The Anticancer Effects of the Garlic Organosulfide Diallyl Trisulfide through the Attenuation of B[a]P-Induced Oxidative Stress, AhR Expression, and DNA Damage in Human Premalignant Breast Epithelial (MCF-10AT1) Cells.

Benzo[a]pyrene (B[a]P) is the most characterized polycyclic aromatic hydrocarbon associated with breast cancer. Our lab previously reported that the organosulfur compound (OSC), diallyl trisulfide (DATS), chemoprevention mechanism works through the induction of cell cycle arrest and a reduction in oxidative stress and DNA damage in normal breast epithelial cells. We hypothesize that DATS will inhibit B[a]P-induced cancer initiation in premalignant breast epithelial (MCF-10AT1) cells. In this study, we evaluated the ability of DATS to attenuate B[a]P-induced neoplastic transformation in MCF-10AT1 cells by measuring biological endpoints such as proliferation, clonogenicity, reactive oxygen species (ROS) formation, and 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA damage levels, as well as DNA repair and antioxidant proteins. The results indicate that B[a]P induced proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing AhR, ARNT/HIF-1ß, and CYP1A1 protein expression compared with the control in MCF-10AT1 cells. B[a]P/DATS's co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, AhR protein expression, and 8-OHdG levels compared with B[a]P alone and attenuated all the above-mentioned B[a]P-induced changes in protein expression, causing a chemopreventive effect. This study demonstrates, for the first time, that DATS prevents premalignant breast cells from undergoing B[a]P-induced neoplastic transformation, thus providing more evidence for its chemopreventive effects in breast cancer.

Novel Therapeutic Combination Targets the Growth of Letrozole-Resistant Breast Cancer through Decreased Cyclin B1.

As breast cancer cells transition from letrozole-sensitive to letrozole-resistant, they over-express epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and human epidermal growth factor receptor 2 (HER2) while acquiring enhanced motility and epithelial-to-mesenchymal transition (EMT)-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Interestingly, glyceollin inhibits the proliferation and tumor progression of triple-negative breast cancer (TNBC) and estrogen-independent breast cancer cells; however, it is unlikely that a single phytochemical would effectively target aromatase-inhibitor (AI)-resistant metastatic breast cancer in the clinical setting. Since our previous report indicated that the combination of lapatinib and glyceollin induced apoptosis in hormone-dependent AI-resistant breast cancer cells, we hypothesized that combination therapy would also be beneficial for hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by decreasing the expression of proteins associated with proliferation and cell cycle progression. While glyceollin + lapatinib treatment caused comparable inhibitory effects on the proliferation and migration in both cell lines, combination treatment selectively induced S and G2/M phase cell cycle arrest of the LTLT-Ca cells, which was mediated by decreased cyclin B1. This phenomenon may represent a unique opportunity to design novel combinatorial therapeutic approaches to target hormone-refractory breast tumors.

A Novel Allosteric Inhibitor Targets PLK1 in Triple Negative Breast Cancer Cells.

While Polo-like kinase 1 (PLK1) inhibitors have shown promise in clinical settings for treating triple-negative breast cancer tumors and other solid tumors, they are limited by their ability to bind non-selectively to the ATP kinase domain. Therefore, we sought to develop a PLK1 allosteric inhibitor targeting the PLK1 T-loop (a switch responsible for activation) and evaluate its effects in triple-negative breast cancer cells. A novel compound, RK-10, was developed based on an in silico model, and its effects on specificity, viability, migration, and cell cycle regulation in MCF-10A and MDA-MB 231 cells were evaluated. When MDA-MB 231 cells were treated with 0−50 µg/mL RK-10, phospho-PLK1 (Thr-210) was decreased in cells cultured adherently and cells cultured as mammospheres. RK-10 significantly inhibited viability after 24 h; however, by 48 h, 25−50 µM RK-10 caused >50% reduction. RK-10 attenuated wound healing by up to 99.7% and caused S and G2/M cell cycle arrest, which was associated with increased p21 expression. We developed a novel allosteric inhibitor which mediates anti-proliferative and anti-migratory properties through targeting phospho-PLK1 (Thr-210) in mammospheres and causing S phase and G2/M cell cycle arrest. Further development of PLK1 allosteric inhibitors may be a promising approach for TNBC treatment.

Glyceollins Trigger Anti-Proliferative Effects in Hormone-Dependent Aromatase-Inhibitor-Resistant Breast Cancer Cells through the Induction of Apoptosis.

Aromatase inhibitors (AIs) are standard treatment for estrogen-dependent postmenopausal breast tumors; however, resistance develops leading to tumor relapse and metastasis. We previously demonstrated that glyceollin inhibits proliferation, survival, and migration of hormone-independent letrozole-resistant breast cancer. Since many AI-resistant tumors remain hormone-dependent, identifying distinctions between estrogen-receptor-positive (ER+) and ER-negative (ER-) AI-resistant tumor response to therapy is critical. We hypothesize that treating ER+ letrozole-resistant T47D breast cancer cells (T47DaromLR) with a combination of 10 µM glyceollin and 0.5 µM lapatinib (a dual EGFR/HER2 inhibitor) will decrease cell proliferation through induction of apoptosis. The T47DaromLR cells were found to overexpress HER2 and MAPK while maintaining aromatase and ER levels compared to their letrozole-sensitive (T47Darom) counterparts. In the absence of estrogen stimulation, glyceollin ± lapatinib had no effect on the proliferation of the T47Darom cells, while glyceollin treatment caused 46% reduction in the proliferation of T47DaromLR cells, which was further diminished when combined with lapatinib. While neither agent influenced cell migration, glyceollin and lapatinib reduced S and G2/M phase cell entry and exclusively induced apoptosis by 1.29-fold in the T47DaromLR cells. Taken together, these results suggest that glyceollins and lapatinib may have potential as a novel combination therapeutic approach for hormone-dependent, letrozole-resistant tumors.

Diallyl Sulfide Attenuation of Carcinogenesis in Mammary Epithelial Cells through the Inhibition of ROS Formation, and DNA Strand Breaks.

Garlic has long been used medicinally for many diseases, including cancer. One of the active garlic components is diallyl sulfide (DAS), which prevents carcinogenesis and reduces the incidence rate of several cancers. In this study, non-cancerous MCF-10A cells were used as a model to investigate the effect of DAS on Benzo (a)pyrene (BaP)-induced cellular carcinogenesis. The cells were evaluated based on changes in proliferation, cell cycle arrest, the formation of peroxides, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, the generation of DNA strand breaks, and DNA Polymerase ß (Pol ß) expression. The results obtained indicate that when co-treated with BaP, DAS inhibited BaP-induced cell proliferation (p < 0.05) to levels similar to the negative control. BaP treatment results in a two-fold increase in the accumulation of cells in the G2/M-phase of the cell cycle, which is restored to baseline levels, similar to untreated cells and vehicle-treated cells, when pretreated with 6 µM and 60 µM DAS, respectively. Co-treatment with DAS (60 µM and 600 µM) inhibited BaP-induced reactive oxygen species (ROS) formation by 132% and 133%, respectively, as determined by the accumulation of H2O2 in the extracellular medium and an increase in 8-OHdG levels of treated cells. All DAS concentrations inhibited BaP-induced DNA strand breaks through co-treatment and pre-treatment methods at all time points evaluated. Co-Treatment with 60 µM DAS increased DNA Pol ß expression in response to BaP-induced lipid peroxidation and oxidative DNA damage. These results indicate that DAS effectively inhibited BaP-induced cell proliferation, cell cycle transitions, ROS, and DNA damage in an MCF-10A cell line. These results provide more experimental evidence for garlic's antitumor abilities and corroborate many epidemiological studies regarding the association between the increased intake of garlic and the reduced risk of several types of cancer.

Mammospheres of letrozole-resistant breast cancer cells enhance breast cancer aggressiveness.

Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and ß-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

Quantitative Proteomic Profiling Identifies a Potential Novel Chaperone Marker in Resistant Breast Cancer.

Development of aromatase inhibitor resistant breast cancer among postmenopausal women continues to be a major clinical obstacle. Previously, our group demonstrated that as breast cancer cells transition from hormone-dependent to hormone-independent, they are associated with increased growth factor signaling, enhanced cellular motility, and the epithelial to mesenchymal transition (EMT). Given the complexity of cancer stem cells (CSC) and their implications on endocrine resistance and EMT, we sought to understand their contribution towards the development of aromatase inhibitor resistant breast cancer. Cells cultured three dimensionally as mammospheres are enriched for CSCs and more accurately recapitulates tumors in vivo. Therefore, a global proteomic analysis was conducted using letrozole resistant breast cancer cells (LTLT-Ca) mammospheres and compared to their adherent counterparts. Results demonstrated over 1000 proteins with quantitative abundance ratios were identified. Among the quantified proteins, 359 were significantly altered (p < 0.05), where 173 were upregulated and 186 downregulated (p < 0.05, fold change >1.20). Notably, midasin, a chaperone protein required for maturation and nuclear export of the pre-60S ribosome was increased 35-fold. Protein expression analyses confirmed midasin is ubiquitously expressed in normal tissue but is overexpressed in lobular and ductal breast carcinoma tissue as well as ER+ and ER- breast cancer cell lines. Functional enrichment analyses indicated that 19 gene ontology terms and one KEGG pathway were over-represented by the down-regulated proteins and both were associated with protein synthesis. Increased midasin was strongly correlated with decreased relapse free survival in hormone independent breast cancer. For the first time, we characterized the global proteomic signature of CSC-enriched letrozole-resistant cells associated with protein synthesis, which may implicate a role for midasin in endocrine resistance.

Acquisition of Letrozole Resistance Through Activation of the p38/MAPK Signaling Cascade.

BACKGROUND/AIM: Previous reports identified a global proteomic signature of estrogen-independent letrozole resistant breast cancer cells, however, it remains unclear how letrozole-resistance is impacted when cells remain estrogen receptor positive (ER+). MATERIALS AND METHODS: To capture the protein expression profile associated with ER+ Aromatase inhibitor (AI) resistance, a global proteomic analysis was conducted using the letrozole-sensitive (T47Darom cells) and letrozole-resistant cells (T47DaromLR cells). To examine the molecular features associated with this phenotype Kaplan- Meier analysis, phospho-antibody arrays, proliferation and apoptosis assays were conducted. RESULTS: MAP3K6 was up-regulated in the T47DaromLR cells by 3.2-fold (p<0.01) which was associated with a decrease in relapse-free survival among breast cancer patients (p=0.0019). Members of the MAPK/p38 pathway (i.e., phospho-MKK6, phospho-p38, phospho-RSK1, phospho-RSK2, and p70S6K MAPK) were also increased in the T47DaromLR cells, while inhibiting p38 led to decreased proliferation and induction of apoptosis. CONCLUSION: Activation of the p38/MAPK pathway leads to ER+ AI-resistance.

A novel and cost-effective ex vivo orthotopic model for the study of human breast cancer in mouse mammary gland organ culture.

Mouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to in vivo carcinogenesis models. Here, we developed a new ex vivo MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics in vivo orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured in vitro with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected ex vivo and in vivo Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15 days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive ex vivo model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.

Phytochemicals: Current strategies for treating breast cancer.

Females with early-stage metastatic, estrogen-dependent breast cancer are generally treated with surgery, radiation and chemotherapy, or with more targeted approaches such as aromatase inhibitors (anastrozole or letrozole) or anti-estrogens (tamoxifen). Despite widespread successful usage of these agents for the treatment of breast cancer, resistance, tumor relapse and metastasis remain the principal causes of mortality for patients with breast cancer. While numerous groups have made major contributions toward an improved understanding of resistance mechanisms, the currently insufficient grasp of the most critical pathways involved in resistance is evident in the inability to adequately treat and drastically improve patient outcomes in females with hormone-refractory breast cancer, including triple negative breast cancer. Therefore, further investigation of novel therapeutic approaches is paramount to reveal previously unconsidered agents that could be utilized to treat metastatic disease. Numerous naturally occurring phytochemicals have recently gained interest as potential therapeutic breast cancer agents appear to directly affect estrogen-dependent and estrogen-independent breast cancer cell proliferation, potentially via affecting breast cancer stem cell populations. While numerous natural compounds have exhibited promise, they are limited by their bioavailability. Therefore, to effectively treat future hormone-refractory breast tumors, it is critical to adequately refine and formulate these agents for effective therapeutic use and delivery. Herein, the literature on the current state of phytochemicals is reviewed, including their limitations and potential as targeted therapies for breast cancer.

Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1.

Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a -3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.

Protein Kinase CK2 Expression Predicts Relapse Survival in ERα Dependent Breast Cancer, and Modulates ERα Expression in Vitro.

The heterotetrameric protein kinase CK2 has been associated with oncogenic transformation, and our previous studies have shown that it may affect estrogenic signaling. Here, we investigate the role of the protein kinase CK2 in regulating ERα (estrogen receptor α) signaling in breast cancer. We determined the correlation of CK2α expression with relapse free breast cancer patient survival utilizing Kaplan Meier Plotter (kmplot.com/analysis/) to mine breast cancer microarrays repositories. Patients were stratified according to ERα status, histological grade, and hormonal therapy. Luciferase reporter assays and flow cytometry were implemented to determine the impact of CK2 inhibition on ERE-mediated gene expression and expression of ERα protein. CK2α expression is associated with shorter relapse free survival among ERα (+) patients with grade 1 or 2 tumors, as well as among those patients receiving hormonal therapy. Biochemical inhibition of CK2 activity results in increased ER-transactivation as well as increased expression among ERα (+) and ERα (-) breast cancer cell lines. These findings suggest that CK2 may contribute to estrogen-independent cell proliferation and breast tumor progression, and may potentially serve as a biomarker and pharmacological target in breast cancer.

Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells.

Estrogen receptors (ERα and ERß) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERß receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERß-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules upregulate ERß activity while down regulating that of ERα.

Proteomic signatures of acquired letrozole resistance in breast cancer: suppressed estrogen signaling and increased cell motility and invasiveness.

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer.

Phytoalexins, miRNAs and breast cancer: a review of phytochemical-mediated miRNA regulation in breast cancer.

There is growing interest in the diverse signaling pathways that regulate and affect breast tumorigenesis, including the role of phytochemicals and the emerging role of microRNAs (miRNAs). Recent studies demonstrate that miRNAs regulate fundamental cellular and developmental processes at the transcriptional and translational level under normal and disease conditions. While there is growing evidence to support the role of phytoalexin-mediated miRNA regulation of cancer, few reports address this role in breast cancer. Recent reports by our group and others demonstrate that natural products, including stilbenes, curcumin, and glyceollins, could alter the expression of specific miRNAs, which may lead to increased sensitivity of cancer cells to conventional anti-cancer agents and, therefore, hormone-dependent and hormone-independent tumor growth inhibition. This review will discuss how dietary intake of natural products, by regulating specific miRNAs, contribute to the prevention and treatment of breast cancer.

In vitro and in vivo evaluation of novel anticancer agents in triple negative breast cancer models.

Triple negative breast cancer (TNBC) is subtype of breast disease devoid of the estrogen, progesterone, and Her2/neu receptors which are targets for pharmacological intervention. There is a need for novel anti-breast cancer agents that target TNBC. Therefore, novel isochalcone DJ52 was evaluated using the alamar blue dye exclusion assay, the luciferase colony assay, and xenograft models to determine its efficacy and potency. DJ52 significantly decreased proliferation of cells measured by using the alamar blue dye method and produced IC50 values of DJ52, DJ56, and DJ82 at 10-6M, 10-5M, and 10-5M, respectively. In vivo studies were conducted by injecting MDA-MB-231 cells into SCID mice to determine tumor regression was measured over 20 days. DJ52 at 50 mg/kg caused significant decrease in tumor volume (p value <.05) by nearly 50% compared with the control with vehicle alone. These data suggest that DJ52 has merit for further evaluation as a novel anticancer agent.

Insulin-like growth factor-1 signaling regulates miRNA expression in MCF-7 breast cancer cell line.

