A novel aspirin prodrug inhibits NFκB activity and breast cancer stem cell properties

INTRODUCTION: Activation of cyclooxygenase (COX)/prostaglandin and nuclear factor κB (NFκB) pathways can promote breast tumor initiation, growth, and progression to drug resistance and metastasis. Thus, anti-inflammatory drugs have been widely explored as chemopreventive and antineoplastic agents. Aspirin (ASA), in particular, is associated with reduced breast cancer incidence but gastrointestinal toxicity has limited its usefulness. To improve potency and minimize toxicity, ASA ester prodrugs have been developed, in which the carboxylic acid of ASA is masked and ancillary pharmacophores can be incorporated. To date, the effects of ASA and ASA prodrugs have been largely attributed to COX inhibition and reduced prostaglandin production. However, ASA has also been reported to inhibit the NFκB pathway at very high doses. Whether ASA prodrugs can inhibit NFκB signaling remains relatively unexplored. METHODS: A library of ASA prodrugs was synthesized and screened for inhibition of NFκB activity and cancer stem-like cell (CSC) properties, an important PGE2-and NFκB-dependent phenotype of aggressive breast cancers. Inhibition of NFκB activity was determined by dual luciferase assay, RT-QPCR, p65 DNA binding activity and Western blots. Inhibition of CSC properties was determined by mammosphere growth, CD44(+)CD24(−)immunophenotype and tumorigenicity at limiting dilution. RESULTS: While we identified multiple ASA prodrugs that are capable of inhibiting the NFκB pathway, several were associated with cytotoxicity. Of particular interest was GTCpFE, an ASA prodrug with fumarate as the ancillary pharmacophore. This prodrug potently inhibits NFκB activity without innate cytotoxicity. In addition, GTCpFE exhibited selective anti-CSC activity by reducing mammosphere growth and the CD44(+)CD24(−)immunophenotype. Moreover, GTCpFE pre-treated cells were less tumorigenic and, when tumors did form, latency was increased and growth rate was reduced. Structure-activity relationships for GTCpFE indicate that fumarate, within the context of an ASA prodrug, is essential for anti-NFκB activity, whereas both the ASA and fumarate moieties contributed to attenuated mammosphere growth. CONCLUSIONS: These results establish GTCpFE as a prototype for novel ASA-and fumarate-based anti-inflammatory drugs that: (i) are capable of targeting CSCs, and (ii) may be developed as chemopreventive or therapeutic agents in breast cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1868-7) contains supplementary material, which is available to authorized users

Estrogenic effects of herbal medicines from Costa Rica used for the management of menopausal symptoms

Objective: Outcomes from the Women's Health Initiative have demonstrated adverse effects associated with hormone therapy and have prioritized the need to develop new alternative treatments for the management of menopause and osteoporosis. To this end, we have been investigating natural herbal medicines used by Costa Rican women to manage menopausal symptoms. Methods: Seventeen plant species were collected and extracted in Costa Rica. To establish possible mechanisms of action and to determine their potential future use for menopause or osteoporosis, we investigated the estrogenic activities of the herbal extracts in an estrogen-reporter gene estrogen receptor (ER) β-Chemically Activated Luciferase Expression assay in U2-OS cells and in reporter and endogenous gene assays in MCF-7 cells. Results: Six of the plant extracts bound to the ERs. Four of the six extracts stimulated reporter gene expression in the ER-β-Chemically Activated Luciferase Expression assay. All six extracts modulated expression of endogenous genes in MCF-7 cells, with four extracts acting as estrogen agonists and two extracts, Pimenta dioica and Smilax domingensis, acting as partial agonist/antagonists by enhancing estradiol-stimulated pS2 mRNA expression but reducing estradiol-stimulated PR and PTGES mRNA expression. Both P. dioica and S. domingensis induced a 2ERE-luciferase reporter gene in transient transfected MCF-7 cells, which was inhibited by the ER antagonist ICI 182,780. Conclusions: This work presents a plausible mechanism of action for many of the herbal medicines used by Costa Rican women to treat menopausal symptoms. However, it further suggests that studies of safety and efficacy are needed before these herbs should be used as alternative therapies to hormone therapy.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

An Integrated Bioinformatics Approach Identifies Elevated Cyclin E2 Expression and E2F Activity as Distinct Features of Tamoxifen Resistant Breast Tumors

Approximately half of estrogen receptor (ER) positive breast tumors will fail to respond to endocrine therapy. Here we used an integrative bioinformatics approach to analyze three gene expression profiling data sets from breast tumors in an attempt to uncover underlying mechanisms contributing to the development of resistance and potential therapeutic strategies to counteract these mechanisms. Genes that are differentially expressed in tamoxifen resistant vs. sensitive breast tumors were identified from three different publically available microarray datasets. These differentially expressed (DE) genes were analyzed using gene function and gene set enrichment and examined in intrinsic subtypes of breast tumors. The Connectivity Map analysis was utilized to link gene expression profiles of tamoxifen resistant tumors to small molecules and validation studies were carried out in a tamoxifen resistant cell line. Despite little overlap in genes that are differentially expressed in tamoxifen resistant vs. sensitive tumors, a high degree of functional similarity was observed among the three datasets. Tamoxifen resistant tumors displayed enriched expression of genes related to cell cycle and proliferation, as well as elevated activity of E2F transcription factors, and were highly correlated with a Luminal intrinsic subtype. A number of small molecules, including phenothiazines, were found that induced a gene signature in breast cancer cell lines opposite to that found in tamoxifen resistant vs. sensitive tumors and the ability of phenothiazines to down-regulate cyclin E2 and inhibit proliferation of tamoxifen resistant breast cancer cells was validated. Our findings demonstrate that an integrated bioinformatics approach to analyze gene expression profiles from multiple breast tumor datasets can identify important biological pathways and potentially novel therapeutic options for tamoxifen-resistant breast cancers

Prolactin signaling in the regulation of estrogen receptor gene expression.

Prolactin signaling in the regulation of estrogen receptor gene expression

Emerging Roles of Ceramides in Breast Cancer Biology and Therapy

One of the classic hallmarks of cancer is the imbalance between elevated cell proliferation and reduced cell death. Ceramide, a bioactive sphingolipid that can regulate this balance, has long been implicated in cancer. While the effects of ceramide on cell death and therapeutic efficacy are well established, emerging evidence indicates that ceramide turnover to downstream sphingolipids, such as sphingomyelin, hexosylceramides, sphingosine-1-phosphate, and ceramide-1-phosphate, is equally important in driving pro-tumorigenic phenotypes, such as proliferation, survival, migration, stemness, and therapy resistance. The complex and dynamic sphingolipid network has been extensively studied in several cancers, including breast cancer, to find key sphingolipidomic alterations that can be exploited to develop new therapeutic strategies to improve patient outcomes. Here, we review how the current literature shapes our understanding of how ceramide synthesis and turnover are altered in breast cancer and how these changes offer potential strategies to improve breast cancer therapy

Estrogen Receptor-Regulated Gene Signatures in Invasive Breast Cancer Cells and Aggressive Breast Tumors

Most metastatic breast cancers arise from estrogen receptor α (ER)-positive disease, and yet the role of ER in promoting metastasis is unclear. Here, we used an ER+ breast cancer cell line that is highly invasive in an ER- and IKKβ-dependent manner. We defined two ER-regulated gene signatures that are specifically regulated in the subpopulations of invasive cells. The first consists of proliferation-associated genes, which is a known function of ER, which actually suppress rather than enhance invasion. The second signature consists of genes involved in essential biological processes, such as organelle assembly and vesicle trafficking. Importantly, the second subpopulation-specific signature is associated with aggressive disease and poor patient outcome, independently of proliferation. These findings indicate a complex interplay between ER-driven proliferation and invasion, and they define new ER-regulated gene signatures that are predictive of aggressive ER+ breast cancer

Histone deacetylase inhibitor-based chromatin precipitation for identification of targeted genomic loci

Histone deacetylase (HDAC) catalyzes the removal of acetyl marks from histones, effectively regulating gene expression. Genome wide chromatin immunoprecipitation (ChIP) studies have shown HDACs are present on numerous active and repressed genes. However, HDAC inhibitors (HDACi) only regulate a small subset of this population in a cell type dependent fashion. To determine genomic locations directly targeted by HDACi, we developed a chromatin precipitation method using a photoreactive HDAC inhibitor probe (photomate). We validate this method by analyzing several canonical HDACi regulated genes, CDKN1A and FOSL1, and compare it to traditional ChIP using HDAC1 antibodies. We show that HDACi target HDACs bound at the promoter regions but not gene bodies, differing from HDAC1 antibody-based ChIP in the case of CDKN1A. This approach is anticipated to be useful for genome wide studies to identify the subset of genes directly regulated by an HDACi in a given cell type

A Protective Role for Triacylglycerols during Apoptosis

Triacylglycerols (TAGs) are one of the major constituents of the glycerolipid family. Their main role in cells is to store excess fatty acids, and they are mostly found within lipid droplets. TAGs contain acyl chains that vary in length and degree of unsaturation, resulting in hundreds of chemically distinct species. We have previously reported that TAGs containing polyunsaturated fatty acyl chains (PUFA-TAGs) accumulate via activation of diacylglycerol acyltransferases during apoptosis. In this work, we show that accumulation of PUFA-TAGs is a general phenomenon during this process. We further show that the accumulated PUFA-TAGs are stored in lipid droplets. Because membrane-residing PUFA phospholipids can undergo oxidation and form reactive species under increased levels of oxidative stress, we hypothesized that incorporation of PUFAs into PUFA-TAGs and their localization within lipid droplets during apoptosis limit the toxicity during this process. Indeed, exogenous delivery of a polyunsaturated fatty acid resulted in a profound accumulation of PUFA phospholipids and rendered cells more sensitive to oxidative stress, causing reduced viability. Overall, our results support the concept that activation of TAG biosynthesis protects cells from lipid peroxide-induced membrane damage under increased levels of oxidative stress during apoptosis. As such, targeting triacylglycerol biosynthesis in cancer cells might represent a new approach to promoting cell death during apoptosis