with other targeted therapeutic drugs to treat breast cancer.

BreastDefend™ prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer

We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers.