Noriko Kanaya

Inhibition of the proliferation of acquired aromatase inhibitor-resistant breast cancer cells by histone deacetylase inhibitor LBH589 (panobinostat)

Aromatase inhibitors (AIs) are important drugs for treating postmenopausal patients with hormone receptor-positive breast cancer. However, acquired resistance to AI therapies is a significant problem. Our study has revealed that the histone deacetylase inhibitor LBH589 treatment abrogated growth of AI-resistant cells in vitro and in vivo, causing cell cycle G2/M arrest and induced apoptosis. LBH589 treatment also reduced the level of NF-κB1 which is overexpressed when AI resistance develops. Analyzing paired tumor specimens from 12 patients, we found that NF-κB1 expression was increased in recurrent AI-resistant tumors as compared to the paired primary tumors before AI treatment. This finding was consistent with up-regulated NF-κB1 expression seen in a collection of well-established AI-resistant cell lines. Furthermore, knockdown of NF-κB1 expression significantly suppressed the proliferation of AI-resistant cells. Treatment of AI-resistant cell lines with LBH589 suppressed NF-κB1 mRNA and protein expression. In addition, LBH589 treatment abrogated growth of AI-resistant tumors in mice, and was associated with significantly decreased levels of NF-κB1 in tumors. In all, our findings strongly support further investigation of LBH589 as a novel therapeutic strategy for patients with AI-resistant breast cancer, in part by suppressing the NF-κB1 pathway.

Protective effects of white button mushroom (agaricus bisporus) against hepatic steatosis in ovariectomized mice as a model of postmenopausal women

Nonalcoholic fatty liver disease (NAFLD) includes various hepatic pathologies ranging from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Estrogen provides a protective effect on the development of NAFLD in women. Therefore, postmenopausal women have a higher risk of developing NAFLD. Hepatic steatosis is an early stage of fatty liver disease. Steatosis can develop to the aggressive stages (nonalcoholic steatohepatitis, fibrosis and cirrhosis). Currently, there is no specific drug to prevent/treat these liver diseases. In this study, we found that white button mushroom (WBM), Agaricus Bisporus, has protective effects against liver steatosis in ovariectomized (OVX) mice (a model of postmenopausal women). OVX mice were fed a high fat diet supplemented with WBM powder. We found that dietary WBM intake significantly lowered liver weight and hepatic injury markers in OVX mice. Pathological examination of liver tissue showed less fat accumulation in the livers of mice on WBM diet; moreover, these animals had improved glucose clearance ability. Microarray analysis revealed that genes related to the fatty acid biosynthesis pathway, particularly the genes for fatty acid synthetase (Fas) and fatty acid elongase 6 (Elovl6), were down-regulated in the liver of mushroom-fed mice. In vitro mechanistic studies using the HepG2 cell line showed that down-regulation of the expression of FAS and ELOVL6 by WBM extract was through inhibition of Liver X receptor (LXR) signaling and its downstream transcriptional factor SREBP1c. These results suggest that WBM is protective against hepatic steatosis and NAFLD in OVX mice as a model for postmenopausal women.

Targeting breast cancer stem cells in triple-negative breast cancer using a combination of LBH589 and salinomycin.

The aim of this study is to investigate the efficacy of combining a histone deacetylase inhibitor (LBH589) and a breast cancer stem cells (BCSC)-targeting agent (salinomycin) as a novel combination therapy for triple-negative breast cancer (TNBC). We performed in vitro studies using the TNBC cell lines to examine the combined effect. We used the mammosphere and ALDEFLUOR assays to estimate BCSC self-renewal capacity and distribution of BCSCs, respectively. Synergistic analysis was performed using CalcuSyn software. For in vivo studies, aldehyde dehydrogenase 1 ALDH1-positive cells were injected into non-obese diabetic/severe combined immunodeficiency gamma (NSG) mice. After tumor formation, mice were treated with LBH589, salinomycin, or in combination. In a second mouse model, HCC1937 cells were first treated with each treatment and then injected into NSG mice. For mechanistic analysis, immunohistochemistry and Western blot analysis were performed using cell and tumor samples. HCC1937 cells displayed BCSC properties including self-renewal capacity, an ALDH1-positive cell population, and the ability to form tumors. Treatment of HCC1937 cells with LBH589 and salinomycin had a potent synergistic effect inhibiting TNBC cell proliferation, ALDH1-positive cells, and mammosphere growth. In xenograft mouse models treated with LBH589 and salinomycin, the drug combination effectively and synergistically inhibited tumor growth of ALDH1-positive cells. The drug combination exerted its effects by inducing apoptosis, arresting the cell cycle, and regulating epithelial–mesenchymal transition (EMT). Combination of LBH589 and salinomycin has a synergistic inhibitory effect on TNBC BCSCs by inducing apoptosis, arresting the cell cycle, and regulating EMT; with no apparent associated severe toxicity. This drug combination could therefore offer a new targeted therapeutic strategy for TNBC and warrants further clinical study in patients with TNBC.

Whole Blueberry Powder Inhibits Metastasis of Triple Negative Breast Cancer in a Xenograft Mouse Model Through Modulation of Inflammatory Cytokines

Triple negative breast cancer (TNBC) comprises approximately 15% of breast cancers and is associated with a poor prognosis. Many patients with TNBC relapse quickly and commonly develop metastases. There are no individualized targeted adjuvant or induction treatments for TNBC, and the current treatments are highly toxic. Development of chemoprevention methods using natural products would be beneficial to patients at risk of TNBC. To investigate the inhibitory effect of blueberries on inflammation-induced TNBC and identify the mechanism underlying modulation of inflammatory proteins by blueberries, we induced a proinflammatory microenvironment by feeding female MDA-MB-231 tumor-bearing mice a high fat western diet (W) with 5% whole blueberry powder (BB) and studied the effect on tumor formation and metastasis. We showed that mice fed a BB diet had significantly smaller tumors, less ulceration, and significantly less metastasis to the inguinal lymph nodes than mice fed a W diet. In BB-fed mice, serum levels of specific antiinflammatory cytokines were increased and specific cytokine expression was also altered. Together, these results suggest that blueberries may inhibit TNBC and TNBC-related metastasis by reducing inflammation via specific cytokine-driven pathways and thus reduce tumor growth and metastasis.

AroER Tri-Screen Is a Biologically Relevant Assay for Endocrine Disrupting Chemicals Modulating the Activity of Aromatase and/or the Estrogen Receptor

Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17β-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.

A phase I trial of mushroom powder in patients with biochemically recurrent prostate cancer: Roles of cytokines and myeloid-derived suppressor cells for Agaricus bisporus-induced prostate-specific antigen responses.

Each year in the United States, nearly 50,000 prostate cancer patients exhibit a rise in prostate‐specific antigen (PSA) levels, which can indicate disease recurrence. For patients with biochemically recurrent prostate cancer, we evaluated the effects of white button mushroom (WBM) powder on serum PSA levels and determined the tolerability and biological activity of WBM.

Identification of Estrogen-Related Receptor α Agonists in the Tox21 Compound Library

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

Use of dual mTOR inhibitor MLN0128 against everolimus-resistant breast cancer

PurposeHR+/HER2− aromatase inhibitor-resistant metastatic breast cancer can be treated with everolimus and a second AI until the cancer recurs. Targeting these everolimus-resistant patients with the latest standard of care, CDK4/6 inhibitors, has not been clearly addressed. Understanding the signaling transduction pathways, which everolimus resistance activates, will elucidate the mechanisms and offer treatment strategies of everolimus resistance.MethodsTo mimic the clinical setting, letrozole-resistant cells were used to generate an everolimus-resistant model (RAD-R). Reverse phase protein array (RPPA) was performed to reveal changes in the signaling transduction pathways, and expression levels of key proteins were analyzed. Inhibitors targeting the major signaling pathways, a CDK4/6 inhibitor palbociclib and a mTORC1/2 inhibitor (MLN0128), were evaluated to establish resistance mechanisms of RAD-R.ResultsRPPA results from RAD-R indicated changes to significant regulatory pathways and upregulation of p-AKT expression level associating with everolimus resistance. MLN0128, that inhibits the AKT phosphorylation, effectively suppressed the proliferation of RAD-R cells while treatment with palbociclib had no effect.ConclusionAmong the many signaling transduction pathways, which are altered post everolimus resistance, targeting dual mTORC1/2 is a possible option for patients who have recurrent disease from previous everolimus treatment.

AroER tri-screen™ is a novel functional assay to estimate both estrogenic and estrogen precursor activity of chemicals or biological specimens

Abstract The purpose of the study is to define AroER tri-screen’s utility for identifying endocrine-disrupting chemicals (EDCs) that target aromatase and/or estrogen receptor (ER), and to measure the total estrogenic activity in biological specimens. ER-positive, aromatase-expressing MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)-driven luciferase reporter plasmid to yield a new high-throughput screening platform—the AroER tri-screen. AroER tri-screen was capable of identifying estrogen precursors, such as cortodoxone, which function as estrogens through a two-step conversion process in aromatase-expressing tissue. Furthermore, the system proved useful for assessing EDC activity in biologically relevant samples. Estimating these activities is critical because natural estrogens and estrogenic EDCs are important factors in ER-positive breast cancer risk. As our research demonstrates, incorporating functionally active aromatase into the AroER tri-screen produces a powerful and unique tool to (1) identify new EDCs targeting aromatase and/or ER; (2) discover novel EDCs activated by aromatase; and (3) estimate overall estrogenic activities in biological samples as a potential intermediate risk factor for breast cancer.

Abstract P6-04-06: Targeting cancer stem cells in triple negative breast cancer by a combination treatment of LBH589 and salinomycin

Background and purpose: Triple-negative breast cancer (TNBC) is aggressive with poorer prognosis compared to breast cancer that is positive for hormone receptors or HER2. This can be explained further due in part to the existence of breast cancer stem cells (BCSCs). Our lab has found that histone deacetylase (HDAC) inhibitors are effective in the growth suppression of TNBC in vitro and in vivo. To understand the mechanisms involved, we performed RNA-seq analysis for TNBC cells treated with two HDAC inhibitors (LBH589 and Entinostat). Our RNA-seq analysis and studies from other laboratories have found that HDAC inhibitors may modify multiple signaling pathways with undesired effects due to its broad reactivity. To improve the efficacy of HDAC inhibitors in the treatment of TNBCs, we have examined the effects of LBH589 in combination with several drugs. Among them, Salinomycin works effectively with LBH589. It has been used as an antibiotic for farm animals, but has been identified from a screen of a large library of chemicals to target BCSCs (Gupta et al., Cell, 2009). The purpose of this study is to examine the synergistic effect between LBH589 and Salinomycin in the anticipation of their clinical utility and to evaluate a new target therapy to treat TNBC more efficiently and completely. Material and method: Two TNBC cell lines (HCC1937, MDA-MB-231) were used as models to examine the combined effects of Salinomycin and LBH589. Cell proliferation studies were performed through 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To evaluate the effects on BCSC population, mammosphere assay was used for estimating self-renewal capacity and ALDEFLUOR analysis was used for monitoring the distribution of BCSCs in treated samples. For tumor induction experiments, ALDH+ cells were injected into NSG mice. As a therapeutic study, single or combined use of LBH589 (10mg/kg) and Salinomycin (5mg/kg) were peritoneally injected into non-obese diabetic scid gamma (NSG) mice 3 days a week. Tumor volume, body weight and food intake were checked weekly. For mechanistic analysis, qPCR, Western blotting and IHC were performed for gene and protein expression using tumors from mice from different treatments. Results: Salinomycin and LBH589 worked synergistically in the suppression of the proliferation of TNBCs (IC50 was 68.8nM and 13.1nM, respectively). Similarly, both drugs inhibited mammosphere formation and ALDH positive population in a synergistic manner. The combination of LBH589 (16nM) with Salinomycin (60nM) reduced 60% of mammosphere formation and 94% of ALDH positive population compared with DMSO treatment. In a mouse model, the combination of LBH589 and Salinomycin had an inhibitory effect on tumor growth compared with the control group and the groups treated with single drug. In all groups, no side effect was seen. The results from mechanistic studies showed that the combination of LBH589 and Salinomycin regulated the Wnt/b-catenin pathway. Discussion: We have evidence that the combination of LBH589 and Salinomycin has a synergistic effect on TNBC through Wnt pathway and suggested this combination could potentially be a new therapeutic strategy for exploring targeting therapy in TNBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-06.