In breast carcinomas, increased levels of insulin-like growth factor 1 (IGF-1) can act as a mitogen to augment tumorigenesis through the regulation of MAPK and AKT signaling pathways. Signaling through these two pathways allows IGF-1 to employ mechanisms that favor proliferation and cellular survival. Here we demonstrate a subset of previously described tumor suppressor and oncogenic microRNAs (miRNAs) that are under the direct regulation of IGF-1 signaling. Additionally, we show that the selective inhibition of either the MAPK or AKT pathways prior to IGF-1 stimulation prevents the expression of previously described tumor suppressor miRNAs that are family and cluster specific. Here we have defined, for the first time, specific miRNAs under the direct regulation of IGF-1 signaling in the estrogen receptor positive MCF-7 breast cancer cell line and demonstrate kinase signaling as a modulator of expression for a small subset of microRNAs. Taken together, these data give new insights into mechanisms governing IGF-1 signaling in breast cancer.

Glyceollins as novel targeted therapeutic for the treatment of triple-negative breast cancer.

The purpose of this study was to investigate the effects of glyceollins on the suppression of tumorigenesis in triple-negative breast carcinoma cell lines. We further explored the effects of glyceollins on microRNA and protein expression in MDA-MB-231 cells. Triple-negative (ER-, PgR- and Her2/neu-) breast carcinoma cells were used to test the effects of glyceollins on tumorigenesis in vivo. Following this procedure, unbiased microarray analysis of microRNA expression was performed. Additionally, we examined the changes in the proteome induced by glyceollins in the MDA-MB-231 cells. Tumorigenesis studies revealed a modest suppression of MDA-MB-231 and MDA-MB-468 cell tumor growth in vivo. In response to glyceollins we observed a distinct change in microRNA expression profiles and proteomes of the triple-negative breast carcinoma cell line, MDA-MB-231. Our results demonstrated that the glyceollins, previously described as anti-estrogenic agents, also exert antitumor activity in triple-negative breast carcinoma cell systems. This activity correlates with the glyceollin alteration of microRNA and proteomic expression profiles.

Endocrine disruptor regulation of microRNA expression in breast carcinoma cells.

BACKGROUND: Several environmental agents termed "endocrine disrupting compounds" or EDCs have been reported to bind and activate the estrogen receptor-α (ER). The EDCs DDT and BPA are ubiquitously present in the environment, and DDT and BPA levels in human blood and adipose tissue are detectable in most if not all women and men. ER-mediated biological responses can be regulated at numerous levels, including expression of coding RNAs (mRNAs) and more recently non-coding RNAs (ncRNAs). Of the ncRNAs, microRNAs have emerged as a target of estrogen signaling. Given the important implications of EDC-regulated ER function, we sought to define the effects of BPA and DDT on microRNA regulation and expression levels in estrogen-responsive human breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the cellular effects of DDT and BPA, we used the human MCF-7 breast cancer cell line, which is ER (+) and hormone sensitive. Our results show that DDT and BPA potentiate ER transcriptional activity, resulting in an increased expression of receptor target genes, including progesterone receptor, bcl-2, and trefoil factor 1. Interestingly, a differential increase in expression of Jun and Fas by BPA but not DDT or estrogen was observed. In addition to ER responsive mRNAs, we investigated the ability of DDT and BPA to alter the miRNA profiles in MCF-7 cells. While the EDCs and estrogen similarly altered the expression of multiple microRNAs in MCF-7 cells, including miR-21, differential patterns of microRNA expression were induced by DDT and BPA compared to estrogen. CONCLUSIONS/SIGNIFICANCE: We have shown, for the first time, that BPA and DDT, two well known EDCs, alter the expression profiles of microRNA in MCF-7 breast cancer cells. A better understanding of the molecular mechanisms of these compounds could provide important insight into the role of EDCs in human disease, including breast cancer.

Environmental signaling and reproduction: a comparative biological and chemical perspective.

Reproduction is a critical element of life. Self-propagation in all living organisms ranging from bacteria to humans involves numerous common strategies. Underlying all reproductive strategies is the essential need for signaling molecules to initiate and maintain the process. In this paper we use comparative biological and chemical approaches to explore the origins and distribution of estrogen signaling as a pathway common to many life forms. In the process we illuminate the mechanisms whereby environmental agents alter reproduction and development. These mechanisms involve altered signaling pathways within cells and shifts in the targets of the signaling pathways to include regulators of gene transcription normally associated with other pathways. We also stress the role of signal cross talk in mediating hormone action.